Regulatory T-cell therapy

Abstract

The present invention relates to a lipid nanoparticle comprising an mRNA encoding FOXP3 and at least one second polypeptide wherein the lipid nanoparticle comprises a moiety capable of specifically binding to a molecule expressed on CD4 + T cells, and wherein the mRNA is modified to an increased stability compared to an unmodified mRNA.

Description

Technical Field

[0001] This disclosure and invention relates to targeted lipid nanoparticles for in vivo delivery of mRNA encoding a polypeptide / protein of interest to target cells. Specifically, the invention relates to targeted lipid nanoparticles comprising mRNA encoding FOXP3 and another protein of interest, typically an antigen or ligand-binding receptor. The invention extends to methods for producing targeted lipid nanoparticles and their in vivo use. Background Technology

[0002] Adoptive cell therapy (ACT), which involves administering functional immune cells to a subject, has become a mature and evolving immunotherapy approach for a variety of medical conditions, particularly malignant or infectious diseases. Tumor-infiltrating lymphocytes were initially shown to be effective in treating metastatic melanoma, followed by the development and clinical application of redirected T cells or NK cells that express chimeric antigen receptors (CARs) or heterologous T cell receptors (TCRs) to target different cellular target molecules. While initial approaches used immune cells with cytotoxic properties (e.g., cytotoxic T cells or NK cells) to target and kill unwanted or harmful cells in the body, regulatory T cells (Tregs) have recently been developed for ACT. Tregs possess immunosuppressive functions, modulate cellular pathological immune responses, and are crucial for maintaining immune tolerance. The suppressive properties of Tregs can be used therapeutically, for example, to improve and / or prevent inflammatory diseases, autoimmune diseases, and immune-mediated organ damage during transplantation.

[0003] The production of Treg immunotherapy typically involves the isolation, culture, and expansion of Tregs, followed by infusion into the patient. As part of this process, Tregs may be incubated with cytokines, drugs, other cells, or antigens to enhance their viability and function and / or increase their responsiveness to specific antigens. This process often further includes a Treg transduction step to introduce exogenous nucleic acid molecules that can encode antigen-specific receptors into cells, thereby achieving cell targeting. Typically, Tregs can be genetically engineered using chimeric antigen receptors (CARs) or T-cell receptors (TCRs) to target predetermined antigens.

[0004] The number of Tregs in the blood is low (approximately 5% to 10% of circulating lymphocytes), and since many immunotherapies require doses of hundreds of millions or even billions of cells, the isolation, culture, and expansion of Tregs are a crucial part of producing effective drugs for patients. Compared to effector T-cell therapies, Treg therapies typically require large-scale sorting and selection of cells from PBMCs and extended expansion in culture. The production cycle for a single therapy is usually at least 14 days, resulting in high commodity costs. Several different methods for Treg isolation and expansion have been reported in the literature. While some methods are used only in R&D environments, others have been used for large-scale manufacturing under GMP conditions. In particular, isolation under GMP conditions is performed using the CliniMACS system (CliniMACS™ instrument, Miltenyi Biotechnology), which provides clinical-scale magnetic enrichment of cells in a closed and sterile environment. Reported protocols include performing an enrichment step on CD25+ cells, which can be performed prior to a removal step (e.g., CD8+ cells). However, it has been reported that the cell purity of this method can be as low as 80%, which could be problematic because the presence of non-Treg cells can be detrimental, especially when Tregs are genetically engineered to achieve specificity for target antigens. Other groups have used flow cytometry-sorted Tregs in clinical trials, for example, using CD4+, CD25+, and CD127+ for sorting. However, current flow cytometry separation methods are performed using open systems, which are difficult to maintain aseptically, thus posing a problem for commercial manufacturing. Therefore, new methods for manufacturing Treg therapies that address the challenges of ex vivo cell manufacturing are needed.

[0005] The use of mRNA as a therapeutic agent is well documented in the literature, with its safety profile and rapid production capabilities making it highly promising in several therapeutic areas. In 2006, in vitro transduction of T cells using in vitro transcribed (IVT) mRNA encoding a CD19-specific chimeric antigen receptor demonstrated the cells' in vitro functionality (Rabinovich et al., 2006, Gene Ther., 17, 1027-1035). Numerous examples now exist of T cell transduction (electroporation) using CAR-encoding mRNA, targeting mesothelin, MCSP, Her-2 / neu, ErbB2, folate receptor, CD33, CD123, GD2, EGRF, VEGFR2, CD20, EpCAM, NY-ESO, or MART1. Furthermore, several groups have developed lipid nanoparticle (LNP) technology to enhance mRNA stability. Veiga et al. (2018, Nature Comm., 9, 4493, 1-9) used LNPs to encapsulate IL10-encoding mRNA and transduced leukocytes, while Billingsley et al. (2020, Nano Lett., 20, 1578-1589) reported the use of C14-4 LNPs to generate CAR-T cells in vitro. Additionally, in 2022, Rurik et al. (Science, 375, 91-96) generated CAR-T cells in vivo by encapsulating mRNA in LNPs targeting CD5.

[0006] However, despite significant progress in using mRNA to engineer cells for effector T immunotherapy, there have been no reports to date on how this technology has been applied to the field of regulatory T cells. Given the small proportion of Treg cells in the blood and the lack of Treg-specific markers, targeting mRNA / LNP is difficult, so it is not surprising that there are no documented cases of using mRNA for Treg immunotherapy. Summary of the Invention

[0007] This invention enables the in vivo production of genetically engineered Tregs using mRNA technology, thereby addressing the problems associated with in vitro production discussed above and overcoming the barriers to targeting Tregs with mRNA in vivo. Therefore, this invention enables the production of a cost-effective, safe, and effective Treg therapy for treating patients with conditions associated with adverse inflammatory responses, autoimmune diseases, or for the prevention of HvG / GvHD. This invention overcomes the problems and barriers of the prior art by specifically targeting CD4+ T cells with mRNA encoding FOXP3 (“FOXP3 mRNA”) in vivo. FOXP3 is a transcription factor that acts as a master regulator in the developmental and functional pathways of regulatory T cells. Transducing CD4+ T cells with FOXP3 can convert any CD4+ effector cells into cells with Treg-like repressive function. Using FOXP3 mRNA in this way requires targeting only CD4+ cells, rather than the smaller fraction of Treg cells in the blood. Furthermore, FOXP3 mRNA provided within the LNP containing the targeting portion can specifically target CD4+ cells and transform them into cells with Treg-like functions. It should be understood that the LNP technology can be used in this invention to target any one or more CD4+ T cell-specific markers, thereby achieving specific delivery of FOXP3 mRNA. The LNP of this invention further comprises mRNA encoding at least one additional protein of interest, particularly an antigen-specific receptor, which allows transduced and transformed CD4+ cells in vivo to specifically home to the desired location. The mRNA used in this invention is typically modified or contains modifications to increase its stability and reduce degradation.

[0008] Therefore, the present invention provides a lipid nanoparticle comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the lipid nanoparticle comprises a moiety capable of specifically binding to a molecule expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0009] Lipid nanoparticles (LNPs) can be any LNP capable of being formulated with mRNA and delivering it to target cells. Therefore, LNPs provide a delivery system for mRNA and can protect it from extracellular degradation while allowing cellular uptake and subsequent mRNA release. LNPs are formed from lipids and include LNPs based on cationic lipids or ionizable lipids (especially ionizable lipid derivatives), as well as LNPs containing other types of lipids (such as phospholipids, cholesterol, and PEG lipids), as discussed in further detail below. LNPs contain a moiety capable of specifically binding to molecules expressed on CD4+ T cells. This moiety can be any part or molecule capable of binding to CD4+ cells (e.g., CD4+ effector T cells or CD4+ Treg cells), but is typically an antibody, antibody fragment, ligand expressing a receptor, or aptamer. The moiety should bind specifically to its target, i.e., preferably not to other targets, or with reduced specificity for other targets. In one specific implementation, the portion is capable of binding to CD4, CD62L, CD25, CCR7, CTLA4, and / or CD45RA. The LNP may contain one or more different portions that can bind to any one or more molecules expressed on CD4+ T cells.

[0010] It should be understood that an LNP may contain multiple moieties (whether of the same or different types) capable of binding to one or more molecules expressed by CD4+ T cells. At least one of the moieties should be present on or on the LNP to enable that moiety to bind to its CD4+ T cell target. In particular, the CD4+ T cell binding domain of at least one of the moieties should be present on or on the LNP. Therefore, although this is possible, it is not necessary for all the moieties contained in the LNP to be present on the surface.

[0011] In one specific embodiment, the present invention provides a lipid nanoparticle comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the lipid nanoparticle comprises a moiety capable of specifically binding to CD4, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0012] As discussed above, the LNP of the present invention comprises mRNA encoding FOXP3 and at least one second polypeptide. The FOXP3 mRNA will be translated within the transduced cells to produce the FOXP3 protein, which will convert any transduced non-Treg CD4+ cells into cells with a Treg-like phenotype and function. Those skilled in the art will understand that the transformed cells may not be exactly the same as native Tregs, but have been shown to have inhibitory function. The second polypeptide can be any polypeptide that may need to be expressed in Treg therapy. In particular, the second polypeptide can enable transduced cells to target specific sites, such as tissues in a subject. Thus, the second polypeptide can be an antigen-binding receptor, such as a TCR or CAR. Alternatively, the second polypeptide can be another transcription factor that may help convert CD4+ effector cells into Treg cells (e.g., Helios), a polypeptide associated with persistence (i.e., increasing the persistence of transduced cells), a cytokine, a polypeptide acting as a safety switch, a polypeptide that promotes tissue repair, or a polypeptide that promotes inhibitory function. It should be understood that the LNP of the present invention may contain mRNA encoding two or more polypeptides, and therefore the mRNA contained in the LNP may encode three, four, five or six different polypeptides. Thus, the mRNA may encode a combination of the aforementioned polypeptides.

[0013] Therefore, in another embodiment, the present invention provides a lipid nanoparticle comprising mRNA encoding FOXP3 and an antigen-binding receptor (particularly CAR or TCR), wherein the lipid nanoparticle comprises a moiety capable of specifically binding to a molecule expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0014] Although LNPs provide protection against extracellular degradation of mRNA, it is well known that mRNA is unstable and can still be susceptible to degradation. Therefore, the mRNA used in this invention is modified to improve its stability (extracellular and / or intracellular stability), thereby providing potent therapy. Any mRNA modification that achieves the desired effect can be performed, including modifications to the 5' cap and 3' polyA tail. Further modifications to the mRNA can be made, for example, to enhance the translation of the encoded polypeptide.

[0015] The LNP of the present invention may further include other additional molecules, such as miRNA, siRNA, or shRNA, which can reduce the endogenous expression of peptides that provide inhibitory function or are detrimental to the Treg phenotype.

[0016] While the LNPs of the present invention are suitable for in vivo use to provide inhibitory Treg therapy, the present invention also covers in vitro applications. In particular, the present invention covers the in vitro production of inhibitory cell therapy by combining CD4+ T cells with the LNPs described above.

[0017] Therefore, the present invention provides a composition comprising CD4+ T cells and LNPs, the LNPs comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the lipid nanoparticles comprise a moiety capable of specifically binding to molecules expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0018] Furthermore, the present invention provides a method for in vitro transduction of CD4+ T cells, comprising the step of combining CD4+ T cells with an LNP comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP comprises a moiety capable of specifically binding to a molecule expressed on the CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0019] Additionally, there is a method for obtaining or acquiring transduced CD4+ T cells by incubating CD4+ T cells with an LNP containing mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP contains a moiety capable of specifically binding to a molecule expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0020] Alternatively, the present invention provides a CD4+ T cell comprising an LNP, the LNP comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP comprises a moiety capable of specifically binding to a molecule expressed on the CD4+ T cell, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0021] The present invention further provides a cell population comprising: CD4+ T cells of the present invention, namely CD4+ T cells obtainable by incubating CD4+ T cells with an LNP, the LNP comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP comprises a moiety capable of specifically binding to a molecule expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA; and / or CD4+ T cells containing an LNP, the LNP comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP comprises a moiety capable of specifically binding to a molecule expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA.

[0022] The present invention also provides a method for producing an LNP comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP comprises a moiety capable of specifically binding to a molecule expressed on CD4+ T cells, and wherein the mRNA is modified to have increased stability compared to unmodified mRNA. The method includes the step of incubating the mRNA with an ionizable lipid or a cationic lipid to prepare an LNP-mRNA formulation. The method may further include the steps of: coupling a moiety capable of specifically binding to a molecule expressed on CD4+ T cells to the LNP, and / or modifying the LNP and / or the moiety such that each comprises a homologous binding partner in a binding partner pair.

[0023] As described above, the LNPs, or ex vivo transduced cells or cell populations of the present invention can be used to deliver inhibitory cell therapy in vivo to subjects in need (e.g., subjects with undesirable inflammatory responses, subjects with autoimmune diseases, or subjects with GvHD or HvG).

[0024] Therefore, the present invention provides a pharmaceutical composition comprising the LNP, CD4+ T cells or cell population of the present invention.

[0025] The pharmaceutical composition may further comprise one or more excipients.

[0026] In this regard, the present invention provides a method for treating or preventing inflammation, autoimmune diseases, or HvG / GvHD in a subject, comprising the step of administering the LNP, CD4+ T cells, cell population, or pharmaceutical composition of the present invention to the subject. Alternatively, the present invention provides a method for promoting tissue remodeling or repair in a subject, comprising the step of administering the LNP, CD4+ T cells, cell population, or pharmaceutical composition of the present invention to the subject.

[0027] The LNPs, cells, cell populations, or pharmaceutical compositions of the present invention can be administered in one or more doses. It should be understood that, due to the transient nature of treatment, treatment of diseases requiring long-term maintenance of suppressive therapy may require administration of more than one dose over a period of time, such as at least two, three, or four doses.

[0028] From another perspective, the present invention provides an LNP, CD4+ cells, cell population or pharmaceutical composition for treatment.

[0029] More specifically, the present invention further provides an LNP, CD4+ cells, cell population or pharmaceutical composition for treating or preventing inflammation, autoimmune diseases or HvG / GvHD or promoting tissue remodeling or repair in a subject.

[0030] In another aspect, the present invention provides the use of LNPs, CD4+ cells, cell populations, or pharmaceutical compositions in the manufacture of medicaments for treating or preventing inflammation, autoimmune diseases, or HvG / GvHD in a subject, or for promoting tissue remodeling or repair in a subject. Attached Figure Description

[0031] Figure 1 The illustration shows an illustrative LNP included in this invention, which has a moiety capable of binding to CD4 and contains modified mRNA encoding FOXP3 and CAR.

[0032] Figure 2 The structures of representative lipids (DOTMA, ePC, DLin-MC3-DMA, ALC-0315, and Lipid H) that can be used in LNPs according to the present invention are shown, as well as two additional lipid compounds (cholesterol and DOPE) that can be included in the formulation. Detailed Implementation

[0033] The subject of this article, regarding products, methods, and uses, is an LNP containing mRNA encoding FOXP3 and a second polypeptide, which can be used in vivo or ex vivo to generate suppressive cell therapy by utilizing the mRNA to transduce CD4+ T cells. The LNP and mRNA modifications protect and stabilize the mRNA, enabling its delivery to specific target cells (CD4+ T cells).

[0034] In particular, the in vivo application of LNPs, as described herein, can provide cost-effective inhibitory cell therapies that do not require the production of ex vivo cell therapies, thereby addressing many of the problems associated with the manufacture of current Treg or inhibitory cell therapies.

[0035] Lipid nanoparticles or LNPs are used interchangeably herein and refer to nanoparticles formed from one or more lipids, as described below. When LNPs are mentioned, they include lipid-like nanoparticles or nanomaterials (LLNs). Typically, LNPs contain several different lipids (e.g., cationic or ionizable lipids), as well as one or more zwitterionic lipids, cholesterol, and PEG-lipids.

[0036] "Nanoparticles" refer to particles with a size less than 1 micrometer. Therefore, the particle size (e.g., diameter) of nanoparticles can be less than 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. Nanoparticles can have any shape, such as spherical, rod-shaped, wire-shaped, disc-shaped, cage-like, core-shell-shaped, etc. A group or plurality of LNPs of the present invention may include LNPs having substantially the same or similar shape and / or size, or may include LNPs with different shapes and / or different sizes. Those skilled in the art will understand that the shape / size of nanoparticles can depend on the process used to manufacture them.

[0037] "Lipids" are amphiphilic molecules containing three domains: a polar head group, a hydrophobic tail region, and a linker between the two domains. Lipids are generally insoluble in water but soluble in many organic solvents and are typically classified into at least three classes: simple lipids (including fats and oils), complex lipids (including phospholipids), and derived lipids (such as steroids). As used herein, "lipids" includes lipid derivatives and lipid-like substances, which typically contain more hydrophobic side chains than natural lipids. Typically, the LNPs described herein can be formulated from cationic or ionizable lipids, which may require encapsulation of RNA via electrostatic interactions.

[0038] "Catonic lipids" refer to lipids with a permanently positively charged head group. Cationic lipids that can be used in the LNP of this invention can be quaternary ammonium salt lipids. Cationic lipids include: 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) (and its analogues, such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)); or dimethyl bis(octadecyl)ammonium bromide (DDAB); 2,3-dioleoyloxy-N-[2-(sperminecarbamoyl)-ethyl]-N,N-dimethyl-1-propylaminetrifluoroacetate (DOPSA); ethylphosphatidylcholine (ePC) and 2-(( (((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentanop[a]phenanthrene-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methyl-1-ammonium bromide butane (BHEM-cholesterol).

[0039] Cationic lipids can be combined with other compounds for use in the LNPs of the present invention, such as with 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and / or carbonate apatite. It should be understood that the inclusion of additional compounds can improve or enhance the properties or function of the LNP. For example, the inclusion of carbonate apatite can increase the interaction between the LNP and the cell membrane.

