mRNA transfection of immune cells

Abstract

To provide mRNA transfection of immune cells.SOLUTION: The present disclosure relates to methods of modifying immune cells by delivering a modified messenger RNA (mRNA) encoding a chimeric antigen receptor (CAR) and modified immune cells comprising the CAR. The present disclosure encompasses, inter alia, methods, systems, and compositions for modifying immune cells, including monocytes, macrophages, and / or dendritic cells. In some embodiments, the provided methods, systems, and / or compositions provide for enhanced production and / or enhanced properties of modified immune cells.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 044,855, filed June 26, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Immunotherapy has been investigated for many diseases and disorders, including cancer, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), systemic amyloidosis, prion diseases, cardiovascular disease, atherosclerosis, and fibrosis, but the functional limitations encountered still need to be addressed.

[0003] Therefore, there is a need to develop optimized therapeutic strategies to enhance expression, survival, and function. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure encompasses, inter alia, methods, systems, and compositions for modifying immune cells, including monocytes, macrophages, and / or dendritic cells. In some embodiments, the provided methods, systems, and / or compositions provide enhanced production and / or enhanced properties of modified immune cells. Surprisingly, the present disclosure encompasses the recognition that the use of modified mRNA (e.g., 5'-capped or uridine-modified mRNA encoding a transgene of interest, such as a chimeric antigen receptor (CAR)) can result in significantly increased expression levels, persistence of expression, and viability of human monocytes, macrophages, and / or dendritic cells. Furthermore, the present disclosure encompasses the recognition that the use of interferon beta can result in significantly increased expression levels, persistence of expression, and function of CAR monocytes, macrophages, and / or dendritic cells transfected with mRNA encoding a CAR or other transgene. The present disclosure also encompasses the recognition that the use of RNAse inhibitors (e.g., RNAse L inhibitors) can result in significantly increased expression levels of desired polynucleotides and / or polypeptides (e.g., transgenes).

[0005] In one aspect, the present disclosure provides a method of modifying an immune cell, the method comprising modifying a messenger RNA (mRNA) encoding a chimeric antigen receptor (CAR), purifying the mRNA, and delivering the mRNA to an immune cell, wherein the immune cell comprises a macrophage, monocyte, or dendritic cell, and wherein the modified immune cell comprises the CAR.

[0006] In some embodiments, the modifying step comprises including modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, and / or a poly(A) tail in the mRNA. In some embodiments, the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof. In some embodiments, the purifying step comprises silica membrane purification and / or high performance liquid chromatography (HPLC). In some embodiments, the delivering step comprises transfection.

[0007] In some embodiments, the modifying step includes including AGCap1 and 5moU in the mRNA, the purifying step includes silica membrane purification, and the delivering step includes electroporation. In some embodiments, the modifying step includes including AGCap1 and PsU in the mRNA, the purifying step includes HPLC, and the delivering step includes electroporation. In some embodiments, the modifying step includes including AGCap1 and N1mPsU in the mRNA, the purifying step includes HPLC, and the delivering step includes electroporation. In some embodiments, the modifying step includes including m6-AGCap1 and N1mPsU in the mRNA, the purifying step includes HPLC, and the delivering step includes electroporation. In some embodiments, the modifying step includes including m6-AGCap1 and PsU in the mRNA, the purifying step includes HPLC, and the delivering step includes electroporation. In some embodiments, the modifying step comprises modifying the mRNA to include AGCap1 and PsU, the purifying step comprises HPLC, and the delivering step comprises transfection. In some embodiments, the modifying step comprises including m6-AGCap1 and PsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises transfection. In some embodiments, the modifying step comprises including m6-AGCap1 and N1mPsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises transfection. In some embodiments, the modifying step comprises including AGCap1 and 5moU in the mRNA. In some embodiments, the modifying step comprises including m6AGCap1 and 5moU in the mRNA.

[0008] In some embodiments, the methods of the present invention further comprise treating the immune cells with an RNaseL inhibitor. In some embodiments, the RNaseL inhibitor comprises sunitinib. In some embodiments, the RNaseL inhibitor comprises ABCE1.

[0009] In some embodiments, the processing step occurs before the delivering step.

[0010] In some embodiments, the methods of the present invention further comprise culturing the immune cells with a cytokine or an immunostimulatory recombinant protein. In some embodiments, the cytokine comprises IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB receptor, TLR agonist, beta-glucan, IL-4, IL-13, IL-10, TGF-β, glucocorticoid, immune complex, or a combination thereof. In some embodiments, the cytokine comprises IFN-β.

[0011] In some embodiments, the culturing step occurs after the delivering step.

[0012] In some embodiments, the methods of the present invention result in modified immune cells that express a CAR. In some embodiments, CAR expression is increased compared to CAR expression in modified immune cells of the same type to which unmodified mRNA encoding the CAR has been delivered. In some embodiments, the modified immune cells exhibit increased effector activity compared to the effector activity in modified immune cells of the same type to which unmodified mRNA encoding the CAR has been delivered.

[0013] In another aspect, the disclosure provides modified immune cells produced by the methods of the present invention. In some embodiments, the modified immune cells exhibit increased survival compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit increased expression of the mRNA encoding a CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit increased CAR expression compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit increased longevity of the mRNA encoding a CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit increased longevity of the CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit increased effector activity compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit increased M1 polarization compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR.

[0014] In another aspect, the disclosure provides compositions comprising one or more modified mRNAs, the one or more modified mRNAs comprising modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, a polyA tail, or a combination thereof, and one or more RNase L inhibitors.

[0015] In some embodiments, the cap structure comprises AGCap1 or m6AGCap1. In some embodiments, the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), or N1-methyl-pseudouridine (N1mPsU). In some embodiments, the one or more RNaseL inhibitors comprise sunitinib. In some embodiments, the one or more RNaseL inhibitors comprise ABCE1.

[0016] The drawings are for purposes of illustration only and not limitation. [Brief explanation of the drawings]