[0040] "Ionizable lipids" refer to lipids that protonate at low pH, thus acquiring a positive charge, but remain neutral at physiological pH. Therefore, ionizable lipids are typically pH-sensitive. Normally, pH sensitivity results in less interaction with the anion exchange membranes of blood cells at physiological pH, but the low pH environment within endosomes promotes positively charged lipids, leading to membrane instability and endosome escape. Examples of ionizable lipids used in this invention include: (2S)-2,5-bis(3-aminopropylamino)-N-[2-(bisoctadecylamino)acetyl]pentanamide (DOGS; Transfectam); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxylamino)ethyl]-3,4-di[oleoyloxy]-benzamide (MV15), DC-cholesterol, N4- Cholesterol-spermine (GL67); 1,2-dilinoleoyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); 2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3). Other compounds that can be used in this invention include TT3, 5A2-SC8, A18-Iso5-2DC18, LP-01, and cKK-E12. Specific lipid derivatives include N 1 N 3 N 5 - Tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) derivatives, especially TT3.

[0041] Ionizable lipids can be further improved by incorporating biodegradable lipids, whereby the biodegradability of lipids can be enhanced by introducing ester structures. For example, MC3 can be modified by inserting ester bonds into the linker and lipid tail to produce lipid di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanedioate (L319). Other biodegradable lipids that can be used to produce the LNP of the present invention include: heptadecanedio-9-yl 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxoethyl)amino)octanoate (lipid 5); heptadecanedio-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (Lipid H(SM-102)) or ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0042] Exemplary lipids that can be used in the LNP of the present invention are shown in WO2021 / 077067 (incorporated herein by reference), for example, as shown in formulas I-XV.

[0043] Amphoteric ionizable lipids can be further used to form the LNPs of the present invention, such as DOPE.

[0044] The LNP of the present invention may comprise one or more cations and / or ionizable lipids. Specifically, the LNP may comprise one or more lipids in a concentration range of at least 0.1 mol% to 100 mol%, more specifically at least 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol%.

[0045] In addition to cationic or ionizable lipids, the LNPs of the present invention may also typically contain other lipid components, such as phospholipids (e.g., phosphatidylcholine or phosphatidylethanolamine, DOPE, DSPC, DSPE, SOPC, SOPE, etc.), neutral or anionic lipids (anionic lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutamate phosphatidylethanolamine, lysylphosphatidylglycerol or palmitoylphosphatidylglycerol), cholesterol lipids, steroids, steroid analogs, stabilized lipids or polyethylene glycol (PEG) functionalized lipids (e.g., PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEF-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified diacylglycerol, especially such as PEG-c-DOMG, PEG-c-DMA or PEG-s-DMG). Specifically, the LNP may contain at least one additional or other lipid component (other than cationic or ionizable lipids). Therefore, the LNP may contain two or three additional lipid components. Those skilled in the art will understand that the inclusion of other lipid components can alter or enhance the function or properties of the LNP, and that they should be able to select suitable combinations for use in this invention. In particular, the inclusion of PEG lipids can provide colloidal stability and prevent protein binding to the LNP; the use of neutral or anionic lipids can provide structural stability and determine biodistribution; and cholesterol lipids can regulate the bilayer density, fluidity, and uptake of the LNP. Additional or other lipids may be present in the LNP at concentrations ranging from 0.01 mol% to 95 mol%, specifically at least 10 mol%, 20 mol%, 30 mol%, 40 mol%, or 50 mol%.

[0046] The lipids, or additional lipid components used to form LNPs, are commercially available (e.g., available at Sigma / Merck) or can be synthesized using known methods, such as by reacting an epoxy-terminated alkyl chain with a polyamine core (available at Avanti Polar Lipids and Enamine, respectively) using Michael addition chemistry. Purification of the lipids can be performed using a known chromatographic system (e.g., CombiFlash) by fractionation.

[0047] The LNPs(s) of this invention can be manufactured by a variety of different methods, many of which are known and available in the art, such as sonication, stepwise mixing of an ethanol lipid solution with an aqueous mRNA solution, or microfluidic focusing. In particular, the LNPs(s) can be formed by ethanol dilution, as reported by Chen et al. in the *Journal of the American Chemical Society* (JACS, 2012, 134, 6948-6951) (incorporated herein by reference), i.e., by rapidly mixing an ethanol solution of lipids and other excipients with an equal volume of aqueous mRNA solution to form mRNA-LNPs. The mRNA-LNPs can be diluted with a buffered aqueous solution to reduce the ethanol content, wherein, in particular, both steps are performed in a microfluidic channel, especially one made of polydimethylsiloxane. Microfluidic systems for LNP formation are commercially available, for example… Microfluidic mixing system (Precision Nanosystems). Billingsley et al. have further reported the method for forming LNPs in the Journal of the American Chemical Society (JACS, 2020, 20, 1578-1589). The LNPs(multiple) of the present invention can be further characterized by dynamic light scattering, for example using a Zetasizer Nano ZS instrument or by using transmission electron microscopy.

[0048] It should be understood that the amount of mRNA contained in the LNP of this invention can vary and generally depends on the amount of mRNA used during LNP formation. The mRNA content of the LNP can be determined using standard assays, such as the Quant-iTRiboGreen RNA assay kit (Invitrogen).

[0049] LNPs may contain moieties capable of specifically binding to one or more portions of a molecule expressed on CD4+ T cells. Typically, an LNP may contain multiple moieties. Different moieties present in an LNP are within the scope of this invention. Therefore, an LNP may contain moieties capable of binding to different molecules or different epitopes within a molecule expressed by CD4+ T cells. Thus, the LNP of this invention may contain at least one, two, three, four, or five different moieties.

[0050] It should be understood that for an LNP to bind to molecules expressed by CD4+ T cells, at least one moiety of the LNP must be present on the surface of the LNP, or at least the binding domain of that moiety (i.e., the part that binds to molecules expressed by CD4+ T cells) must be present on the surface of the LNP. Typically, the surface of an LNP will have multiple mocies (or at least the binding domains of these mocies) to enable the LNP to bind to CD4+ T cells.

[0051] One or more moiety can bind directly to molecules expressed by CD4+ T cells, or indirectly, for example, through one or more other molecules.

[0052] One or more moieties can be coupled to the LNP of the present invention using any known technique. In particular, the LNP and the moieties can be modified with functional groups to bind one or more moieties to the LNP surface. Thus, typically, corresponding binding groups are introduced into the LNP and the moieties. For example, the LNP can be functionalized to introduce maleimide groups into the surface. Functionalization can typically occur across the entire surface of the LNP, but those skilled in the art will understand that functionalization of any site on the LNP surface can achieve moiety conjugation, thereby producing a target-specific LNP. One or more moieties can be functionalized to, for example, introduce thiol groups (e.g., using N-succinimide-S-acetylthioacetate (SATA)). When using SATA-maleimide chemistry, thioester coupling chemistry can be used to couple one or more moieties to the surface of the LNP.

[0053] Alternatively, the domains that bind to the moiety used in this invention can be linked to lipid-bound peptides to insert them into the LNP. One particular system involves using the CDQSSS peptide NlpA motif, which is lipid-bound in bacteria, and can be coupled to any domain capable of binding to the moiety of interest (e.g., a domain capable of binding to the Fc domain of an antibody moiety). Kedmi et al. reported an exemplary system in Nature Nanotech (2018, 13, 214-219) (incorporated herein by reference).

[0054] This moiety can bind to any molecule expressed by CD4+ T cells (i.e., any molecule present on the cell surface of CD4+ T cells). Specifically, it can bind to endogenously expressed molecules (i.e., molecules naturally expressed by CD4+ T cells), such as CD4, CD25, CD62L, CCR7, CTLA4, GITR, CD39, CD73, and / or CD45RA. In one most specific embodiment, the moiety can bind to CD4. In another specific embodiment, the molecule expressed by CD4+ T cells can be CD4+ T cell-specifically expressed (i.e., expressed only by CD4+ T cells and not by another cell type, or expressed by CD4+ T cells at significantly higher levels compared to other cell types, e.g., at least 50%, 60%, 70%, 80%, or 90% higher). The expression level of the molecule can be determined using standard methods, such as flow cytometry.

[0055] As used in this article, "CD4" refers to the immunoglobulin co-receptor glycoprotein that assists in communication between the TCR and antigen-presenting cells. CD4 is present on the surface of immune cells such as helper T cells and Tregs, and includes four immunoglobulin domains (D1 to D4) expressed on the cell surface. During antigen presentation, CD4 is recruited together with the TCR complex and binds to different regions of MHC class II molecules (CD4 binds to MHC IIβ2, while the TCR complex binds to MHC IIαI / βI). Human CD4 typically contains the sequence SEQ ID NO:36.

[0056] As discussed above, LNPs can bind to one or more molecules expressed by CD4+ T cells, such as CD4, and one or more of CD25, CD62L, CCR7, CD45RA, CTLA4, GITR, CD73, or CD39. Therefore, in one embodiment, LNPs can bind to CD4 and CD25, CD4 and CD62L, CD4 and CCR7, or CD4 and CD45RA.

[0057] The LNP used in this invention comprises a moiety capable of specifically binding to molecules expressed on CD4+ T cells. This moiety can be any moiety capable of binding to molecules expressed on CD4+ T cells. As previously mentioned, this moiety can be selected from, but is not limited to, antibodies, ligands of CD4+ T cell receptors, or aptamers.

[0058] As used herein, “antibody” refers to antibody structures of various forms, including but not limited to monoclonal and polyclonal antibodies (including full-length antibodies), multispecific antibodies (e.g., bispecific antibodies that can bind to two different molecules expressed on the surface of CD4+ T cells or two different epitopes within the same molecule), antibody fragments, immunoadhesins, and antibody-immunoadhesin chimeras that specifically recognize (i.e. bind) target antigens (molecules expressed on CD4+ T cells (e.g., Treg cells)). As long as the antibody can still bind to the target antigen, it can be a humanized antibody, a chimeric antibody, or an antibody that has undergone further genetic engineering. “Antibody fragment” includes a portion of a full-length antibody, particularly its variable domain, or at least its antigen-binding site. Examples of antibody fragments include Fab (antigen-binding fragment), scFv (single-chain variable region fragment), single-domain antibodies, diabody, dsFv, Fab', single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0059] A "single-chain antibody" (scFv) is an engineered antibody composed of light chain variable regions and heavy chain variable regions that are directly linked to each other or linked to each other via peptide linker sequences. Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., glycine) to 30 amino acids, for example, any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to any one of 12, 15, 18, 20, 21, 25, or 30 amino acids, for example, 5 to 30, 5 to 25, 6 to 25, 10 to 15, 12 to 25, 15 to 25, etc.

[0060] The "heavy chain variable region" or "VH" refers to the heavy chain segment of the antibody, which contains three CDRs intercalated between flanking segments called framework regions. These framework regions are more conserved than the CDRs and form a scaffold supporting the CDRs. The "light chain variable region" or "VL" refers to the light chain segment of the antibody, which contains three CDRs intercalated between the framework regions.

[0061] For members of the immunoglobulin superfamily (such as antibodies or TCRs), the "complementarity-determining region" or "CDR" refers to a highly variable loop within the variable region of the heavy or light chain of the immunoglobulin superfamily member. CDRs can interact with antigen conformation and largely determine binding to the antigen (although some framework regions are also known to be involved in binding). Both the heavy and light chain variable regions contain three CDRs (heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, numbered from the amino terminus to the carboxyl terminus).

[0062] CDRs of the heavy and light chain variable regions of immunoglobulin superfamily members can be predicted based on the heavy and light chain variable region sequences of an antibody using prediction software available in the art (e.g., the Abysis algorithm) or IMGT / V-QUEST software (e.g., the IMGT algorithm (ImMunoGeneTics) available at www.IMGT.org) (see, for example, Lefranc et al., 2009 NAR 37:D1006-D1012 and Lefranc 2003, Leukemia 17:260-266). CDR regions identified by either algorithm are considered equally applicable to this invention. The length of the CDRs can vary depending on the immunoglobulin superfamily member from which the CDRs are predicted and the differences between the heavy and light chains. Thus, the three heavy chain CDRs of a complete antibody can have different lengths (or the same length), and the three light chain CDRs of a complete antibody can have different lengths (or the same length). For example, the length of a CDR can range from 2 or 3 amino acids to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In particular, the length of a CDR can be from 3 to 14 amino acids, for example, at least 3 amino acids and less than 15 amino acids.

[0063] It should be noted that this paper follows Kabat nomenclature where necessary in order to define the location of the CDR (Kabat et al., 1991, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669).

[0064] Antibodies and their derivatives and fragments that specifically bind to target molecules can be prepared using methods well known to those skilled in the art. Such methods include phage display, methods for generating human antibodies or humanized antibodies, or methods for generating human antibodies using engineered transgenic animals or plants. Phage display libraries of partially or fully synthesized antibodies are available, and antibodies or fragments thereof capable of binding to target molecules can be screened. Phage display libraries of human antibodies are also available. Once identified, the amino acid or polynucleotide sequence encoding the antibody (or its derivatives or fragments) can be isolated and / or determined. The antibody sequence can be used to design suitable derivatives or fragments thereof.

[0065] The term "humanized antibody" refers to an antibody in which the frame region and / or core radix (CDR) are modified to contain CDRs of immunoglobulins from a species different from the parental immunoglobulin. Specifically, CDRs from rodents (e.g., mice) are transplanted into the frame region of a human antibody to prepare a humanized antibody. The humanized antibody binds to the same or similar antigens to the donor antibody that provides the CDR.

[0066] Antibodies that bind to molecules expressed on CD4+ T cells (e.g., antibodies that bind to CD4, CD25, CD62L, CTLA4, GITR, CD39, CD73, CCR7 and / or CD45RA) are well known in the art and are widely available commercially, such as those from Thermo Fisher Scientific, Miltenyi Biotechnology, Invitrogen, etc.

[0067] The sequence of an antibody that binds to CD4 is also provided in US8399621 (incorporated herein by reference), and includes antibodies containing VH with the amino acid sequence SEQ ID NO:1 and VL with the amino acid sequence SEQ ID NO:2.

[0068] The sequence of the antibody that binds to CD25 is provided in EP3216804 (incorporated herein by reference), and also includes an antibody having a heavy chain of SEQ ID NO:3 and a light chain of SEQ ID NO:4.

[0069] The sequences of antibodies that bind to CCR7 can be found in any of WO2007 / 00326, WO2012 / 043533, WO2014 / 151834 or WO2017 / 025569 (incorporated herein by reference).

[0070] The sequence of an antibody that binds to CD62L can be found in WO1994012215 (incorporated herein by reference), or alternatively, an antibody that binds to CD62L may comprise a heavy chain as shown in SEQ ID NO:47 and a light chain as shown in SEQ ID NO:48.

[0071] The sequences of antibodies that bind to CTLA4 can be found in WO2017106372, those that bind to GITR can be found in WO2015184099, those that bind to CD39 can be found in WO2017157948, and those that bind to CD73 can be found in WO2016081748 (all of which are incorporated herein by reference).

[0072] The ligand for the CD4+ T cell receptor can include a ligand that binds to any receptor expressed by CD4+ T cells, such as IL2R. In this respect, the ligand can be a cytokine or chemokine, or particularly a variant thereof, that binds specifically to CD4+ T cells. In this embodiment, the ligand can include a mutant IL2 protein, particularly an IL2 mutant protein with enhanced selectivity for binding to Tregs compared to other cell types expressing IL2R. Therefore, the IL2 mutant protein may exhibit increased selectivity for binding to CD25 (IL2α), decreased selectivity for binding to CD122 (IL2Rβ), and / or decreased selectivity for binding to CD132 (IL2Rγ). Compared to unmodified IL2 (e.g., IL2 comprising the sequence SEQ ID NO:38), the IL2 mutant protein may exhibit an increased selectivity for binding to CD25 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Alternatively or additionally, the selectivity of IL2 mutant proteins for binding to CD122 and / or CD132 may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to unmodified IL2 (e.g., IL2 comprising the sequence of SEQ ID NO:38). In particular, the IL2 mutant protein may contain only epitopes capable of binding to CD25, and not epitopes capable of binding to CD122 or CD132. Specific IL2 mutant proteins used in this invention include IL2 mutant proteins having amino acid substitutions of L18R, Q22E, and Q126H in SEQ ID NO:38.

[0073] As used in this article, "aptamer" refers to a single-stranded oligonucleotide (single-stranded DNA or RNA molecule) capable of specifically binding to a target molecule (a molecule expressed by CD4+ T cells). This is typically achieved through intramolecular folding of the single-stranded aptamer, thus exhibiting a 3D structure for target binding. Using well-known and established methods, aptamers can be readily developed to specifically bind to any target. In particular, aptamers can be selected using the magnetic bead-based exponential enrichment ligand systematic evolution technique (SELEX), where random DNA libraries containing a large number of sequences can be screened for binding to the target molecule. Aptamers can be of any length, but are typically 20 to 60 nucleotides, more typically 30 to 50, or approximately 40 nucleotides in length.

[0074] An exemplary DNA sequence encoding an anti-CD4 aptamer used in this invention may comprise or consist of the sequence of SEQ ID NO:5.

[0075] As discussed above, this moiety is capable of specifically binding to molecules expressed on CD4+ T cells. The term "specifically" or "specifically binding" as used herein refers to a moiety that recognizes and binds to a specific target antigen (a molecule expressed on CD4+ T cells) but substantially does not recognize or bind to other antigens; for example, its binding affinity is significantly lower than that of its target molecule (e.g., at least 10, 50, 100, 500, 1000, or 10000 times lower than its affinity for the target molecule). Therefore, the binding affinity of the moiety referred to herein to the target molecule expressed on CD4+ T cells can be at least 10, 50, 100, 500, 1000, or 10000 times greater than its affinity for binding to other proteins. The binding affinity of this portion can be determined using methods known in the art, such as the Lineweaver-Burk method, or commercially available binding modeling software, such as the 1:1 binding model in the BIAcore 1000 evaluation software. Suitablely, an HBS-P buffer system (0.01M Hepes, pH 7.4, 0.15M NaCl, 0.05% surfactant P20) is used. A portion that specifically binds to a target antigen from one species can also bind to an antigen from another species. A portion that specifically binds to a target antigen can also bind to different allelic forms of the antigen (alleloids, splice variants, isomers, etc.).

[0076] As described above, the LNP is complexed with mRNA encoding FOXP3 and at least one second polypeptide. Therefore, the LNP can encapsulate mRNA, wherein the mRNA can be encapsulated within the lipid portion of the LNP or within an aqueous space encapsulated by part or all of the lipid portion of the LNP.

[0077] The terms “mRNA” or “mRNA polynucleotide” used interchangeably herein refer to messenger ribonucleic acid, which contains multiple (i.e., two or more) linked nucleotides typically selected from uracil, cytosine, guanine, and adenine, or analogues thereof. Those skilled in the art should understand that, due to the degeneracy of the genetic code, many different mRNA polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the mRNA as defined herein, to reflect the codon usage for the polypeptide to be expressed in any particular host organism.

[0078] The mRNA molecules used in this invention may contain any one or more of the following: a 5' cap, a 5' UTR and a 3' UTR, a poly-A tail, and a nucleotide sequence / coding sequence that encodes a polypeptide of interest (e.g., FOXP3) and / or at least a second polypeptide. Further, the mRNA may be circularized.

[0079] The 5' cap is an essential structure for mRNA maturation and for ribosome recognition of mRNA for efficient protein translation. The cap further provides stabilization for the mRNA. Specifically, mRNA may contain a 5'cap1 structure or an analogue, which can be produced using vaccinia capping enzymes and 2'-O-methyltransferases, as discussed below. Commercial kits for introducing the 5' cap are available, such as Clean Cap (TriLink, San Diego, CA).

[0080] UTRs are located in the upstream (5') and downstream (3') domains of the mRNA coding region and can affect translation efficiency, localization, and stability. UTRs can range in length from 0 to 3000 nucleotides and can be added to the coding sequence using various methods, including primers annealed to different regions of the UTR. The coding sequence encodes the polypeptide of interest, and poly-A tails are generally essential for protein translation and mRNA stability. The 5' and 3' UTRs can be native endogenous 5' and 3' UTRs for the gene of interest, or they can be non-endogenous and can be incorporated as described above. UTRs can be optionally modified to improve mRNA stability, for example, by using UTRs with reduced AU content. Alternatively or additionally, Kozak sequences found within the 5' UTR that can affect translation can be modified. Therefore, modifications can be made that enhance or increase translation efficiency by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (determined by measuring the protein level of the gene of interest). Furthermore, the use of nucleotide analogs in the 5'UTR or 3'UTR can improve mRNA stability by inhibiting exonuclease degradation.

[0081] PolyA tails can be incorporated into mRNA by introducing polyT into the DNA template prior to in vitro transcription (IVT), as described below. Alternatively, polyA tails can be further extended after IVT using a polyadenylate polymerase (such as E. coli polyadenylate polymerase). PolyA tails can contain at least 5, 10, 20, 30, 50, 100, or 200 nucleotides. PolyA tails can be further modified to improve stability, for example by incorporating ATP analogs.

[0082] As described herein, the mRNA contained in the LNP is modified mRNA. Compared to an unmodified equivalent (e.g., identical or similar) mRNA molecule (unmodified mRNA), the mRNA is modified to improve its stability, particularly when exposed to the same environmental conditions. Specifically, stability can be improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the unmodified equivalent mRNA molecule. mRNA stability can be determined by any standard method known in the art, including measuring mRNA levels under identical conditions using well-known techniques such as Northern blotting. Improved mRNA stability can refer to the stability of the mRNA when it is complexed with or present in the LNP, the intracellular stability of the mRNA, or the extracellular stability of the mRNA. In other words, modified mRNA can have a longer half-life or a lower degradation rate compared to the unmodified equivalent mRNA. Specifically, compared with the unmodified equivalent mRNA, the half-life of the modified mRNA can be increased by at least 2, 3, 4 or 5 times, or the degradation rate can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared with the unmodified equivalent mRNA.

[0083] Any one or more modifications can be made to the mRNA to produce the desired effect, namely, enhanced stability. It should be understood that different modifications can be made to the mRNA to improve stability, or, from another perspective, to provide protection against degradation. Therefore, the mRNA molecules used in this invention can be modified with at least one, two, three, four, or five different types of modifications. It should be understood that, typically, the mRNA is modified before binding to or complexing with LNPs.

[0084] It should be understood that while multiple modifications to mRNA improve its stability, these modifications may also produce other desired effects, such as reducing the immunogenicity of the mRNA (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). Further modifications to mRNA to provide other beneficial properties are also within the scope of this invention. Therefore, mRNA may contain modifications that do not contribute to improved stability but, for example, reduce immunogenicity, provided that at least one modification according to the invention is present to improve mRNA stability.

[0085] Specifically, the mRNA may contain one or more of the modifications described above (e.g., a 5' cap or a poly-A tail), or may contain one or more modifications to these structures (as discussed). Additionally or alternatively, the 5' and / or 3' UTR may be modified as discussed, or the mRNA may be circularized.

[0086] The mRNA of the present invention may further or alternatively contain one or more nucleotide modifications. Nucleotide modification refers to the mRNA containing one or more modified nucleotides, nucleosides, or nucleobases, particularly chemically modified nucleotides, nucleosides, or nucleobases. Nucleotide modifications that can be performed on mRNA are well known in the art, for example in WO2011 / 012316 (incorporated herein by reference), wherein such modifications include the use of chemically modified nucleotides, such as pseudouridine, 5-methoxyuridine, 5-methylcytidine, N1-methylpseudouridine, N6-methyladenosine, 2-thiouridine, or 5-methyluridine. As previously stated, the mRNA molecules used in the present invention may contain any one or more of these chemically modified nucleotides / nucleosides. Typically, uridine and / or cytidine nucleotides / nucleosides within the mRNA molecule are chemically modified.

[0087] Other examples of chemically modified nucleotides / nucleosides that can be used in the mRNA of this invention include thiouridine, N1-methylpseuuridine, 5-hydroxymethylcytidine, 5-hydroxymethyluridine, 5-methylcytidine, 5-methoxyuridine, 5-methoxycytidine, 5-carboxymethyl esteruridine, 5-formylcytidine, 5-carboxycytidine, 5-hydroxycytidine, thiopheneguanidine, and / or 5-formyluridine.

[0088] Specifically, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the uridine nucleotide / nucleoside residues in the mRNA molecule used herein may be chemically modified. In one embodiment, all uridine nucleotide / nucleoside residues in the mRNA molecule used herein may be chemically modified. Additionally or alternatively, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cytosine nucleotide / nucleoside residues in the mRNA molecule used herein may be chemically modified. In one embodiment, all cytosine nucleotide / nucleoside residues in the mRNA molecule used herein may be chemically modified.

[0089] The mRNA molecules used in this invention can be modified using any known method, including by incorporating modified nucleotides during in vitro transcription, such as any of the modified nucleotides described above, particularly 2-thio-UTP and / or 5-methyl-CTP. The modified nucleotides are commercially available; for example, 2-thio-UTP and 5-methyl-CTP are available from TriLInk Bio Technologies. Different proportions of modified nucleotides can be incorporated into the mRNA of this invention.

[0090] The mRNA used in this invention can be generated by any method known in the art. For example, the mRNA can be generated from a pDNA template containing a DNA-dependent RNA polymerase promoter and a corresponding mRNA construct sequence. The pDNA can be amplified in bacterial cells (e.g., *E. coli*) and purified to obtain a pure, concentrated circular pDNA, which is then linearized to serve as a template for RNA polymerase transcription of mRNA. Linearization can be achieved by mixing plasmid DNA with restriction endonucleases in a reaction buffer according to well-established techniques. Impurities can be removed by tangential flow filtration or chromatography before in vitro transcription using RNA polymerase and nucleotide triphosphates (modified nucleotides can be incorporated, as discussed above). The 5' cap can be added to the resulting mRNA via co-transcription or enzymatic addition. Co-transcriptional capping can be achieved by adding a cap analog and guanosine triphosphate (GTP) to the transcription mixture. Enzymatic capping can be performed after purifying the mRNA from the in vitro transcription mixture, and the capping structure can be added to the mRNA structure using a vaccinia virus capping enzyme. After in vitro transcription, mRNA can be purified from impurities using any suitable technique (such as tangential flow filtration and / or chromatography). Commercial kits are available for mRNA production, such as the MEGAScript T7 kit (Invitrogen).

[0091] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or polypeptide should contain at least two amino acids, and there is no limit to the maximum number of amino acids that may be present.

[0092] As discussed above, the mRNA encodes FOXP3 to be expressed in the target cells (CD4+ T cells) to which the mRNA is delivered. Delivery of the mRNA encoding FOXP3 (“FOXP3 mRNA”) used in this article typically results in increased expression levels of FOXP3 in target cells (CD4+ T cells) transduced using the mRNA. “FOXP3” is short for Forkhead Box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and plays a master regulator role in the developmental and functional pathways of regulatory T cells. The term “FOXP3” as used in this article encompasses variants, isoforms, and functional fragments of FOXP3.

[0093] "Enhancing FOXP3 expression" means increasing the level of FOXP3 mRNA and / or protein in cells (or cell populations) compared to unmodified corresponding cells (or cell populations). For example, the level of FOXP3 mRNA and / or protein in cells (or such cell populations) modified according to the present invention can be increased to at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, or at least 150 times the level in corresponding cells (or such cell populations) not modified according to the present invention. Preferably, the cells are CD4+ cells (e.g., Tregs), or the cell population is a CD4+ T cell population (e.g., Tregs), as discussed in further detail below. Suitably, the level of FOXP3 mRNA and / or protein in cells (or such cell populations) modified according to the present invention can be increased to at least 1.5 times, 2 times, or 5 times the level in corresponding cells (or such cell populations) not modified according to the present invention.

[0094] Techniques for measuring specific mRNA and protein levels are well known in the art. mRNA levels in cell populations (e.g., Treg cells) can be measured using techniques such as Affymetrix ebioscience PrimeFlow RNA assay, Northern blotting, sequenced gene expression analysis (SAGE), or quantitative polymerase chain reaction (qPCR). Protein levels in cell populations can be measured using techniques such as flow cytometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0095] "FOXP3" or "FOXP3 polypeptide" is a polypeptide with FOXP3 activity, that is, a polypeptide capable of binding to FOXP3 target DNA and acting as a transcription factor regulating Treg development and function. Specifically, the FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO:6), for example, it may have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of the wild-type FOXP3 polypeptide. Therefore, the activity of the FOXP3 polypeptide encoded by the mRNA described herein may be increased or decreased compared to wild-type FOXP3. Techniques for measuring transcription factor activity are well known in the art. For example, the DNA-binding activity of a transcription factor can be measured by ChIP. The transcriptional regulatory activity of a transcription factor can be measured by quantifying the expression level of the genes it regulates. Gene expression can be quantified by measuring the levels of mRNA and / or protein produced by the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al., Annu. Rev. Immunol. 2006, 24:209-26, incorporated herein by reference). As discussed in detail below, FOXP3 or FOXP3 peptides include their functional fragments, variants, and isoforms, such as SEQ ID NO:6.

[0096] "Functional fragments of FOXP3" can refer to a portion or region of a FOXP3 polypeptide or an mRNA encoding a FOXP3 polypeptide with the same or similar activity as the full-length FOXP3 polypeptide or mRNA. Functional fragments can have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the activity of the full-length FOXP3 polypeptide or mRNA. Those skilled in the art will be able to generate functional fragments based on the known structural and functional characteristics of FOXP3. These are described, for example, in the following literature: Song X. et al., 2012. Cell reports, 1(6), pp. 665-675; Lopes JE et al., 2006. The Journal of Immunology, 177(5), pp. 3133-3142; and Lozano T. et al., 2013. Frontiers in oncology, 3, p. 294. Furthermore, WO2019 / 241549 (incorporated herein by reference) describes an N-terminal and C-terminal truncated FOXP3 fragment, for example, having the sequence SEQ ID NO:10 discussed below.

[0097] "FOXP3 variants" may include an amino acid sequence or nucleotide sequence that has at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity with the FOXP3 polypeptide or the mRNA encoding the FOXP3 polypeptide (e.g., SEQ ID NO: 6), preferably at least 95%, at least 97%, or at least 99%. FOXP3 variants may have the same or similar activity as wild-type FOXP3 polypeptides or mRNA, for example, they may have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of wild-type FOXP3 polypeptides or mRNA. Those skilled in the art will be able to generate FOXP3 variants based on the known structural and functional characteristics of FOXP3 and / or using conserved substitutions. Compared to wild-type FOXP3, FOXP3 variants may have similar or identical turnover times (or degradation rates) in Treg cells, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the turnover time (or degradation rate) of wild-type FOXP3 in Tregs. Some FOXP3 variants may have reduced turnover times (or degradation rates) compared to wild-type FOXP3, for example, the FOXP3 variants described in WO2019 / 241549 (incorporated herein by reference) with amino acid substitutions (e.g., S418E and / or S422A) at amino acids 418 and / or 422 of SEQ ID NO:6.

[0098] Suitablely, the FOXP3 polypeptide encoded by the mRNA molecule described herein may comprise or consist of a human FOXP3 polypeptide sequence (such as UniProtKB accession number Q9BZS1 (SEQ ID NO:6)) or a functional fragment or variant thereof.

[0099] In some embodiments of the invention, the FOXP3 polypeptide comprises or consists of an amino acid sequence or functional fragment thereof having at least 70% identity with SEQ ID NO:6. Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence or functional fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:6. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO:6 or a functional fragment thereof.

[0100] In some implementations, as discussed above, the FOXP3 peptide may contain a mutation at residues 418 and / or 422 of SEQ ID NO:6, as follows:

[0101] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSS

[0102] SLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVL

[0103] QVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKD

[0104] STLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMV

[0105] QSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGP

[0106] REAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQR

[0107] TLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR

[0108] EQRPSRCSNPTPGP(SEQ ID NO:7);

[0109] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSS

[0110] SLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVL

[0111] QVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKD

[0112] STLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMV

[0113] QSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGP

[0114] REAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQR

[0115] TLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR

[0116] SQRPARCSNPTPGP (SEQ ID NO:8); or

[0117] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSS

[0118] SLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVL

[0119] QVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKD

[0120] STLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMV

[0121] QSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGP

[0122] REAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQR

[0123] TLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR

[0124] EQRPARCSNPTPGP (SEQ ID NO:9).

[0125] In some embodiments of the present invention, the FOXP3 polypeptide may be truncated at its N-terminus and / or C-terminus to produce functional fragments. Specifically, the N-terminal and C-terminal truncated functional fragments of FOXP3 may comprise or consist of an amino acid sequence or a functional variant thereof having at least 80%, 85%, 90%, 95%, or 99% identity with it.

[0126] GGAHASSSL NPMPPSQLQL PTLPLVMVAPSGARLGPLPH LQALLQDRPH

[0127] FMHQLSTVDA HARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP

[0128] GINVASLEWV SREPALLCTF PNPSAPRKDS TLSAVPQSSY PLLANGVCKW

[0129] PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ CLLQREMVQS LEQQLVLEKE

[0130] KLSAMQAHLA GKMALTKASS VASSDKGSCC IVAAGSQGPV VPAWSGPREA

[0131] PDSLFAVRRH LWGSHGNSTF PEFLHNMDYF KFHNMRPPFT YATLIRWAIL

[0132] EAPEKQRTLN EIYHWFTRMF AFFRNHPATW KNAIRHNLSL HKCFVRVESE

[0133] KGAVWTVDEL EF (SEQ ID NO:10).

[0134] Suitablely, the FOXP3 peptide can be a variant of SEQ ID NO:6, such as a native variant. Suitablely, the FOXP3 peptide is an isomer of SEQ ID NO:6. For example, the FOXP3 peptide may contain a deletion of amino acids 72 to 106 relative to SEQ ID NO:6. Alternatively, the FOXP3 peptide may contain a deletion of amino acids 246 to 272 relative to SEQ ID NO:6.

[0135] Suitable, the FOXP3 polypeptide comprises SEQ ID NO:11 or a functional variant or functional fragment thereof.

[0136] Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence or functional fragment thereof having at least 70% identity with SEQ ID NO:11. Suitably, the FOXP3 polypeptide comprises an amino acid sequence or functional fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:11. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO:11 or a functional fragment thereof.

[0137] Suitablely, the FOXP3 peptide can be a variant of SEQ ID NO:11, such as a native variant. Suitablely, the FOXP3 peptide is an isomer of SEQ ID NO:11 or a functional fragment thereof. For example, the FOXP3 peptide may contain a deletion of amino acids 72 to 106 relative to SEQ ID NO:11. Alternatively, the FOXP3 peptide may contain a deletion of amino acids 246 to 272 relative to SEQ ID NO:11.

[0138] Unless otherwise stated, any variant of the amino acid sequence provided herein may have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the reference sequence (i.e., with the reference SEQ ID NO. specified herein). In particular, such variants retain the desired or necessary properties of their source parent molecule (i.e., the reference sequence). Thus, variant sequences may have the stated percentages of sequence identity provided that the variant sequence is functional.