[0017] [Figure 1A] 1 shows an exemplary graph depicting macrophage viability after electroporation or transfection with mCherry mRNA containing various modifications. [Figure 1B] 1 shows an exemplary graph depicting the mean fluorescence intensity after electroporation or transfection with mCherry mRNA containing various modifications. [Figure 1C] 1 shows an exemplary graph demonstrating persistence after electroporation or transfection by mCherry mRNA containing various modifications. [Figure 2A] 1 shows an exemplary graph depicting macrophage viability after electroporation or transfection with CAR mRNA containing various modifications. [Figure 2B] 1 shows an exemplary graph showing CAR expression after electroporation or transfection with CAR mRNA containing various modifications. [Figure 2C] 1 shows an exemplary graph showing CAR expression after electroporation or transfection with CAR mRNA containing various modifications. [Figure 3A] 1 shows exemplary graphs demonstrating the effect of CAR mRNA modification on macrophage function. 2 shows tumor growth curves 2 days after electroporation of macrophages with CAR mRNA. [Figure 3B] 1 shows an exemplary graph illustrating the effect of CAR mRNA modification on macrophage function. Tumor growth curves are shown when cancer cells are co-cultured with CAR macrophages at a 4:1 effector (CAR macrophages) to target (cancer cells) ratio. [Figure 3C]1 shows an exemplary graph illustrating the effect of CAR mRNA modification on macrophage function. Tumor growth curves are shown when cancer cells are co-cultured with CAR macrophages at a 2:1 effector (CAR macrophages) to target (cancer cells) ratio. [Figure 3D] 1 shows an exemplary graph illustrating the effect of CAR mRNA modification on macrophage function. Tumor growth curves are shown when cancer cells are co-cultured with CAR macrophages at a 1:1 effector (CAR macrophages) to target (cancer cells) ratio. [Figure 4A] 1 shows an exemplary graph depicting CAR macrophage viability after treatment with cytokines. [Figure 4B] The mean fluorescence intensity of surface markers after treatment with cytokines is shown. [Figure 4C] 1 shows an exemplary graph depicting CAR macrophage viability after treatment with cytokines. [Figure 4D] The mean fluorescence intensity of surface markers after treatment with cytokines is shown. [Figure 4E] The mean fluorescence intensity of surface markers after treatment with cytokines is shown. [Figure 4F] The mean fluorescence intensity of surface markers after treatment with cytokines is shown. [Figure 5A] 1 shows an exemplary graph depicting macrophage viability after transfection with CAR mRNA containing various modifications and treatment with interferon cytokines. [Figure 5B] 1 shows an exemplary graph depicting CAR expression after transfection with CAR mRNA containing various modifications and treatment with interferon cytokines. [Figure 5C] 1 shows an exemplary graph depicting mean fluorescence intensity after transfection of CAR mRNA containing various modifications and treatment with interferon cytokines. [Figure 6] 1 shows an exemplary graph demonstrating the persistence of CAR expression in macrophages treated with interferon cytokines. [Figure 7A]1 shows exemplary graphs depicting CAR macrophage viability, CAR expression and mean fluorescence intensity after transfection with CAR mRNA and treatment with various IFN-β concentrations. [Figure 7B] 1 shows an exemplary graph depicting the induction of M1 markers after transfection with CAR mRNA and treatment with various IFN-β concentrations. [Figure 8A] 1 shows an exemplary graph depicting CAR macrophage M2 and M1 marker mean fluorescence intensity 2 days after electroporation with CAR mRNA and treatment with IFN-β. [Figure 8B] 1 shows an exemplary graph depicting CAR macrophage M2 and M1 marker mean fluorescence intensity 7 days after electroporation with CAR mRNA and treatment with IFN-β. [Figure 9A] 1 shows an exemplary graph illustrating the anti-tumor function of CAR macrophages. Results are shown for macrophages transfected with CAR mRNA with or without sensitization with various concentrations of IFN-β. [Figure 9B] 1 shows an exemplary graph demonstrating the anti-tumor function of CAR macrophages. Results are shown for macrophages transfected with CAR mRNA containing various modifications and treated with IFN-β. [Figure 9C] 1 shows an exemplary graph demonstrating the anti-tumor function of CAR macrophages. Results are shown for macrophages transfected with CAR mRNA and treated with interferon cytokines. [Figure 10] 1 shows an exemplary graph depicting the effect of treatment of CAR macrophages with interferon on cytokine secretion. [Figure 11A] 1 shows exemplary graphs demonstrating the effect of treatment with interferon on the persistence of CAR mRNA in macrophages and the duration of CAR macrophage function. 1 shows results from a study of viability and CAR expression in CAR macrophages treated with interferon cytokines. [Figure 11B]1 shows exemplary graphs demonstrating the effect of treatment with interferon on the persistence of CAR mRNA in macrophages and the duration of CAR macrophage function. 1 shows the results of tumor growth from cancer cells electroporated with CAR mRNA and cultured with macrophages treated with interferon cytokines. [Figure 11C] 1 shows exemplary graphs demonstrating the effect of treatment with interferon on the persistence of CAR mRNA in macrophages and the duration of CAR macrophage function. 1 shows the results of tumor growth from cancer cells electroporated with CAR mRNA and cultured with macrophages treated with interferon cytokines. [Figure 12A] 1 shows exemplary graphs showing the effect of treatment with interferon on macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. Shown are the viability, CAR expression, and M1 marker expression of macrophages transfected with CAR mRNA and treated with interferon. [Figure 12B] 1 shows exemplary graphs depicting the effect of treatment with interferon on macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. Results of tumor killing from cancer cells cultured with macrophages electroporated with CAR mRNA and treated with interferon cytokines are shown. [Figure 12C] 1 shows exemplary graphs depicting the effect of treatment with interferon on macrophage viability, CAR expression, M1 marker expression, and CAR macrophage functionality. Results of tumor killing from cancer cells cultured with macrophages electroporated with CAR mRNA and treated with interferon cytokines are shown. [Figure 13] 1 shows an exemplary graph depicting the effect of IFN-γ on transfected macrophages. [Figure 14A]1 shows exemplary graphs depicting the effect of RNaseL inhibitors on CAR macrophages. 1 shows mCherry expression in transfected macrophages after treatment with IFN-γ and the RNaseL inhibitor sunitinib. [Figure 14B] 1 shows exemplary graphs illustrating the effect of RNaseL inhibitors on CAR macrophages. 2 shows tumor growth curves of cancer cells cultured with CAR macrophages treated with sunitinib. [Figure 14C] 1 shows exemplary graphs illustrating the effect of RNaseL inhibitors on CAR macrophages. 2 shows the tumor-killing activity of CAR macrophages treated with sunitinib. [Figure 15] 1 shows exemplary graphs depicting macrophage viability and mCherry expression in macrophages co-transfected with mRNA encoding mCherry and mRNA encoding the RNaseL inhibitor ABCE1. [Figure 16] 1 shows an exemplary graph showing CAR expression in macrophages co-transfected with mRNA encoding CAR and mRNA encoding the RNaseL inhibitor NS1. [Figure 17] 1 shows an exemplary graph showing the stability of CAR mRNA in macrophages co-transfected with mRNA encoding CAR and either the RNaseL inhibitor ABCE1 or the RNaseL inhibitor NS1. [Figure 18A] Graph showing tumor killing capacity after incubation of macrophages and CAR macrophages with CD40 ligand (CD40L). [Figure 18B] Graph showing induction of M1 and M2 marker expression after incubation of macrophages and CAR macrophages with CD40 ligand (CD40L). [Figure 19A] 1 is a graph showing tumor killing capacity after incubation of macrophages and CAR macrophages with 4-1BB. [Figure 19B]1 is a graph showing the induction of expression of M1 and M2 markers after incubation of macrophages and CAR macrophages with 4-1BB. [Figure 20A] 1 is a graph showing tumor killing capacity after incubation of macrophages and CAR macrophages with 4-1BB ligand (4-1BBL). [Figure 20B] 1 is a graph showing the induction of expression of M1 and M2 markers after incubation of macrophages and CAR macrophages with 4-1BB ligand (4-1BBL). [Figure 21] 1 shows an exemplary graph showing CAR expression in human monocytes electroporated with CAR mRNA. [Figure 22] 1 shows an exemplary graph demonstrating the efficacy of CAR macrophages generated via mRNA electroporation with and without IFN-β sensitization in a xenograft solid tumor mouse model. [Figure 23] 1 shows an exemplary graph demonstrating the efficacy of CAR macrophages generated via mRNA electroporation with and without IFN-β sensitization in a syngeneic solid tumor mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of these terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide further detail regarding its practice are incorporated herein by reference.