[0139] The term "derivative" or "variant," used interchangeably herein in relation to the proteins or peptides of this invention, includes any substitution, variation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues in the sequence, provided that the resulting protein or peptide retains the desired function. For example, when the derivative or variant is a transcription factor, the desired function may be the ability of the protein to induce transcription of a specific gene, or when the derivative or variant is an antigen-binding receptor (such as a CAR or TCR), the desired function may be the activation of cells expressing the antigen upon binding. Alternatively, variants or derivatives as referred to herein are generally functional variants or derivatives. For example, a variant or derivative may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function compared to the corresponding reference sequence. A variant or derivative may have a similar or identical level of function compared to the corresponding reference sequence, or it may have a higher level of function (e.g., an increase of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%).

[0140] Typically, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs. For example, variants or derivatives may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity or ability compared to the corresponding reference sequence. Variant or derivatives may have similar or the same level of activity or ability compared to the corresponding reference sequence, or they may have a higher level of activity or ability (e.g., an increase of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%).

[0141] Proteins or peptides can also exhibit deletions, insertions, or substitutions of amino acid residues, resulting in silent changes and producing functionally equivalent proteins. As long as the intrinsic function is preserved, amino acid substitutions can be made cautiously based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0142] Conservative substitution can be carried out, for example, according to Table 1 below.

[0143] Table 1

[0144]

[0145] Derivatives may be homologs. As used herein, the term "homologous" refers to an entity that shares some degree of homology with wild-type amino acid and nucleotide sequences. The term "homology" may be equated with "identity."

[0146] Homologous or variant sequences may comprise amino acid sequences that share at least 80%, 85%, or 90% identity with the target sequence, preferably at least 95%, 96%, 97%, 98%, or 99%. Typically, variants will contain the same active sites, etc., as the target amino acid sequence. Although homology can also be considered from the perspective of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of this document, homology is preferably expressed from the perspective of sequence identity.

[0147] Homology comparisons can be performed visually, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0148] The percentage of homology or sequence identity can be calculated using consecutive sequences, i.e., by aligning one sequence to another, where each amino acid in one sequence is directly compared to its corresponding amino acid in the other sequence, one residue at a time. This is called "vacancy-free" alignment. Typically, this vacancy-free alignment is only performed on a relatively small number of residues.

[0149] While this is a very simple and consistent approach, it doesn't account for situations where, for example, an insertion or deletion in one nucleotide sequence within a pair of originally identical sequences can lead to subsequent codon misalignments, potentially causing a significant drop in the homology percentage during global alignment. Therefore, most sequence alignment methods are designed to produce optimal alignments that account for possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology as much as possible.

[0150] However, these more sophisticated methods assign a "vacancy penalty" to each vacancy encountered in the alignment, so that for the same number of identical amino acids, sequence alignments with fewer vacancies (reflecting a higher correlation between the two compared sequences) will receive a higher score than those with more vacancies. "Affine vacancy cost" is typically used to charge a relatively high cost for the presence of a vacancy and impose a smaller penalty on each subsequent residue within the vacancy. This is the most commonly used vacancy scoring system. Higher vacancy penalties will naturally result in optimized alignments with fewer vacancies. Most alignment programs allow modification of the vacancy penalty. However, when using such software for sequence alignment, the default values ​​are preferred. For example, when using the GCG Wisconsin Bestfit package, the default vacancy penalties for amino acid sequences are: vacancy: -12 points, each extension: -4 points.

[0151] Therefore, calculating the maximum homology / sequence identity percentage first requires generating the best alignment, taking into account gap penalties. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acid Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST software package (see Ausubel et al. (1999) ibid., Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), and the GNEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al. (1999) ibid., pp. 7-58 to 7-60). However, for certain applications, the GCG Bestfit program is preferred. Another tool called BLAST 2Sequences can also be used to compare protein sequences and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0152] While the final percentage of homology can be measured from the perspective of identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​usually used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix—the default matrix in the BLAST program suite. The GCG Wisconsin program typically uses common defaults or a custom symbol lookup table (if provided) (see the user manual for more details). For some applications, it is preferred to use the common defaults of the GCG package, or in the case of other software, the default matrix, such as BLOSUM62, is used. Suitablely, the percentage of identity is determined across the entire reference sequence and / or query sequence. Once the software has generated the best alignment, the percentage of homology, preferably the sequence identity percentage, can be calculated. The software typically includes this as part of the sequence comparison and generates numerical results.

[0153] A “fragment” typically refers to a selected region of a polypeptide or polynucleotide that has functional significance, such as a region that has a functional or coding segment. Therefore, a “fragment” refers to an amino acid or nucleic acid sequence that is part of (or a segment of) a full-length polypeptide or polynucleotide.

[0154] Such variants, derivatives, and fragments can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. In the case of insertion, synthetic DNA encoding the insertion, along with 5' and 3' flanking regions corresponding to the natural sequence on either side of the insertion site, can be prepared. The flanking regions will contain convenient restriction enzyme sites corresponding to the sites in the natural sequence, allowing the sequence to be cleaved using appropriate enzyme(s) and the synthetic DNA to be ligated into the cleavage. The DNA is then expressed according to the invention to produce a protein-encoding protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences; other known techniques may also be used.

[0155] Suitablely, the FOXP3 mRNA used in this invention encodes the FOXP3 polypeptide and may specifically comprise or consist of SEQ ID NO:12.

[0156] In some embodiments of the invention, the mRNA polynucleotide sequence encoding the FOXP3 polypeptide or variant may comprise a sequence or functional fragment thereof having at least 70% identity with SEQ ID NO:12. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or functional fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:12. In some embodiments of the invention, the mRNA encoding the FOXP3 polypeptide or variant may comprise or consist of SEQ ID NO:12 or a functional fragment thereof.

[0157] Suitable, the mRNA encoding the FOXP3 polypeptide may contain or consist of the polynucleotide sequence shown in SEQ ID NO:13.

[0158] In some embodiments of the invention, the mRNA polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or a functional fragment thereof having at least 70% identity with SEQ ID NO:13. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or a functional fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:13. In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises or consists of SEQ ID NO:13 or a functional fragment thereof.

[0159] Suitablely, the mRNA polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof can be codon-optimized. Suitablely, the polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof can be codon-optimized for expression in human cells.

[0160] One or more mRNA molecules contained in an LNP further encode at least one second polypeptide. Therefore, (multiple) mRNA molecules may encode at least one, two, three, four, or five polypeptides or additional polypeptides.

[0161] In one specific implementation, (multiple) mRNA molecules may encode a polypeptide that enables cells transduced using the mRNA to target specific locations within the subject, such as specific tissues within the subject. In particular, the mRNA may encode an antigen-binding receptor, such as a CAR or TCR.

[0162] As used herein, "chimeric antigen receptor" or "(multiple) CARs" refers to engineered receptors that confer cellular antigen specificity, such as CD4+ T cells, particularly Tregs. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs typically contain an extracellular domain (containing an antigen-specific targeting region referred to herein as the antigen-binding domain), a transmembrane domain, and an intracellular domain (optionally including one or more co-stimulatory domains and intracellular signaling domains). The antigen-binding domain is typically linked to the transmembrane domain via a hinge domain. The design of CARs and the various domains they may contain are well known in the art.

[0163] When a CAR binds to its target antigen, this results in the delivery of an activation signal to cells expressing that CAR. Therefore, the CAR specifically directs engineered cells toward the target antigen, particularly cells expressing the target antigen.

[0164] The antigen-binding domain of a CAR can be derived from or obtained from any protein or polypeptide that binds to (i.e., has affinity for) a desired target antigen or, more generally, a desired target molecule. This can be, for example, a ligand or receptor, or a physiologically binding protein of the target molecule or a portion thereof, or a synthetic or derived protein. The target molecule can typically be expressed on the surface of a cell (e.g., the target cell or cells near the target cell (for the bystander effect)), but is not required to be. Depending on the nature and specificity of the antigen-binding domain, the CAR can recognize soluble molecules, such as when the antigen-binding domain is based on or derived from a cellular receptor.

[0165] Antigen-binding domains most commonly originate from antibody variable chains (e.g., which are typically in the form of scFv), but can also be generated from T cell receptor variable domains or other molecules as described above (such as ligand receptors or other binding molecules).

[0166] CARs are typically expressed as peptides that also contain signal sequences (also known as leader sequences), specifically signal sequences that target the CAR to the cell membrane. These are usually located adjacent to or near the antigen-binding domain, often upstream of it. Therefore, the extracellular domain or extracellular region of a CAR can contain both a signal sequence and an antigen-binding domain.

[0167] The antigen-binding domain provides CARs with the ability to bind to predetermined antigens of interest. The antigen-binding domain preferably targets antigens of clinical interest or antigens at disease sites.

[0168] As described above, an antigen-binding domain can be any protein or peptide capable of specifically recognizing and binding to biomolecules (e.g., cell surface receptors or components thereof). Antigen-binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinant binding coupler of the biomolecule of interest. Illustrative antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands of cell surface molecules / receptors or their receptor-binding domains, and tumor-binding proteins. Although antigen-specific targeting domains may preferably be antibodies or antibody-derived domains, as discussed below, other antigen-specific targeting domains are also included, such as antigen-specific targeting domains formed by combinations of antigen peptides / MHC or HLA that can bind to the TCRs of Tcon cells active at transplantation, inflammation, or disease sites.

[0169] CARs can target any desired antigen or molecule. This can be selected based on the intended therapy and the condition to be treated. For example, the target antigen or molecule can be an antigen or molecule associated with a specific condition, or an antigen or molecule associated with the cells to be targeted for the treatment of the condition. Typically, this antigen or molecule is a cell surface antigen or molecule.

[0170] The term “targeting” is synonymous with “specific to” or “anti-”. In other words, a CAR recognizes a target molecule. Therefore, this means that a CAR can specifically bind to a particular or given antigen or target. Specifically, the antigen-binding domain of a CAR can specifically bind to a target molecule or antigen (more specifically, when the CAR is expressed on the surface of cells, especially immune effector cells). Specific binding can be distinguished from non-specific binding to non-target molecules or antigens. Therefore, cells expressing a CAR will be directed or redirected to specifically bind to target cells expressing target molecules or antigens, particularly target cells expressing the target antigen or molecule on their cell surface. Appropriately, as used herein, “specific binding” means that the antigen-binding domain does not bind to other proteins, or that its affinity is significantly reduced compared to its affinity for its target molecule (e.g., at least 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold lower than its affinity for the target molecule). Therefore, the binding affinity of the antigen-binding domain to the target molecule, as referred to herein, can be at least 10, 50, 100, 500, 1000, or 10000 times greater than its binding affinity to other proteins. The binding affinity of this antigen-binding domain can be determined using methods known in the art, such as the Lineweaver-Burk method, or commercially available binding modeling software, such as the 1:1 binding model in the BIAcore1000 evaluation software. Suitablely, an HBS-P buffer system (0.01 M Hepes, pH 7.4, 0.15 M NaCl, 0.05% surfactant P20) is used.

[0171] The antigens that this CAR can target include, but are not limited to, antigens expressed on cells associated with transplanted organs, autoimmune diseases, allergic diseases, and inflammatory diseases (e.g., neurodegenerative diseases). Those skilled in the art will understand that when the cells engineered to express the CAR are Treg cells, due to the bystander effect of Treg cells, the antigen may be present and / or expressed only at the transplant site, the site of inflammation, or the site of disease.

[0172] Antigens expressed on cells associated with neurodegenerative diseases include antigens presented on glial cells, such as MOGs.

[0173] Organ transplantation-related antigens and / or transplant organ-related cells include, but are not limited to, HLA antigens present in the transplant organ but not in the patient's body, or antigens upregulated during transplant rejection, such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, and CXCL11.

[0174] In one implementation, the CAR targets HLA antigens, particularly the HLA-A2 antigen.

[0175] Antibodies against this antigen are known in the art, and scFv can be readily obtained or generated based on known or available antibodies. In this regard, VH and VL, as well as CDR sequences, are publicly available to facilitate the preparation of such antibody-binding domains, for example in WO 2020 / 044055, the disclosure of which is incorporated herein by reference. Any antigen-binding domain or CDR, VH, and / or VL sequences disclosed in WO 2020 / 044055 may be used.

[0176] For example, a CAR can contain an antigen-binding domain that can bind to HLA-A2 (HLA-A2 may also be referred to as HLA-A*02, HLA-A02, and HLA-A*2 in this paper). HLA-A*02 is a specific group of class I major histocompatibility complex (MHC) alleles at the HLA-A locus.

[0177] A representative scFv that can be used for CARs in combination with HLAA2 may contain or consist of the sequence shown in SEQ ID NO:14 or a functional variant thereof.

[0178] CARs can contain hinged domains (also known as “spacer domains”), which are the extracellular portions of the CAR that separate the antigen-binding domain from the transmembrane domain. The hinge provides flexibility to access the target antigen. For example, long spacer regions provide additional flexibility to the CAR and allow for better access to proximal membrane epitopes.

[0179] Suitable hinge domains will be apparent to those skilled in the art (e.g., Guedan S. et al., 2018, Molecular Therapy-Methods & Clinical Development, 12, 145-156). Suitable hinge domains include, but are not limited to, CD28 hinge domains, CD8 hinge domains, IgG hinge domains, and IgD hinge domains. Preferably, the hinge domain is a CD8 hinge domain or a CD28 hinge domain.

[0180] Most preferably, the hinge domain is a CD8 hinge domain. Suitably, the hinge domain may contain an amino acid sequence as shown in SEQ ID NO:15, or a variant having at least 80% identity with SEQ ID NO:15.

[0181] Suitablely, the hinge domain is a CD28 hinge domain. Suitablely, the hinge domain may contain an amino acid sequence as shown in SEQ ID NO:16, or a variant having at least 80% identity with SEQ ID NO:16.

[0182] A CAR may contain a transmembrane domain, which refers to the CAR portion that anchors the CAR to the cell membrane. Therefore, the transmembrane domain can span or be present within the cell membrane. The transmembrane domain can originate from proteins containing extracellular and / or intracellular portions; thus, in addition to portions within or across the cell membrane, the transmembrane domains used herein can also attach to extracellular and / or intracellular residues derived from the original protein. For example, a transmembrane domain can attach to a hinge domain derived from the original protein; for instance, a transmembrane domain derived from CD8 can attach to a hinge domain derived from CD8. Further, a transmembrane domain derived from CD8 or CD28 can, for example, attach to a CD8 or CD28 co-stimulatory domain. Those skilled in the art will understand that transmembrane domains can also be synthetic (e.g., de novo designed) rather than derived from proteins possessing transmembrane domains. The presence of transmembrane domains in the cell membrane can be assessed using any suitable method known in the art, including fluorescent labeling using fluorescence microscopy.

[0183] Suitable transmembrane domains will be apparent to those skilled in the art. A transmembrane domain may contain a transmembrane sequence from any protein possessing a transmembrane domain (including any type I, II, or III transmembrane protein). The transmembrane domain of a CAR may also contain an artificial hydrophobic sequence. The transmembrane domain may be selected to be non-dimerizable. Examples of transmembrane (TM) domains used in CAR constructs include: 1) CD28 TM domain (Pule et al., Mol Ther, 2005, Nov; 12(5):933-41; Brentjens et al., CCR, 2007, Sep 15; 13(18Pt 1):5426-35; Casucci et al., Blood, 2013, Nov 14; 122(20):3461-72.); 2) OX40 TM domain (Pule et al., Mol Ther, 2005, Nov; 12(5):933-41); 3) 41BB TM domain (Brentjens et al., CCR, 2007, Sep 15; 13(18Pt 1):5426-35); 4) CD3ζ TM domain (Pule et al., Mol Ther, 2005, Nov; 12(5):933-41); 5) CD3ζ TM domain (Pule et al., Mol Ther, 2007, Sep 15; 13(18Pt 1):5426-35); 6) CD3ζ TM domain (Pule et al., Mol Ther, 2005, Nov; 12(5):933-41); 7) CD3ζ TM domain (Pule et al., Mol Ther, 2005, Nov; 12(5):933-41); 8) CD3ζ TM domain (Pule et al., Mol Ther, 2005, Nov; 12(5):933-41); 9) CD28 ... Ther, 2005, Nov; 12(5):933-41; Savoldo B, Blood, 2009, Jun 18; 113(25):6392-402.); 5) CD8α™ domain (Maher et al., Nat Biotechnol, 2002, Jan; 20(1):70-5.; Imai C, Leukemia, 2004, Apr; 18(4):676-84; Brentjens et al., CCR, 2007, Sep 15; 13(18Pt 1):5426-35; Milone et al., Mol Ther, 2009, Aug; 17(8):1453-64.; 6) ICOS™ domain; 7) CD4™ domain.

[0184] Most preferably, the CAR may comprise a CD8 transmembrane domain. Suitably, the transmembrane domain may comprise an amino acid sequence as shown in SEQ ID NO:17, or a variant having at least 80% identity with SEQ ID NO:17.

[0185] Suitablely, the CAR may include a CD28 transmembrane domain. Suitablely, the transmembrane domain may include an amino acid sequence as shown in SEQ ID NO:18, or a variant having at least 80% identity with SEQ ID NO:18.

[0186] Technicians should understand that variants of transmembrane domains must still be able to exist across or traverse the cell membrane, i.e., possess the properties of transmembrane domains.

[0187] CARs may contain an endodomain, which includes one or more intracellular signal transduction domains and optionally one or more co-stimulatory domains.

[0188] The “intracellular signal transduction domain” used in this article refers to the intracellular portion of CAR, which is involved in transducing information about effective CAR binding into the cell to trigger cellular functions, such as immunosuppression.

[0189] Suitable intracellular signal transduction domains will be apparent to those skilled in the art. Intracellular signal transduction domains are essential for transducing effector functional signals and guiding Tregs to perform their specific functions upon antigen binding. Examples of intracellular signal transduction domains include, but are not limited to, the ζ chain of the T cell receptor or any homolog thereof (e.g., the η chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction (such as CD2, CD5, and CD28 or their signal transduction domains). Intracellular signal transduction domains can be human CD3ζ signal transduction domains, FcyRIII, FcsRI, the cytoplasmic tail region of the Fc receptor, cytoplasmic receptors with an immune receptor tyrosine activation motif (ITAM), or combinations thereof.