[0019] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0020] Approximately or About: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from the context, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (above or below) of the stated reference value (except where such number exceeds 100% of the possible values).

[0021] Activated: As used herein, the term "activated" refers to the state of a cell, e.g., a monocyte, macrophage, or dendritic cell, that has been sufficiently stimulated to induce detectable cell proliferation or to exert its effector function. Activation may also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, and / or antigen processing and presentation.

[0022] Activated Monocyte / Macrophage / Dendritic Cell: As used herein, the term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a monocyte / macrophage / dendritic cell that is undergoing cell division or exerting an effector function. The term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a cell that is performing an effector function or exhibiting some activity not found in the resting state, including phagocytosis, cytokine secretion, proliferation, changes in gene expression, changes in metabolism, and other functions.

[0023] Drug: As used herein, the term "drug" (or "biological agent" or "therapeutic agent") refers to a molecule that can be expressed, released, secreted, or delivered to a target by the modified cells described herein. Drugs include, but are not limited to, nucleic acids, antibiotics, anti-inflammatory agents, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, proteins (e.g., RNAse inhibitors), peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. Drugs can bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on the target or target cell. Drugs can diffuse or be transported into the cell and act within the cell.

[0024] Antibody: As used herein, the term "antibody" refers to a polypeptide containing sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As known in the art, intact antibodies, as produced in nature, are tetrameric agents of approximately 150 kD, composed of two identical heavy chain polypeptides (approximately 50 kD each) and two identical light chain polypeptides (approximately 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each approximately 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (located at the base tip of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain consists of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch." An intact antibody tetramer consists of two heavy-light chain dimers, in which the heavy and light chains are linked to each other by one disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to form a tetramer. Naturally produced antibodies are typically glycosylated in the CH2 domain. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold," formed by two beta sheets (e.g., three-, four-, or five-stranded sheets) packed together into an antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a natural antibody folds, the FR regions form beta sheets, providing a structural framework for the domain, and the CDR loop regions of both the heavy and light chains join in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to components of the complement system and also to receptors on effector cells, e.g., effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention (e.g., as components of CARs) comprise a glycosylated Fc domain, e.g., an Fc domain with modified or engineered glycosylation. In some embodiments, any polypeptide, or complex of polypeptides, comprising a sufficient immunoglobulin domain sequence as found in a natural antibody can be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., by an animal responding to an antigen) or produced by recombinant genetic engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody," as used herein, can refer, in appropriate embodiments (unless otherwise stated or clear from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative embodiments.For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single-chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced in nature. In some embodiments, the antibody can include a covalent modification (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0025] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may contain one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may contain one or more antibody sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative embodiments.For example, in some embodiments, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), small modular antibodies (e.g., IgG, IgE, IgM, IgA, IgB, IgC, IgD, IgE ... ImmunoPharmaceuticals ("SMIPs™"), single-chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody agent may lack covalent modifications (e.g., glycan attachment) that it would have if produced in nature. In some embodiments, an antibody agent can include a covalent modification (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs).In some embodiments, an antibody agent is a polypeptide comprising at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody, or comprises a polypeptide comprising at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are either identical in sequence or contain one to five amino acid substitutions compared to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs are deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR in that one to five amino acids within the included CDR are deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain, or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is not a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain, and / or does not comprise a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent can be or comprise a molecule or composition that does not comprise immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule comprising at least one antigen-binding domain).

[0026] Antibody fragment: As used herein, the term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments, and human and humanized versions thereof.

[0027] Antibody heavy chain: As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0028] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0029] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0030] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include either antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen," as the term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be synthetically produced or derived from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or body fluids.

[0031] Anti-tumor effect: As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention in preventing the development of an initial tumor.

[0032] Autologous: As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual.

[0033] Allogeneic: As used herein, the term "allogeneic" refers to any material (e.g., a population of cells) derived from a different animal of the same species.

[0034] Xenogeneic: As used herein, the term "xenogeneic" refers to any material (e.g., a population of cells) derived from an animal of a different species.

[0035] Cancer: As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream or lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. In certain embodiments, the cancer is medullary thyroid cancer.

[0036] Conservative sequence modification: As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies for various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for their ability to bind to antigen using the functional assays described herein.

[0037] Costimulatory Ligand: As used herein, the term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal that mediates a monocyte / macrophage / dendritic cell response, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages / dendritic cells, including, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0038] Cytotoxic: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging cells. In one embodiment, metabolically enhanced cellular cytotoxicity is improved, e.g., macrophage cytolytic activity is increased.