[0190] Most preferably, the intracellular signal transduction domain may comprise an intracellular signal transduction domain of the human CD3ζ signal transduction domain. Suitably, the intracellular signal transduction domain may comprise an amino acid sequence as shown in SEQ ID NO:19, or a variant having at least 80% identity with SEQ ID NO:19.

[0191] CARs may also contain one or more costimulatory domains, which are intracellular portions of the CAR that can promote cellular function (e.g., immunosuppressive function), expansion, and / or persistence.

[0192] Therefore, a CAR can contain a complex domain formed by the fusion of one or more co-stimulatory domains with an intracellular signaling domain (e.g., CD3ζ). This complex domain can be referred to as a second-generation CAR, which can simultaneously deliver activation and co-stimulatory signals upon antigen recognition. The most commonly used co-stimulatory domain is the CD28 domain. This provides the most effective co-stimulatory signal—immune signal 2—which triggers proliferation. Suitable co-stimulatory domains will be apparent to those skilled in the art.

[0193] Therefore, the CAR preferably contains the CD28 co-stimulatory domain as described in SEQ ID NO:20 or a variant having at least 80% identity with SEQ ID NO:20.

[0194] Suitable, one or more co-stimulatory domains may include one or more TNF receptor family signaling domains, such as the signaling domains of OX40, 4-1BB, ICOS, or TNFRSF25.

[0195] Variants of intracellular signal transduction domains and / or costimulatory domains may have the same or similar functions as the compared wild-type intracellular signal transduction domains and / or costimulatory domains, for example, they may have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the wild-type domain function (e.g., the signal transduction capacity of the wild-type domain).

[0196] In some implementations, the CAR contains one or more signal peptides.

[0197] CARs may contain a leader sequence that targets the endoplasmic reticulum pathway for expression on the cell surface. An illustrative leader sequence is the CD8 leader sequence, for example, as shown in SEQ ID NO:21.

[0198] "TCR" or "T cell receptor" refers to a member of the immunoglobulin superfamily containing a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail. TCRs specifically bind to antigenic peptides encoded by receptors of the major histocompatibility complex (MHC). TCRs can be present on the surface of T cells or exist in a soluble form in the extracellular environment. TCRs are heterodimers, typically composed of α-β or γ-δ heterodimers, each chain having a constant region and a highly polymorphic variable region containing three CDRs.

[0199] Like other antigen-binding members of the immunoglobulin superfamily, the extracellular portion of the TCR chain comprises two immunoglobulin domains, an N-terminal variable domain (e.g., the α-chain variable domain or Vα, the β-chain variable domain or Vβ, typically amino acids 1-116 based on Kabat numbering), and a constant domain adjacent to the cell membrane (e.g., the α-chain constant domain or Cα, typically amino acids 117-259 based on Kabat; the β-chain constant domain or Cβ, typically amino acids 117-295 based on Kabat). As discussed above, the variable domain contains CDRs separated by a frame.

[0200] The CD4+ cells targeted by the LNP of this invention typically express endogenous TCRs. However, the mRNAs used in this invention can encode exogenous TCRs, particularly exogenous TCRs comprising both α and β chains. As used herein, the term "exogenous" refers to a polypeptide or protein not naturally expressed by the cell. Typically, the α and β chains of the TCRs encoded by the mRNAs used in this invention may each contain three CDRs, which may be able to bind to a combination of MHC / HLA and peptides expressed at the site of interest, thereby providing targeting capability to cells expressing exogenous TCRs. Many exogenous TCRs are known in the art and can be used according to this invention to provide transduced cells with targeting capability. For example, WO2019 / 202322 (incorporated herein by reference) discloses TCRs that bind to the myelin basic protein:HLA complex, which can be used in this invention. Self-reactive TCRs isolated from islets of pancreas from type 1 diabetic organ donors can also be used in this invention (as reported by Seay et al. in JCI insight, 2016, 1(20), e88242), and in particular, TCRs specific to GAD65 (555-567) presented in the HLA-DR*02:01 background, especially containing the CDR3β amino acid sequence of SEQ ID NO:39, can also be used.

[0201] mRNA can further encode transcription factors that may be associated with the transformation of CD4+ effector T cells into cells with a Treg phenotype. For example, mRNA may encode one or more of Helios, Sp1, NFAT, AP1, STAT5, CREB, ATF, FOXO1, or Smad.

[0202] In particular, Helios may be encoded by the mRNA molecule described herein, wherein Helios may comprise or consist of the amino acid sequence described in SEQ ID NO:22 or a functional variant or fragment thereof (e.g., having at least 80% sequence identity with SEQ ID NO:22 and having at least 50%, 60%, 70%, 80%, or 90% of the Helios transcription factor function).

[0203] The mRNA used in this invention may additionally or alternatively encode a polypeptide associated with the persistence of transduced cells, i.e., a polypeptide that increases the persistence of transduced cells (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to cells that do not express persistence-related polypeptides. When the polypeptide is expressed, the persistence of CD4+ T cells (e.g., Treg cells) is increased compared to CD4+ T cells (e.g., Treg cells) that do not express the polypeptide. As used herein, “persistence” is defined as the length of time a cell survives in a specific environment, such as in vivo (e.g., in a human patient or animal model). The persistence of CD4+ T cells disclosed herein is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to CD4+ cells that do not express the polypeptide. Persistence can be measured, for example, by determining the amount or number of transduced cells in a subject or patient that change over time, where cells expressing the peptide are compared to an equivalent cell type that does not express the peptide, or to unengineered cells. Transduced cells can be tracked, for example, using marker proteins (e.g., CD34 for cells that simultaneously express the RQR8 safety switch).

[0204] T cell persistence can also be determined by measuring the level of pSTAT5 signaling in cells or cell populations. Enhanced pSTAT5 signaling is associated with increased persistence, for example, an enhancement of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. pSTAT5 signaling can be measured using standard assays.

[0205] STAT5 is a transcription factor involved in the IL-2 signaling pathway, playing a crucial role in the function, stability, and survival of Tregs by promoting the expression of genes such as FOXP3, IL2RA, and BCLXL. To function and translocate to the nucleus, STAT5 requires phosphorylation. The IL-2-IL2R linkage phosphorylates STAT5 via specific signaling domains located in the IL-2Rβ and IL-2Rγ chains, activating Jak1 / Jak2 and Jak3 kinases, respectively. While JAK1 (or JAK2) can phosphorylate STAT5 without JAK3, transphosphorylation of JAK1 / JAK2 with JAK3 enhances STAT5 activity, thereby stabilizing kinase activity. Phosphorylation of STAT5 enables homodimerization, resulting in a functional transcription factor.

[0206] Several techniques for enhancing the persistence of CD4+ T cells, particularly Treg cells, have been reported. For example, techniques based on delivering constitutive STAT5 signaling to transduced cells have been reported. Such techniques involve using modified JAK and / or STAT5 molecules, which can be constitutively phosphorylated or dimerized, for example. WO2017 / 218850 (incorporated herein by reference) discloses a modified STAT5b molecule, STAT5bCA, that allows constitutive STAT5 signaling in Treg cells expressing it. Further, WO2019 / 053420 (incorporated herein by reference) discloses the use of STAT5 molecules engineered to include dimerizing domains, thereby allowing spontaneous intermolecular dimerization (e.g., STAT5 molecules can be engineered to include a leucine zipper), or alternatively, can be engineered to include functionally gaining mutations, such as the S710F mutation in STAT5.

[0207] Other reported persistence technologies are based on chimeric peptides containing STAT5-associated motifs and JAK1 and / or JAK2 binding motifs.

[0208] As used in this article, the “STAT5-associated motif” refers to an amino acid motif containing a tyrosine residue that, upon phosphorylation, binds to the STAT5 peptide. The STAT5-associated motif can be endogenously present in the cytoplasmic domains of transmembrane proteins, such as cytokine receptors, particularly IL2R. Specifically, the inner domain (or a truncated portion thereof) of the IL2R β chain contains both the STAT5-associated motif and the JAK1-binding motif and can be used in persistent techniques to provide pSTAT5 signaling.

[0209] As used herein, “JAK1 binding motif” refers to a BOX motif that allows the tyrosine kinase JAK1 to bind. Similarly, as used herein, “JAK2 binding motif” refers to a BOX motif that allows the tyrosine kinase JAK2 to bind. Suitable JAK1 binding motifs and JAK2 binding motifs are described, for example, by Ferraro & Lupardus (Frontiers in Endocrinology; 2017; 8(71), which are incorporated herein by reference).

[0210] WO2020 / 044055 (incorporated herein by reference) relates to a chimeric antigen receptor comprising a STAT5-associated motif and a JAK1-binding motif within a CAR intima. Specifically, the CAR intima comprises an intima or a truncated intima derived from an IL2RB polypeptide. Other techniques that may be used to encode mRNA for the purposes of this invention include chimeric cytokine receptors comprising, for example, a STAT5-associated motif and a JAK1 and / or JAK2-binding motif within an intima, and an exodomain that may be derived from a protein different from the intima (e.g., the exodomain may comprise an exodomain of a cytokine receptor different from that derived from the intima). Examples of chimeric cytokine receptors that may be used in the present invention are described in WO2018 / 038945, WO2017 / 029512, and WO2012 / 138858 (incorporated herein by reference). Persistent techniques based on the use of exogenous small molecule-induced signals have also been reported and can be used in this invention, such as the techniques disclosed in WO2019 / 169290 (incorporated herein by reference).

[0211] The mRNA used in this invention can encode one or more cytokines. As used herein, "cytokine" refers to cellular signaling molecules that regulate the immune system's response to inflammation and infection and facilitate intercellular communication in the immune response. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Specific cytokines that can be encoded include any one or more of IL10, IL35, IL33, TGFβ, IL37, and IL22.

[0212] Therefore, the mRNA can encode IL10 that includes or consists of the sequence described in SEQ ID NO:23 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:23).

[0213] The mRNA may further or alternatively encode IL35 (IL12α), which comprises or consists of the sequence described in SEQ ID NO:24 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:24).

[0214] The mRNA may encode TGFβ, which comprises or consists of the sequence described in SEQ ID NO:25 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:25).

[0215] The mRNA may encode IL33, which comprises or consists of the sequence described in SEQ ID NO:40 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:40).

[0216] The mRNA may encode IL37, which comprises or consists of the sequence described in SEQ ID NO:49 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:49).

[0217] The mRNA may encode IL22, which comprises or consists of the sequence described in SEQ ID NO. 50 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO. 50).

[0218] The mRNA can encode a safety switch. This safety switch peptide provides a suicide moiety to cells expressing the peptide. This is very useful as a safety mechanism, allowing the removal of cells administered to a subject when needed or more generally according to will or need (e.g., once the cells have exerted or completed their therapeutic effect).

[0219] Suicide moieties possess the ability to induce cell death, or more generally, to lead to cell elimination or loss. An example of a suicide moiety is a suicide protein encoded by a suicide gene that can be expressed intracellularly or on the cell along with a desired transgene. When expressed, the suicide protein causes cell elimination, thereby shutting down transgene expression. In this paper, the suicide moieties are suicide peptides, which are peptides that, under permissible conditions (i.e., conditions that are induced or activated), cause cell elimination.

[0220] The suicide moiety can be a polypeptide or amino acid sequence that can be activated by an activator administered to the subject to perform cell clearance activities, or be active in the presence of a substrate that can be administered to the subject, thereby performing cell clearance activities. In a particular embodiment, the suicide moiety can serve as a target for a separate cell scavenger administered to the subject. By binding to the suicide moiety, the cell scavenger can target the cells to be cleared. In particular, the suicide moiety can be recognized by an antibody, and when expressed on the cell surface, the binding of the antibody to the safety switch polypeptide results in the elimination or clearance of the cell.

[0221] The suicide moiety can be HSV-TK or iCasp9. However, it is preferred that the suicide moiety is or contains an epitope recognized by cell clearance antibodies or other binding molecules capable of inducing cell clearance. In this embodiment, the safety switch peptide is expressed on the cell surface.

[0222] In the context of cell clearance, the term “clearance” is synonymous with “removal,” “ablation,” or “elimination.” This term is used to encompass cell killing or inhibition of cell proliferation, thereby reducing the number of cells in a subject’s body. 100% complete removal may be ideal but may not necessarily be achievable. Reducing the number of cells in a subject or inhibiting their proliferation may be sufficient to produce beneficial effects.

[0223] Specifically, the suicide portion can be a CD20 epitope recognized by the rituximab antibody. Therefore, in a safety switch peptide, the suicide portion can contain a minimal epitope based on a CD20 epitope recognized by the rituximab antibody. Biosimilars of rituximab are available and usable. Those skilled in the art can readily prepare antibodies with rituximab binding specificity using conventional methods from the available amino acid sequence of rituximab.

[0224] Cells that also express a safety switch peptide containing this sequence can be selectively killed using rituximab or antibodies with rituximab binding specificity. The safety switch peptide is expressed on the cell surface, and cell death occurs when the expressed peptide is exposed to or comes into contact with rituximab or an antibody with the same binding specificity.

[0225] For example, suicide constructs of WO2013 / 153391 or PCT / EP2021 / 064053 (both incorporated herein by reference) can be used in cells or cell populations as described herein (e.g., CD4+ T cells, Tregs, or Treg populations).

[0226] The mRNA used in this invention can encode one or more polypeptides that can promote tissue repair. Such polypeptides include areg, CCN2 or CCN3, or functional variants or fragments thereof. The mRNA can encode an Areg that comprises or consists of the sequence described in SEQ ID NO:26 or functional variants or fragments thereof (e.g., having at least 80% identity with SEQ ID NO:26).

[0227] The mRNA may encode CCN2, which contains or consists of the sequence described in SEQ ID NO:41 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:41).

[0228] The mRNA may encode CCN3, which comprises or consists of the sequence described in SEQ ID NO:27 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:27).

[0229] The mRNA used in this invention can encode one or more peptides that can enhance the inhibitory function of cells expressing the peptide. Such peptides include CTLA4, LAG3, PDL1 / PDL2, ICOS, CD73, or CD39.

[0230] Therefore, the mRNA can encode CTLA4, which contains or consists of the sequence described in SEQ ID NO:42 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:42).

[0231] Therefore, the mRNA can encode LAG3, which contains or consists of the sequence described in SEQ ID NO:43 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:43).

[0232] Therefore, the mRNA can encode PDL1, which contains or consists of the sequence described in SEQ ID NO:44 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:44).

[0233] Therefore, the mRNA can encode PDL2, which contains or consists of the sequence described in SEQ ID NO:45 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:45).

[0234] Therefore, mRNA can encode ICOS, which contains or consists of the sequence described in SEQ ID NO:46 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:46).

[0235] Therefore, the mRNA may encode CD73, which contains or consists of the sequence described in SEQ ID NO:28 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:28).

[0236] Alternatively or additionally, the mRNA may encode CD39, which comprises or consists of the sequence described in SEQ ID NO:29 or a functional variant or fragment thereof (e.g., having at least 80% identity with SEQ ID NO:29).

[0237] The LNP of the present invention comprises mRNA encoding FOXP3 and at least one second polypeptide. FOXP3 and the at least one second polypeptide may be encoded by a single mRNA molecule or by a separate mRNA molecule contained within the LNP. Thus, in one embodiment, the LNP may comprise a single mRNA molecule encoding FOXP3 and at least one second polypeptide (e.g., an antigen-binding receptor such as CAR or TCR), or it may comprise two or more different mRNA molecules, each encoding a separate polypeptide (e.g., the LNP may comprise an mRNA molecule encoding FOXP3 and a second mRNA molecule encoding an antigen-binding receptor (e.g., CAR or TCR)). When encoding other polypeptides, additional independent mRNA molecules may be present to encode such polypeptides (e.g., polypeptides encoding persistence-related peptides, cytokines, safety switch polypeptides, etc.), or such polypeptides may be encoded by a single mRNA molecule that also encodes both FOXP3 and the second polypeptide. Alternatively, the mRNA molecule encoding FOXP3 and the second polypeptide may be provided separately, but this mRNA molecule may further encode a third, fourth, or other polypeptide as needed. When individual mRNA molecules are complexed with LNPs, these molecules can be provided in different proportions or amounts if different expression levels of peptides are required.

[0238] When two or more polypeptide sequences are expressed by a single mRNA molecule, these polypeptide sequences can be linked by sequences that allow the co-expression of two or more coding sequences. In particular, co-expression sequences (or co-expression sites) can enable the encoded protein or polypeptide to be expressed as discrete entities.

[0239] In particular, the co-expressed sequence can encode a self-cleaving sequence between encoded peptides. Specifically, the self-cleaving sequence can be a self-cleaving peptide. This sequence is automatically cleaved during protein production. Self-cleaving peptides that can be used are 2A peptides or 2A-like peptides known and described in the art, for example, in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041, which is incorporated herein by reference. 2A and 2A-like peptides are thought to induce ribosome jumping and produce a cleavage pattern in which the ribosome skips the formation of the peptide bond between the 2A peptide terminus and the downstream amino acid sequence. The “cleavage” occurs between a glycine residue and a proline residue at the C-terminus of the 2A peptide, meaning that the upstream cis-trans will add some additional residues at the end, while the downstream cis-trans will start from proline.

[0240] Suitable self-cleaving domains include the P2A, T2A, E2A, and F2A sequences, as shown in SEQ ID Nos. 30-33, respectively. These sequences can be modified to include the amino acid GSG at the N-terminus of the 2A peptide. Therefore, as a possible option, sequences corresponding to SEQ ID Nos. 30-33, but with GSG at their N-terminus, are also included. Such modified alternative 2A sequences are known in the art and have been reported. Alternative 2A sequences that can be used are shown in the literature of Donnelly et al. (ibid.), such as the TaV sequence.

[0241] The self-cutting sequences contained in (multiple) mRNA molecules can be the same or different.

[0242] The self-cleaving sequence may contain additional cleavage sites that can be cleaved by common enzymes present in the cell. This may facilitate complete removal of the 2A sequence post-translation. Such additional cleavage sites may, for example, include furin protease cleavage sites. Such cleavage sites are known in the art and may include, for example, RXXR (SEQ ID NO:34) or RRKR (SEQ ID NO:35).

[0243] The nucleic acid molecules / polynucleotides used in this article are codon-optimized. Codon optimization has previously been described in WO1999 / 41397 and WO 2001 / 79518 (both incorporated herein by reference). Different cells use specific codons differently. This codon bias corresponds to the relative abundance bias of specific tRNAs in a cell type. By altering codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, by intentionally selecting codons known to be less abundant for the corresponding tRNA in a specific cell type, expression can be decreased. Thus, additional levels of translational regulation can be obtained.

[0244] The LNP of the present invention may further comprise additional molecules or components, such as miRNA, siRNA, or shRNA. Specifically, while the mRNA contained in the LNP can enhance the expression of specific peptides in CD4+ T cells, those skilled in the art will understand that it may be necessary to additionally reduce or decrease the expression of specific endogenous peptides / proteins, particularly those that may be detrimental to providing repressive function or a Treg phenotype. This can be achieved using molecules well-known in the art, such as miRNA or siRNA. Typically, miRNA molecules are relatively short (average 22 nucleotides) and can interact with the 3'UTR of the target mRNA to inhibit translation. siRNA is also a short-chain RNA molecule (typically 20-24 nucleotides in length), but it is complementary to the target mRNA molecule and can induce mRNA cleavage. miRNAs or siRNAs that reduce the protein / mRNA levels of Tbet, RORγ, and / or Gata3 (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the absence of introduced miRNAs or siRNAs) may be included in the LNP of the present invention.

[0245] Although the main focus of this invention is to use the LNP of this invention in vivo to generate inhibitory cell therapy, thereby avoiding ex vivo manufacturing, LNPs can also be used to generate ex vivo transduced cells that can be used as inhibitory therapy.

[0246] Therefore, in this respect, the present invention provides a method for in vitro transduction of CD4+ T cells, the method comprising the step of combining CD4+ T cells with an LNP comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein the LNP comprises a moiety capable of specifically binding to a molecule expressed on the CD4+ T cells, and wherein the mRNA is modified to increase stability compared to unmodified mRNA.

[0247] Those skilled in the art will understand that the step of combining CD4+ T cells with the LNP of the present invention takes a sufficiently long time to allow transduction of CD4+ T cells using the mRNA contained in the LNP, for example, at least 1 hour, 2 hours, 5 hours, 24 hours or 48 hours.

[0248] Cells can be activated and / or expanded before or after the introduction of the LNP / mRNA described herein, for example by utilizing anti-CD3 monoclonal antibodies or both anti-CD3 and anti-CD28 monoclonal antibodies. Cell expansion can also be achieved by binding IL-2 in the presence of both anti-CD3 and anti-CD28 monoclonal antibodies. Where appropriate, IL-2 can be replaced by IL-15. Other components that can be used in cell (e.g., Treg) expansion protocols include, but are not limited to, rapamycin, all-trans retinoic acid (ATRA), and TGFβ. As used herein, “activation” means that cells are stimulated to the point of proliferation. As used herein, “expansion” means that cells or cell populations are induced to proliferate. The expansion of a cell population can be measured, for example, by counting the number of cells present in the population. The phenotype of the cells can be determined by methods known in the art, such as flow cytometry.

[0249] The term "CD4+ T cells" as used in this article refers to lymphocytes that endogenously express TCRs on their cell surface. CD4+ T cells also express CD4 on their cell surface. CD4+ T cells include helper T cells, which are activated when MHC class II molecules present peptide antigens on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including Th1, Th2, h3, Th17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses. CD4+ T cells can include memory T cells, a subset of antigen-specific T cells that persist long after the immune response subsides. Memory T cells rapidly proliferate to a great number upon re-exposure to their homologous antigens. Memory T cells comprise three subsets: central memory T cells (TCM cells), TEM cells, and TEMRA cells. Memory cells typically express the marker CD45RO.

[0250] The CD4+ T cells used in this article can be further classified as Treg cells.

[0251] Several distinct Treg subsets have been identified, which can express different specific markers or different levels of specific markers. Tregs typically express CD4, CD25, and FOXP3 (CD4+). + CD25 + FOXP3 + T cells that are biomarkers.

[0252] Treg cells can also express CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-induced TNF receptor).

[0253] Treg cells are present in peripheral blood, lymph nodes, and tissues, and the Tregs used in this article include thymus-derived native Treg cells (nTreg), peripherally generated Treg cells, and induced Treg cells (iTreg).

[0254] On the surface protein CD127 (CD4) + CD25 + CD127 - or CD4 + CD25 + CD127 低 In cases of Treg deficiency or low expression levels, Tregs can be identified using cell surface markers CD4 and CD25. The use of such markers to identify Tregs is known in the art and has been described, for example, by Liu et al. (JEM; 2006; 203; 7(10); 1701-1711).

[0255] Treg can be CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127 - T cells or CD4 + CD25 + FOXP3 + CD127 - / 低 T cells.

[0256] Suitablely, Tregs can be native Tregs (nTregs). As used herein, the term "native Treg" refers to thymic-derived Tregs. Native Tregs are CD4+ Tregs. + CD25 + FOXP3 + Helios + Neurociliin 1 + Compared to iTreg, nTreg showed higher expression of PD-1 (programmed cell death-1, pdcd1), neurocilitin 1 (Nrp1), Helios (Ikzf2), and CD73. nTreg can be distinguished from iTreg based on the expression of either Helios protein or neurocilitin 1 (Nrp1).

[0257] Tregs can possess Treg-specific demethylation regions (TSDRs). TSDRs are important methylation-sensitive elements that regulate Foxp3 expression (Polansky JK et al., 2008. European journal of immunology, 38(6), pp. 1654-1663).

[0258] Other suitable Tregs include, but are not limited to, Tr1 cells (which do not express Foxp3 and have high IL-10 production).

[0259] It is known that different Treg subgroups exist, including the initial Treg (CD45RA). + FoxP3 低 ), effector Treg / memory Treg (CD45RA) - FoxP3 高 ) and Tregs that produce cytokines (CD45RA) - FoxP3 低 "Memory Treg" is a gene that expresses CD45RO and is considered to be CD45RO. +The Tregs are Tregs that, compared to the initial Tregs, have increased CD45RO levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RO) and, preferably, do not express or have low levels of CD45RA (mRNA and / or protein) compared to the initial Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to the initial Tregs). "Cytokine-producing Tregs" refers to Tregs that, compared to the initial Tregs, do not express or have very low levels of CD45RA (mRNA and / or protein) (e.g., at least 80%, 90%, or 95% less CD45RA compared to the initial Tregs) and have low levels of FOXP3 compared to memory Tregs (e.g., 50%, 60%, 70%, 80%, or 90% less FOXP3 compared to memory Tregs). Tregs that produce cytokines can produce interferon-γ, and their in vitro inhibition rates may be lower than those of the initial Treg (e.g., 50%, 60%, 70%, 80%, or 90% lower than the initial Treg). Expression levels referred to herein can refer to mRNA or protein expression. Specifically, for cell surface markers such as CD45RA, CD25, CD4, and CD45RO, expression can refer to cell surface expression, i.e., the amount or relative amount of the labeled protein expressed on the cell surface. Expression levels can be determined by any method known in the art. For example, mRNA expression levels can be determined by Northern blotting / array analysis, and protein expression can be determined by Western blotting, or preferably by staining with antibodies expressing cell surface expression using FACS.

[0260] Specifically, Treg can be the initial Treg ( Tregs (initially regulatory T cells, naive regulatory T cells, or naive Tregs) are used interchangeably in this article to refer to Treg cells that express CD45RA (especially those expressing CD45RA on the cell surface). Therefore, naive Tregs are described as CD45RA-expressing Tregs. +Nascent Tregs typically refer to Tregs that are not activated by peptide / MHC through their endogenous TCR, while effector Tregs / memory Tregs are associated with Tregs that are activated through their endogenous TCR. Typically, nascent Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-nascent Treg cells (e.g., memory Treg cells). Alternatively, nascent Treg cells may express at least 2, 3, 4, 5, 10, 50, or 100 times more CD45RA than non-nascent Treg cells (e.g., memory Treg cells). The expression level of CD45RA can be readily determined by methods in the art (e.g., by flow cytometry using commercially available antibodies). Typically, non-nascent Treg cells do not express CD45RA or express low levels of CD45RA.

[0261] In particular, the initial Treg may not express CD45RO and can be considered as CD45RO. - Therefore, naïve Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less CD45RO than memory Tregs, or alternatively, at least 2, 3, 4, 5, 10, 50, or 100 times less CD45RO than memory Tregs.

[0262] Although initial Tregs express CD25 as described above, the expression level of CD25 may be lower than that in memory Tregs, depending on the source of the initial Treg. For example, for initial Tregs isolated from peripheral blood, the expression level of CD25 may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than that in memory Tregs. Such initial Tregs can be considered to have moderate to low levels of CD25 expression. However, those skilled in the art should understand that initial Tregs isolated from umbilical cord blood may not show this difference.

[0263] Typically, the initial Treg, as defined in this article, can be CD4. + CD25 + FOXP3 + CD127 低 CD45RA + .

[0264] The low expression of CD127 used in this article refers to its low expression compared to CD4 from the same subject or donor. +CD127 expression levels were lower in non-regulatory or Tcon cells compared to other cells. Specifically, it was lower in CD4+ cells from the same subject or donor. + Compared to non-regulatory or Tcon cells, naïve Treg cells may express less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of CD127. CD127 levels can be assessed using standard methods in the art, including flow cytometry of cells stained with an anti-CD127 antibody.

[0265] Typically, naïve Tregs do not express or express low levels of CCR4, HLA-DR, CXCR3, and / or CCR6. In particular, naïve Tregs may express even lower levels of CCR4, HLA-DR, CXCR3, and CCR6 than memory Tregs, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower levels.

[0266] Initial Tregs can further express other markers, including CCR7. + and CD31 + .

[0267] The isolated initial Tregs can be identified by methods known in the art, including by determining the presence of any one or more of the markers discussed above on the surface of the isolated cells. Examples include CD45RA, CD4, CD25, and CD127. 低 This can be used to determine whether cells are naïve Tregs. Methods for determining whether isolated cells are naïve Tregs or have the desired phenotype can be performed as discussed below regarding the additional steps that may be performed, and methods for determining the presence and / or expression levels of cell markers are well known in the art and include, for example, flow cytometry using commercially available antibodies.

[0268] Suitably, the CD4+ T cells (e.g., Tregs) used in the ex vivo method of the present invention are isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject. Suitably, the subject from whom the PBMCs are obtained is a mammal, preferably a human. If the cells are intended to be administered to a subject after transduction, the cells may be type-matched (e.g., HLA matching) or may be autologous cells of the subject to whom the transduced cells are to be administered. Suitably, the subject to be treated is a mammal, particularly a human. The cells can be generated ex vivo from the patient's own peripheral blood (first method), or from donor peripheral blood in the case of hematopoietic stem cell transplantation (second method), or from peripheral blood of an unrelated donor (third method).

[0269] Suitablely, CD4+ T cells (e.g., Tregs) are part of a cell population. It should be understood that although the cell population contains the CD4+ T cells described herein, other cell types may also be present, and therefore the population may not be purely a CD4+ T cell population. However, in one specific embodiment, the population may contain at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% CD4+ T cells. Suitablely, a cell population composed of CD4+ T cells contains at least 70% Tregs, such as at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% Tregs. Such a population may be referred to as an “enriched Treg population.”

[0270] In some aspects of the in vitro method of the present invention, CD4+ T cells (e.g., Tregs) may be derived from the in vitro differentiation of induced progenitor cells (e.g., iPSCs) or embryonic progenitor cells into Tregs. Suitable differentiation methods are known in the art and include those disclosed by Haque et al. in JVis Exp., 2016, 117, 54720 (incorporated herein by reference).

[0271] Therefore, the targeted CD4+ T cells can be non-Treg CD4+ cells or Treg cells as defined above. Non-Treg CD4+ T cells generally do not possess inhibitory activity and are considered CD4+ Tconv cells as defined below. Therefore, according to the present invention, any type or both types of cells can be transduced in vivo or in vitro. The present invention covers the transduction of CD4+ cell populations, wherein such populations may contain both non-Treg CD4+ T cells and Treg cells, or may contain only non-Treg CD4+ T cells or only Treg cells. These populations may, for example, contain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% Treg cells. As discussed herein, transducing non-Treg CD4+ T cells can convert these cells from having conventional T cell function to having inhibitory function by expressing FOXP3. Such inhibitory transdifferentiated cells are referred to herein as Treg-like cells. The inhibitory transduced cells of the present invention can provide inhibitory cell therapy.

[0272] The method of transducing CD4+ T cells in vitro using LNPs according to the present invention can convert non-Treg CD4+ T cells into cells with suppressive function. Those skilled in the art will understand that not all cells incubated with LNPs can be transduced, and only a portion of the cells may be transduced (e.g., at least 30%, 40%, or 50%). It should also be understood that not all transduced non-Treg cells can transdifferentiate into cells with suppressive function. However, preferably, at least 70%, 80%, 90%, or 95% of the transduced non-Treg CD4+ cells can be transdifferentiated into cells with suppressive function. Compared to untransduced Treg cells, the suppressive function of transduced Treg cells may be enhanced or increased (e.g., an increase in suppressive function of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%).

[0273] As used herein, “inhibitory,” “inhibitory function,” or “immunosuppressive function” can refer to the ability of a cell (e.g., Treg cells or Treg-like cells (e.g., CD4+ T cells expressing FOXP3)) to reduce or suppress one or more physiological and cellular effects induced by the immune system in response to stimuli such as pathogens, allogeneic antigens, or autoantigens. Examples of such effects include enhanced proliferation of conventional T cells (Tcon) and secretion of pro-inflammatory cytokines. Any such effect can be used as an indicator of the strength of the immune response. A relatively weak immune response in the presence of a Tconv cell (e.g., Treg cells or Treg-like cells (e.g., CD4+ T cells expressing FOXP3)) indicates that the cell has the ability to suppress the immune response. For example, a relatively reduced secretion of cytokines indicates a weaker immune response, thus indicating that the cell (e.g., Treg cells or Treg-like cells (e.g., CD4+ T cells expressing FOXP3)) has the ability to suppress the immune response. In particular, Tregs can also suppress the immune response by modulating the expression of co-stimulatory molecules on antigen-presenting cells (APCs) such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to assess the inhibitory efficacy of co-cultured, in vitro activated Tregs.

[0274] Methods for measuring indicators of immune response intensity are known in the art, and thus can measure the inhibitory capacity of cells (e.g., Treg cells or Treg-like cells (e.g., CD4+ T cells expressing FOXP3)). Specifically, antigen-specific Tconv cells can be co-cultured with cells, and a peptide corresponding to the antigen is added to the co-culture to stimulate a Tconv cell response. The degree of Tconv cell proliferation in response to the added peptide and / or the amount of IL-2 secreted by Tconv cells in response to the added peptide can be used as indicators of the inhibitory capacity of the co-cultured cells.

[0275] As used herein, the terms “conventional T cell,” Tcon, or Tconv (which are used interchangeably here) refer to T lymphocytes that express the αβ T cell receptor (TCR) and co-receptors (which may be cluster 4 (CD4) or cluster 8 (CD8) and do not have immunosuppressive function). Conventional T cells are found in peripheral blood, lymph nodes, and tissues.

[0276] Antigen-specific Tconv cells co-cultured with transduced cells as defined herein may proliferate by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than the same Tconv cells cultured in the absence of transduced cells. For example, antigen-specific Tconv cells co-cultured with transduced cells may proliferate by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than the same Tconv cells cultured in the presence of unengineered cells (e.g., CD4+ unengineered cells). Cells containing mRNA as defined herein (e.g., Treg) may have enhanced inhibitory activity (e.g., an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of inhibitory activity) compared to unengineered cells (e.g., Treg).

[0277] Antigen-specific Tconv cells co-cultured with the transduced cells described herein express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less of the effector cytokines compared to the corresponding Tconv cells cultured in the absence of transduced cells (e.g., in the presence of unengineered cells). The effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Suitably, the effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0278] The present invention further includes a pharmaceutical composition comprising: (i) the LNP of the present invention, (ii) CD4+ T cells transduced in vitro using the LNP of the present invention available as described above, or a cell population containing said cells, or (iii) CD4+ T cells containing the LNP of the present invention, or a cell population containing said cells.

[0279] The pharmaceutical composition can be prepared by any method known in the art, including combining the active ingredient (i.e., LNP or transduced cells) with a carrier or any desired excipient. The pharmaceutical compositions according to the invention can be developed for administration to any animal subject, including humans and other primates, domestic animals such as cats and dogs, and agricultural animals such as sheep, pigs, and cattle. The LNP can be further used for administration to mice, rats, and rabbits.

[0280] A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent (i.e., LNPs, cells (e.g., Tregs or inhibitory cells), or cell populations). The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington Pharmaceutical Science (ARGennaro, 1985), published by Mack Publishing Co. The choice of a pharmaceutical carrier, excipient, or diluent can be made based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilizer(s) as, or in addition to, a carrier, excipient, or diluent.

[0281] "Pharmaceutically acceptable" includes sterile and pyrogen-free formulations. The carrier, diluent, and / or excipient must be "acceptable" in the sense that they are compatible with cells or the carrier and harmless to their receptors. Typically, the carrier, diluent, and excipient will be sterile and pyrogen-free saline or infusion media, but other acceptable carriers, diluents, or excipients may also be used.

[0282] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, aromatic oils, monoglycerides of fatty acids, diglycerides of fatty acids, petroleum ether fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0283] LNPs, cells, cell populations, or pharmaceutical compositions may be administered in a manner suitable for the treatment and / or prevention of the desired disease or condition. Although appropriate dosages may be determined through clinical trials, the amount and frequency of administration will be determined by factors such as the subject's condition, the type and severity of the subject's disease or condition, etc. Pharmaceutical compositions may be formulated accordingly.

[0284] The LNPs, cells, cell populations, or pharmaceutical compositions described herein can be administered parenterally (e.g., intravenously) or via infusion techniques. The LNPs, cells, cell populations, or pharmaceutical compositions can be administered as a sterile aqueous solution, which may contain other substances, such as sufficient salt or glucose, to make the solution isotonic with blood. The aqueous solution may be appropriately buffered (preferably from pH 3 to pH 9). The pharmaceutical compositions can be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished using standard pharmaceutical techniques known to those skilled in the art.

[0285] The pharmaceutical composition may contain LNPs or cells in an infusion medium, such as a sterile isotonic solution. The pharmaceutical composition may be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0286] LNPs, cells, cell populations, or pharmaceutical compositions can be administered in single or multiple doses. In particular, LNPs, cells, cell populations, or pharmaceutical compositions can be administered in a single, one-time dose. Pharmaceutical compositions can be formulated accordingly.

[0287] The pharmaceutical composition may further comprise one or more active agents. The pharmaceutical composition may further comprise one or more other therapeutic agents, such as lymphocyte depletion agents (e.g., anti-thymocyte globulin, Campas-1H, anti-CD2H antibody, anti-CD3 antibody, anti-CD20 antibody, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), drugs that inhibit co-stimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or drugs that inhibit specific cytokines (IL-6, IL-17, TNFα, IL18).

[0288] Depending on the disease / condition to be treated, the subject, and the route of administration, LNPs, cells, cell populations, or pharmaceutical compositions may be administered in different doses (e.g., per cell / kg or per cell / subject). In any case, the physician will determine the actual dose best suited for any individual subject, and this dose will vary depending on the specific subject's age, weight, and response. However, typically, for the cells described herein, 5 × 10⁶ cells may be administered per subject. 7 Up to 3×10 9 Or 10 8 Up to 2×10 9 The dose per cell.

[0289] The LNPs, cells, cell populations, or pharmaceutical compositions of the present invention can be administered in one or more doses. It should be understood that, due to the transient nature of treatment, treatment of diseases requiring long-term maintenance of suppressive therapy may require administration of more than one dose over a period of time, such as at least two, three, or four doses.

[0290] Cells or cell populations can be appropriately modified for use in pharmaceutical compositions. For example, cells can be cryopreserved and thawed at appropriate times and then infused into a subject.

[0291] As described above, the LNPs, or ex vivo transduced cells or cell populations of the present invention can be used to deliver inhibitory cell therapy in vivo to subjects who need this therapy (e.g., subjects with undesirable inflammatory responses, subjects with autoimmune diseases, subjects with GvHD or HvG, or subjects who require tissue remodeling or repair).

[0292] Therefore, the present invention provides an LNP, CD4+ cells, cell population or pharmaceutical composition for treatment.

[0293] LNPs, cells, and compositions comprising LNPs and cells are used in adoptive cell therapy (ACT). Various conditions can be treated by administering the LNPs, cells, and compositions of the present invention. As mentioned above, these conditions may be those responding to immunosuppression, particularly to the immunosuppressive effects of Treg cells or transdifferentiated Treg-like cells. Therefore, the LNPs, cells, cell populations, and compositions described herein can be used to induce or achieve immunosuppression in a subject. Cells administered or modified in vivo can be targeted by expressing antigen-binding receptors (e.g., CARs). Conditions suitable for such treatment include infectious diseases, neurodegenerative diseases, or inflammatory diseases, or more broadly, conditions associated with any undesirable, unwanted, or harmful immune response. Furthermore, the LNPs, cells, cell populations, and compositions described herein can therefore be used to promote tissue remodeling or repair, such as tissue regeneration.

[0294] Conditions to be treated or prevented include inflammation, or alternatively, conditions related to or involving inflammation. Inflammation can be chronic or acute. Furthermore, inflammation can be low-level or systemic. For example, inflammation can occur in cases of metabolic disorders (e.g., metabolic syndrome), or in conditions such as insulin resistance, type 2 diabetes, or obesity.

[0295] In particular, the conditions to be treated include those that may require rapid and broad-based immune modulation, such as acute liver dysfunction, sepsis, lung infections, autoimmune disease flare-ups (acute exacerbations of chronic diseases), stroke, and vascular diseases.

[0296] Specifically, LNPs, cells, cell populations, and pharmaceutical compositions provide a method for: inducing transplant tolerance; treating and / or preventing cellular and / or humoral transplant rejection; treating and / or preventing graft-versus-host disease (GvHD), autoimmune or allergic diseases; or promoting tissue repair and / or tissue regeneration; or alleviating inflammation. Cells, cell populations, and pharmaceutical compositions can be used in a method comprising the step of administering the LNPs, cells, cell populations, or pharmaceutical compositions described herein to a subject.

[0297] As used in this article, "inducing transplant tolerance" refers to inducing the recipient's tolerance to the transplanted organ. In other words, inducing transplant tolerance means reducing the recipient's immune response to the donor organ. Inducing tolerance to the transplanted organ can reduce the amount of immunosuppressive drugs required by the recipient, or it can discontinue the use of immunosuppressive drugs altogether.

[0298] For example, the LNP, cells, cell populations, or pharmaceutical compositions of the present invention can be administered to a subject suffering from a disease to alleviate, reduce, or improve at least one symptom of the disease, such as jaundice, dark urine, itching, abdominal distension or tenderness, fatigue, nausea or vomiting, and / or loss of appetite. The at least one symptom may be alleviated, reduced, or improved by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the at least one symptom may be completely relieved.

[0299] The LNPs, cells, cell populations, or pharmaceutical compositions of the present invention can be administered to subjects suffering from a disease in order to slow, reduce, or block the progression of the disease. Compared to subjects who have not been administered engineered cells, the progression of the disease can be slowed, reduced, or blocked by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the progression of the disease can be completely stopped.

[0300] In one implementation, the subject is a graft recipient receiving immunosuppressive therapy.

[0301] Appropriately, the subjects were mammals. Appropriately, the subjects were humans.

[0302] Grafts can be selected from liver, kidney, heart, lung, pancreas, intestine, stomach, bone marrow, vascularized composite tissue grafts, and skin grafts.

[0303] Suitable, transduced cells can express a CAR containing an antigen-binding domain that can specifically bind to HLA antigens present in the transplant (graft) donor but not in the transplant (graft) recipient.

[0304] Suitablely, the graft is a liver graft. In an implementation where the graft is a liver transplant, the antigen may be an HLA antigen present in the transplanted liver but not in the patient, a liver-specific antigen (such as NTCP), or an antigen upregulated during rejection (such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, CXCL11).

[0305] As discussed above, in a representative and preferred embodiment, the antigen is HLA-A2.

[0306] A method for treating a disease or condition relates to the therapeutic use of the LNPs, cells, cell populations, or pharmaceutical compositions described herein. In this regard, the LNPs, cells, cell populations, or pharmaceutical compositions of the present invention can be administered to a subject suffering from an existing disease or condition to alleviate, reduce, or improve at least one symptom associated with the disease or condition and / or slow, reduce, or block the progression of the disease.

[0307] Suitablely, treatment and / or prevention of cellular and / or humoral transplant rejection may refer to the application of an effective amount of the LNP, cells, cell populations or pharmaceutical composition of the present invention, such that the amount of immunosuppressive drug required by the graft recipient is reduced, or that the immunosuppressive drug can be discontinued.

[0308] Prevention of disease or condition involves the preventive use of the LNPs, cells, cell populations, or pharmaceutical compositions of the present invention herein. In this regard, the LNPs, cells, cell populations, or pharmaceutical compositions of the present invention may be administered to subjects who have not been infected with or developed the disease or condition and / or who do not exhibit any symptoms of the disease or condition, to prevent the disease or condition, or to reduce or prevent the development of at least one symptom associated with the disease or condition. The subject may be predisposed to the disease or condition, or is considered to be at risk of developing the disease or condition.

[0309] Autoimmune or allergic diseases can be selected from inflammatory skin diseases, including: psoriasis and dermatitis (e.g., atopic dermatitis); reactions associated with inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis); dermatitis; allergic conditions such as food allergies, eczema, and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including lupus nephritis and cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS); chronic inflammatory demyelinating polyneuropathy (CIPD); and juvenile diabetes.

[0310] More specifically, the present invention further provides an LNP, CD4+ cells, cell population or pharmaceutical composition for treating or preventing inflammation, autoimmune diseases or HvG / GvHD or promoting tissue remodeling or repair in subjects.

[0311] In another aspect, the present invention provides the use of LNPs, CD4+ cells, cell populations, or pharmaceutical compositions in the manufacture of medicaments for the treatment or prevention of inflammation, autoimmune diseases, or HvG / GvHD, or for promoting tissue remodeling or repair in subjects.

[0312] Furthermore, the present invention provides a method for treating or preventing inflammation, autoimmune diseases, or HvG / GvHD in a subject, or for promoting tissue remodeling or repair in a subject, the method comprising the step of administering to the subject the LNP, CD4+ T cells, cell population, or pharmaceutical composition of the present invention.

[0313] Prior to administering the LNP or a pharmaceutical composition containing the LNP of the present invention, an additional step may be performed on the subject, namely, administering a low dose of IL2 or, possibly preferably, a CD25-binding mutant IL2 protein (e.g., the mutant protein described above). This administration may increase the percentage or number of Treg cells in the patient prior to LNP administration. In particular, the present invention can provide a method for treating or preventing inflammation, autoimmune diseases, or HvG / GvHD in a subject, comprising the steps of: (i) administering a low dose of IL2, or an enhanced or increased CD25-selective IL2 mutant protein, to the subject; and (ii) administering the LNP of the present invention or a pharmaceutical composition containing the LNP of the present invention, as described herein.

[0314] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described and materials herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the numbers that define the range. Unless otherwise stated, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right, respectively, from amino to carboxyl.

[0315] When a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of the range (in units of one-tenth of the lower limit, unless the context explicitly states otherwise) is also specifically disclosed. Every smaller range between any specified value or intermediate value within the specified range and any other specified value or intermediate value within that specified range is covered in this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and provided that there are any specifically excluded limits within the specified range, each range that includes any one, any two, or both of those limits is also covered in this disclosure. If the specified range includes one or two limits, the range that excludes any one or both of those limits is also included in this disclosure.

[0316] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include the plural meaning.

[0317] As used herein, the terms “comprising, comprises, and comprised of” are synonymous with “including and includes” or “containing and contains”, and are inclusive or open-ended, not excluding additional, unlisted members, elements, or method steps. The terms “comprising, comprises, and comprised of” also include the term “consisting of”.

[0318] The publicly available information discussed herein is limited to that disclosed prior to the filing date of this application. Nothing herein shall be construed as an admission that such publicly available information constitutes prior art as claimed in the appended claims. All references herein are incorporated by way of citation.

[0319] Sequence Listing

[0320] SEQ ID NO:1 VH anti-CD4 antibody

[0321] EVQLVESGGGLVQPGRSLRLSCAASGFTFSNYGMAWVRQAPGKGLEWVATISYDGS

[0322] ITYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREEQYSSWYFDFWGQ

[0323] GTLVTVSS

[0324] SEQ ID NO:2 VL anti-CD4 antibody

[0325] DIQLTQSPSSLSASVGDRVTITCRASQSVSISSHDLMQWYQQKPGKAPKLLIYDAFN

[0326] LASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSKDDPYTFGQGTKLEIK

[0327] SEQ ID NO:3 Heavy chain anti-CD25 antibody

[0328] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPST

[0329] GYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGT

[0330] LVTVSS

[0331] SEQ ID NO:4 Light chain anti-CD25 antibody

[0332] DIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVP

[0333] ARFSGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVK

[0334] SEQ ID NO:5 Anti-CD4 aptamer

[0335] GGGAGACAAGAATAAACGCTCAATGACGTCCTTAGAATTGCGCA

[0336] TTCCTCACACAGGATCTTTTCGACAGGAGGCTCACAACAGGC

[0337] SEQ ID NO:6 FOXP3 amino acid sequence

[0338] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAH

[0339] ASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDA

[0340] HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCT

[0341] FPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE

[0342] KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCC

[0343] IVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMR

[0344] PPFTYATLIRWAILEAPEKQRTNLEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCF

[0345] VRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP

[0346] SEQ ID NO:7 FOXP3 amino acid sequence change

[0347] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAH

[0348] ASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDA

[0349] HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCT

[0350] FPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE

[0351] KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCC

[0352] IVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMR

[0353] PPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCF

[0354] VRVESEKGAVWTVDELEFRKKREQRPSRCSNPTPGP

[0355] SEQ ID NO.8 FOXP3 variant amino acid sequence

[0356] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAH

[0357] ASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDA

[0358] HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCT

[0359] FPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE

[0360] KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCC

[0361] IVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMR

[0362] PPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCF

[0363] VRVESEKGAVWTVDELEFRKKRSQRPARCSNPTPGP

[0364] SEQ ID NO.9 FOXP3 variant amino acid sequence

[0365] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAH

[0366] ASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDA

[0367] HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCT

[0368] FPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE

[0369] KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCC

[0370] IVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMR

[0371] PPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCF

[0372] VRVESEKGAVWTVDELEFRKKREQRPARCSNPTPGP

[0373] SEQ ID NO:10 Truncated FOXP3 amino acid sequence

[0374] GGAHASSSSL NPMPPSQLQL PTLPLVMVAP SGARLGPLPH LQALLQDRPH

[0375] FMHQLSTVDA HARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP

[0376] GINVASLEWV SREPALLCTF PNPSAPRKDS TLSAVPQSSY PLLANGVCKW ' [[ID=%36]]

[0377] PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ CLLQREMVQS LEQQLVLEKE '

[0378] KLSAMQAHLA GKMALTKASS VASSDKGSCC IVAAGSQGPV VPAWSGPREA

[0379] PDSLFAVRRH LWGSHGNSTF PEFLHNMDYF KFHNMRPPFT YATLIRWAIL

[0380] EAPEKQRTLN EIYHWFTRMF AFFRNHPATW KNAIRHNLSL HKCFVRVESE

[0381] KGAVWTVDEL EF

[0382] SEQ ID NO:11 FOXP3 polypeptide

[0383] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAH

[0384] ASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDA

[0385] HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCT

[0386] FPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE

[0387] KGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAG

[0388] SQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTY

[0389] ATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVE

[0390] SEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN

[0391] SEQ ID NO:12——FOXP3 mRNA

[0392] AUG CCCAAC CCCAGG CCU GGC AAG CCC UCG GCC CCU UCC UUG GCC CUU

[0393] GGC CCAUCC CCAGGA GCC UCG CCCAGC UGGAGG GCU GCACCC AAA GCC

[0394] UCA GAC CUG CUG GGG GCC CGG GGC CCA GGG GGA ACC UUC CAG GGC

[0395] CGA GAU CUU CGA GGC GGG GCC CAU GCC UCC UCU UCU UCC UUG AAC

[0396] CCCAUG CCACCAUCG CAG CUG CAG CUG CCCACA CUG CCC CUAGUC AUG

[0397] GUG GCA CCC UCC GGG GCA CGG CUG GGC CCC UUG CCC CAC UUA CAG

[0398] GCA CUC CUC CAG GAC AGG CCA CAU UUC AUG CAC CAG CUC UCA ACG

[0399] GUG GAU GCC CAC GCC CGG ACC CCU GUG CUG CAG GUG CAC CCC CUG

[0400] GAGAGC CCA GCC AUG AUC AGC CUC ACA CCA CCC ACC ACC GCC ACU GGG

[0401] GUC UUC UCC CUC AAG GCC CGG CCU GGC CUC CCA CCU GGG AUC AAC

[0402] GUG GCC AGC CUG GAA UGG GUG UCC AGG GAG CCG GCA CUG CUC UGC

[0403] ACC UUC CCAAAU CCCAGU GCACCCAGGAAG GAC AGCACC CUU UCG GCU

[0404] GUG CCC CAG AGC UCC UAC CCA CUG CUG GCA AAU GGU GUC UGC AAG

[0405] UGG CCC GGA UGU GAG AAG GUC UUC GAA GAG CCA GAG GAC UUC CUC

[0406] AAG CAC UGC CAG GCG GAC CAU CUU CUG GAU GAG AAG GGC AGG GCA

[0407] CAA UGU CUC CUC CAG AGA GAG AUG GUA CAG UCU CUG GAG CAG CAG

[0408] CUG GUG CUG GAG AAG GAG AAG CUG AGU GCC AUG CAG GCC CAC CUG

[0409] GCU GGG AAA AUG GCA CUG ACC AAG GCU UCA UCU GUG GCA UCA UCC

[0410] GAC AAG GGC UCC UGC UGC AUC GUA GCU GCU GGC AGC CAA GGC CCU

[0411] GUC GUC CCA GCC UGG UCU GGC CCC CGG GAG GCC CCU GAC AGC CUG

[0412] UUU GCU GUC CGG AGG CAC CUG UGG GGU AGC CAU GGA AAC AGC ACA

[0413] UUC CCA GAG UUC CUC CAC AAC AUG GAC UAC UUC AAG UUC CAC AAC

[0414] AUG CGA CCC CCU UUC ACC UAC GCC ACG CUC AUC CGC UGG GCC AUC CUG

[0415] GAG GCU CCA GAG AAG CAG CGG ACA CUC AAU GAG AUC UAC CAC UGG

[0416] UUC ACA CGC AUG UUU GCC UUC UUC AGA AAC CAU CCU GCC ACC UGG

[0417] AAG AAC GCC AUC CGC CAC AAC CUG AGU CUG CAC AAG UGC UUU GUG

[0418] CGG GUG GAG AGC GAG AAG GGG GCU GUG UGG ACC GUG GAU GAG CUG

[0419] GAG UUC CGC AAG AAA CGG AGC CAG AGG CCC AGC AGG UGU UCC AAC

[0420] CCU ACA CCU GGC CCC UGA

[0421] SEQ ID NO:13 FOXP3 mRNA

[0422] GAAUUCCACAACCCCAGACCCGGCCAGCCUUGCCCCUUCU

[0423] GGCCUGGGACCUCUCCUGGCGCCUCCCCAUCUUGGAGAGCCGCCCCUAAA

[0424] GCCAGCGAUCUGCUGGGAGCUAGAGGCCCUGGCGGCAUUCCAGGGCAGAG

[0425] AUCUGAGAGGCGGAGCCCACGCCUCUAGCAGCAGCCUGAAUCCCAUGCCCCC

[0426] UAGCCAGCUGCAGCUGCCUACACUGCCUCUCGUGAUGGUGGCCCCUAGCGGA

[0427] GCUAGACUGGCCCUCUGCCUCAUCAGGCUCUGCAGGACCGGCCCC

[0428] ACUUUAUGCACCAGCUGAGCACCGUGGACGCCCACGCCAGAAACCCUGUGCU

[0429] GCAGGUGCACCCCCUGGAAAGCCCUGCCAUGAUCAGCCUGACCCCUCCAACCA

[0430] CAGCCACCGGCGUGUUCAGCCUGAAGGCCAGACCUGGACUGCCCCCUGGCAU

[0431] CAAUGUGGCCAGCCUGGAAUGGGUCUCCCGCAACCUGCCCUGCUGUGCAC

[0432] UUCCCCAAUCCUAGCGCCCCCAGAAAGGACAGCACACAGUGUCUGCCGUGCCCA

[0433] GAGCAGCUAUCCCCUGCUGGCUAACGGCGUGUGCAAGUGGCCUGGCUGCGAG

[0434] AAGGUGUUCGAGGAACCCGAGGACUUCCUGAAGCACUGCCAGGCCGACCAUC

[0435] UGCUGGACGAGAAAGGCAGAGCCCAGUGCCUGCUGCAGCGCGAGUGGUGCA

[0436] GUCCCUGGAACAGCAGCUGGUGCUGGAAAAGAAAAGCUGAGCCGCCAUGCAG

[0437] GCCCACCUGGCCGGAAAGAUGGCCCUGACAAAAGCCAGCGUGGCCAGCU

[0438] CCGACAAGGGCAGCUGUUGUAUCGUGGCCGCUGGCAGCCAGGGACCUGUGGU

[0439] GCCUGCUUGGAGCGGACCUAGAGAGGCCCCCGAUAGCCUGUUUGCCGUGCGG

[0440] AGACACCUGUGGGGCAGCCACGGCAACUCUACCUUCCCCGAGUUCCUGCACA

[0441] ACAUGGACUACUUCAAGUUCCACAACAUGAGGCCCCCCUUCACCUACGCCAC

[0442] CCUGAUCAGAUGGGCCAUUCUGGAAGCCCCCGAGAAGCAGCGGACCCUGAAC

[0443] GAGAUCUACCACUGGUUUACCCGGAUGUUCGCCUUCUUCCGGAACCACCCCG

[0444] CCACCUGGAAGAACGCCAUCCGGCACAAUCUGAGCCUGCACAAGUGCUUCGU

[0445] GCGGGUGGAAAGCGAGAAGGGCGCCGUGUGGACAGUGGACGAGCUGGAAUU

[0446] UCGGAAGAAGCGGUCCCAGAGGCCCAGCCGGUGUAGCAAUCCUACACCUGGC

[0447] CCUGAGGGCAGAGGAAGUCUGCUAACAUGCGGUGACGUCGAGGAGAAUCC

[0448] SEQ ID NO:14 —— anti - HLA - A2 scFv

[0449] QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWVAFIRND

[0450] GSDKYYADSVKGRFTISRDNSEKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWY<^

[0451] LDLWGRGTLVTVSSGGGGSGGGGSGGGGSTDVVMTQSPSSLSASVGDRVTITCQ

[0452] SSLDISHYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTHFTFTISSLQPED

[0453] FATYYCQQYDNLPLTFGGGTKLEIK

[0454] SEQ ID NO:15 CD8 hinge

[0455] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0456] SEQ ID NO.16 CD28 hinge

[0457] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0458] SEQ ID NO:17 CD8 transmembrane

[0459] IYIWAPLAGTCGVLLLSLVIT

[0460] SEQ ID NO.18 CD28 transmembrane

[0461] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0462] SEQ ID NO:19 CD3ζ Intracellular Signal Transduction Domain

[0463] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ

[0464] EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL

[0465] PPR

[0466] SEQ ID NO:20 CD28 costimulatory sequence

[0467] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0468] SEQ ID NO:21 ——CD8 leader sequence

[0469] MALPVTALLLPLALLLHAARP

[0470] SEQ ID NO:22 ——Helios peptide

[0471]

[0472]

[0473] SEQ ID NO:23 IL10 peptide

[0474]

[0475] SEQ ID NO:24 IL35 peptide

[0476]

[0477]

[0478] SEQ ID NO:25 TGFβ polypeptide

[0479]

[0480] SEQ ID NO:26 Areg polypeptide

[0481]

[0482]

[0483] SEQ ID NO:27 ——CCN3 polypeptide

[0484]

[0485] SEQ ID NO.28 ——CD73 polypeptide

[0486]

[0487]

[0488] SEQ ID NO.29 ——CD39 polypeptide

[0489]

[0490] SEQ ID NO:30 P2A peptide – cleavage domain

[0491] ATNFSLLKQAGDVEENPGP

[0492] SEQ ID NO:31 T2A peptide – cleavage domain:

[0493] EGRGSLLTCGDVEENPGP

[0494] SEQ ID NO:32 E2A peptide – cleavage domain:

[0495] QCTNYALLKLAGDVESNPGP

[0496] SEQ ID NO:33 F2A peptide — cleavage domain:

[0497] VKQTLNFDLLKLAGDVESNPGP

[0498] SEQ ID NO:34 furin cleavage site

[0499] RXXR

[0500] SEQ ID NO:35 furin cleavage site

[0501] RRKR

[0502] SEQ ID NO:36 ——CD4

[0503]

[0504]

[0505] SEQ ID NO:37 ——IL2 precursor

[0506]

[0507] SEQ ID NO:38 ——IL2

[0508]

[0509] SEQ ID NO:39 —CDR3β sequence of GAD65(555-567):HLA-DR*04:01

[0510] CASSLVGGPSSEAFF

[0511] SEQ ID NO:40 ——IL33

[0512]

[0513]

[0514] SEQ ID NO:41 ——CCN2

[0515]

[0516] SEQ ID NO:42 ——CTLA 4

[0517]

[0518]

[0519] SEQ ID NO:43 ——LAG3

[0520]

[0521] SEQ ID NO:44 ——PDL1

[0522]

[0523]

[0524] SEQ ID NO:45 ——PDL2

[0525]

[0526] SEQ ID NO:46 ——ICOS

[0527]

[0528] SEQ ID NO:)47 ——Heavy chain of anti-CD62L antibody

[0529] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWIGYIYPYN

[0530] DGTKYNEKFKGRVTITSDESTNTAYMELSSLRSEDTAVYYCAREEYGNYVRYFDVW

[0531] GQGTLVTVSS

[0532] SEQ ID NO:48 ——Light chain of anti-CD62L antibody

[0533] DIQMTQSPSTLSASVGDRVTITCKSSQSLLNSSNQKNYLAWYQQKPGKAPKLLVYF

[0534] ASTRESGVPDRFIGSGSGTDFTLTISSLQPEDFATYFCHQHYSTPLTFGQGTKVEVK

[0535] SEQ ID NO:49 ——IL37

[0536]

[0537] SEQ ID NO:50 ——IL22

[0538]

[0539] SEQ ID NO:51 —Heavy chain of anti-CD62L antibody (alternative)

[0540] EVQLQQSGPDLVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYIYPY

[0541] NDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAREEYGNYVRYFDV

[0542] WGAGTTVTVSS

[0543] SEQ ID NO:52 —Light chain of anti-CD62L antibody (alternative)

[0544] DIVMTQSPSSLAMSVGQKVTMTCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVY

[0545] FASTRESGVPDRFIGSGSGTTDFTLTISSVQAEDLADYFCHQHYSTPLTFGAGTKLELK

[0546] Example

[0547] Example 1

[0548] The mRNA was produced via in vitro transcription using a plasmid containing a T7 promoter, a 120 bp poly-A tail encoding FOXP3 (SEQ ID NO:6), and a chimeric antigen receptor containing scFv (SEQ ID NO:14). The plasmid was linearized and transcribed in vitro using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) incorporating 10% 2-thio-UTP and 5-methyl-CTP (TriLinkBio Technologies).

[0549] Capping of the mRNA will be performed using the trinucleotide cap1 analog Clean Cap (TriLink). The mRNA will be purified by cellulose purification and stored at -20°C.

[0550] The purified mRNA was encapsulated in LNPs using an ethanol lipid mixture consisting of cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol lipids. This mixture was rapidly mixed with an aqueous solution of mRNA at an acidic pH, and the particles were characterized using a Zetasizer Nano ZS (Malvern Instruments).

[0551] To prepare CD4-targeting LNPs, LNPs were conjugated to anti-CD4 antibodies using a SATA-maleimide chemistry. The LNPs were modified with maleimide functional groups, and the antibody was functionalized using SATA to introduce thiol groups. A thioester conjugation chemistry was used to bind the antibody to the LNPs. The LNPs were then purified using a Sepharose CL-4B gel filtration column. The LNPs were stored at 4°C.

[0552] Example 2

[0553] The LNP was then tested in vitro to assess cell specificity and expression efficacy. The CD4-targeting LNP, containing modified mRNA encoding HLA-A2-CAR and FOXP3, was co-incubated with freshly isolated human PBMCs. Cell cultures were activated using anti-CD3 / CD28 magnetic beads to promote T cell activation. Phenotypic and functional characterization of cells was performed at 24, 48, and 72 hours post-exposure to CD4 / LNP-HLAA2-FOXP3. LNP conjugated with an isotype control antibody was used to evaluate non-specific delivery of mRNA to non-CD4 cell types.

[0554] Protein expression of HLA.A2-CAR and FOXP3 will be assessed at multiple time points using flow cytometry to evaluate target specificity and expression efficacy. Additionally, multiple biomarkers associated with regulatory T cell (Treg) function will be analyzed to identify phenotypic changes in target cells (i.e., CTLA4, HELIOS, CD25, CD127, and PD1).

[0555] Functional characterization of cells exposed to CD4 / LNP-HLAA2-FOXP3 will be performed on isolated HLA.A2-CAR-expressing cells. Antigen-specific activation will be tested by culturing isolated cells using K562 cells expressing or not expressing HLA.A2 molecules and assessing CD69 and CD137 activation markers at 24 hours. Cell viability of K562 target cells will be assessed to evaluate and exclude cytotoxicity of transformed cells. Additionally, culture supernatants will be collected to investigate the levels of anti-inflammatory and pro-inflammatory cytokines.

[0556] The inhibitory function of the transformed cells will be further evaluated in vitro when co-cultured with HLA-A2-expressing B cells and CD4+CD25- effector T cells. In short, CD4+CD25- T cells will be isolated and cultured using a B cell line expressing the HLA-A2 molecule. Transformed CD4 / LNP-HLAA2-FOXP3 cells will be added to the culture at varying intercellular ratios, and the proliferation of CD4+CD25- T cells will be assessed after 5 days.

[0557] Example 3 It should be noted that there seems to be a small error in the original text where "SEQ ID NO:)47" should probably be "SEQ ID NO:47". This has been corrected in the translation as much as possible while maintaining the original structure.

[0558] To evaluate the ability of CD4 / LNP-HLAA2-FOXP3 to efficiently reprogram human T cells in vivo, PBMCs from HLA.A2-negative donors were transferred into NSG.HLA.A2 mice, and CD4 / LNP-HLAA2-FOXP3 particles were injected 2 weeks later. Phenotypic characterization of cells from blood and spleen was performed at 24, 48, and 96 hours after intravenous injection of LNP. The expression levels of HLA.A2 CAR and FOXP3 on CD4+ T cells and other cells were assessed to determine their specificity, efficacy, and persistence.

[0559] In addition, this same model will be used to evaluate the immunomodulatory capacity of CD4 / LNP-HLAA2-FOXP3 in regulating the progression of xenoGvHD. Human PBMCs were transferred into three groups of NSG.HLA.A2 mice, and GvHD signs were tracked for 8 weeks. The first group received PBMCs only, while the other two groups received one or two intravenous doses of CD4 / LNP-HLAA2-FOXP3 (one dose in week 2, or one dose in week 2 + one dose in week 3, respectively). All animals were scored daily to assess the progression of xenoGvHD. Additionally, tail blood was collected weekly to track human cell engraftment (i.e., the ratio of human CD45 to mouse CD45 frequency) and to assess the reprogramming of circulating human T cells (i.e., the frequency of CD4+HLA.A2+FOXP3+).

Claims

1. A lipid nanoparticle comprising mRNA encoding FOXP3 and at least one second polypeptide, wherein, The lipid nanoparticles contain a portion capable of specifically binding to molecules expressed on CD4+ T cells, wherein the mRNA is modified to increase stability compared to unmodified mRNA.

2. The lipid nanoparticles according to claim 1, wherein, The portion can be combined with CD4, CD25, CD62L or CD45RA.

3. The lipid nanoparticles according to claim 1 or 2, wherein, The LNP binds specifically to CD4+ T cells.

4. The lipid nanoparticles according to claim 3, wherein, The CD4+ T cells mentioned are Treg cells.

5. The lipid nanoparticles according to any one of claims 1 to 4, wherein, The part in question is an antibody, antibody fragment, or aptamer.

6. The lipid nanoparticles according to any one of claims 1 to 5, wherein, The mRNA encodes at least three, four, or five polypeptides.

7. The lipid nanoparticles according to any one of claims 1 to 6, wherein, The at least one second polypeptide is selected from any one or more of antigen-binding receptors, transcription factors, persistence-related polypeptides, cytokines, safety switch polypeptides, or polypeptides that promote tissue repair or inhibit function.

8. The lipid nanoparticles according to claim 7, wherein, The antigen-binding receptor is either a CAR or a TCR.

9. The lipid nanoparticles according to claim 7 or 8, wherein, The antigen-binding receptor binds to HLA-A2.

10. The lipid nanoparticles according to any one of claims 7 to 9, wherein, The antigen-binding receptor comprises SEQ ID NO:14 or a functional variant thereof.

11. The lipid nanoparticles according to claim 7, wherein, The transcription factors are selected from the group consisting of Helios, Sp1, NFAT, AP1, STAT5, CREB, ATF, or Smad.

12. The lipid nanoparticles according to claim 7, wherein, The persistence-associated peptide is a STAT5 or JAK1 peptide containing a gain-of-function mutation, or a STAT5-associated motif and a JAK1 and / or JAK2 binding motif.

13. The lipid nanoparticles according to claim 7, wherein, The cytokines are IL10, IL35, or TGFβ.

14. The lipid nanoparticles according to claim 7, wherein, The safety switch polypeptide contains a CD20 epitope.

15. The lipid nanoparticles according to claim 7, wherein, The peptide that promotes tissue repair is bimodal protein or CCN3.

16. The lipid nanoparticles according to claim 7, wherein, The polypeptide that promotes the inhibitory function is CD73 or CD39.

17. The lipid nanoparticles according to any one of claims 1 to 16, wherein, The mRNA contains a 5' cap and a poly-A tail.

18. The lipid nanoparticles according to any one of claims 1 to 17, wherein, The mRNA contains one or more modified nucleotides.

19. The lipid nanoparticles according to any one of claims 1 to 18, wherein, The lipid nanoparticles are formulated from ionizable lipids or cationic lipids and may optionally contain DOPE and / or cholesterol.

20. A composition comprising CD4+ T cells and LNPs according to any one of claims 1 to 19.

21. A CD4+ T cell comprising LNP as described in any one of claims 1 to 19.

22. A method for transducing CD4+ T cells in vitro, comprising the step of combining CD4+ T cells with LNPs according to any one of claims 1 to 19.

23. A transduced CD4+ T cell that can be obtained or obtained by the method of claim 22.

24. The cell according to claim 21 or 23, wherein, The cells in question are Treg cells.

25. A population of cells comprising the cells of claim 21, 23 or 24.

26. A pharmaceutical composition comprising any LNP of claims 1 to 19, cells of claims 21, 23 or 24, or a cell population of claim 25, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

27. The LNP of any one of claims 1 to 19, the cell of claim 21, 23 or 24, the cell population of claim 25 or the pharmaceutical composition of claim 26, for use in treatment.

28. The LNP of any one of claims 1 to 19, the cell of claim 21, 23 or 24, the cell population of claim 25 or the pharmaceutical composition of claim 26, for the treatment or prevention of inflammation, autoimmune disease or HvG / GvHD in a subject.

29. Use of the LNP of any one of claims 1 to 19, the cell of claim 21, 23 or 24, the cell population of claim 25 or the pharmaceutical composition of claim 26 in the manufacture of a medicament for treating or preventing inflammation, autoimmune disease or HvG / GVHD in a subject.

30. A method for producing LNP according to any one of claims 1 to 19, comprising the following steps: An LNP-mRNA formulation is prepared by incubating an ionizable lipid or cationic lipid with an mRNA encoding FOXP3 and a second polypeptide, wherein the mRNA is modified to increase intracellular stability compared with unmodified mRNA.

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