[0039] Effective amount: As used herein, "effective amount" and "therapeutically effective amount" are used interchangeably and refer to an amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological result or provides a manufacturing, therapeutic, or prophylactic benefit. Such results include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0040] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity carried out by an immune cell in response to immune cell stimulation. For example, an effector function of a macrophage is to engulf and digest cellular debris, foreign bodies, microorganisms, cancer cells, and other unhealthy cells through phagocytosis.

[0041] Encode: As used herein, "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0042] Endogenous: As used herein, "endogenous" refers to any substance that originates or is produced within a particular organism, cell, tissue, or system.

[0043] Exogenous: As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside a particular organism, cell, tissue, or system.

[0044] Expansion: As used herein, the term "expansion" refers to an increase in number, as in increasing the number of cells, e.g., monocytes, macrophages, and / or dendritic cells. In one embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number compared to the number initially present in the culture. In another embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number compared to other cell types in the culture. In some embodiments, expansion can occur in vivo. As used herein, the term "ex vivo" refers to cells removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0045] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) generation of an RNA template from the DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0046] Expression Vector: As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0047] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that comprises a distinct portion of a whole, but lacks one or more portions found in the whole structure. In some embodiments, the fragment consists of such a distinct portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., nucleic acid) found throughout the nucleotide sequence. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the nucleotide whole. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the whole.

[0048] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, various algorithms are available that allow for comparison of sequences to determine the degree of homology, including, for example, allowing gaps of a specified length in one sequence relative to another sequence when considering which residues in different sequences "correspond" to each other. Calculating the percent homology between two nucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the reference sequence. Next, nucleotides at corresponding nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position; when a position in the first sequence is occupied by a nucleotide that is similar to the corresponding position in the second sequence, the molecules are similar at that position.The percent homology between two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0049] Identity: As used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, for example, if each position in two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid-long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0050] Substantial identity: As used herein, the term "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, BLASTP for amino acid sequences, gapped BLAST, and PSI-BLAST. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of CDRs, references to "substantial identity" typically refer to CDRs having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the reference CDR.

[0051] Immune cell: As used herein, the term "immune cell" refers to a cell that is involved in an immune response, for example, promoting an immune response. Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, or B-lymphocytes. The source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0052] Immune response: As used herein, the term "immune response" refers to a cellular and / or systemic response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen.

[0053] Immunoglobulin: As used herein, the terms "immunoglobulin" or "Ig" refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in most controlled primary immune responses. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but it may function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0054] Isolated: As used herein, the term "isolated" refers to something that has been modified or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0055] Modified: As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0056] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level and / or change in the nature of a response in a subject compared to the level and / or nature of the response in the subject in the absence of the treatment or compound, and / or compared to the level and / or nature of the response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0057] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids include RNA. In some embodiments, nucleic acids are single-stranded. In some embodiments, nucleic acids are double-stranded. In some embodiments, nucleic acids include both single-stranded and double-stranded portions. In some embodiments, nucleic acids include a backbone that includes one or more phosphodiester linkages. In some embodiments, nucleic acids include a backbone that includes both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, nucleic acids can include a backbone that includes one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide linkages, e.g., as in "peptide nucleic acids." In some embodiments, nucleic acids include one or more or all naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, nucleic acids include one or more or all non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide.In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., replication in a recombinant cell or system, in vivo or in vitro, or chemical synthesis). In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length.

[0058] Operably linked: As used herein, the term "operably linked" refers to a functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous, in the same reading frame, and, where necessary, to join two protein-coding regions.

[0059] Overexpressed tumor antigen: As used herein, the term "overexpressed" tumor antigen or "overexpression" of a tumor antigen refers to an abnormal level of expression of the tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0060] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR™, etc., as well as by synthetic means.

[0061] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an amino acid sequence that has been engineered to be designed and / or produced by the action of the hand of man. In some embodiments, a polypeptide can comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide can comprise one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, and the like, e.g., combinations thereof. In some embodiments, a polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides.For each such class, the present specification provides, and / or one of skill in the art will be aware of, exemplary polypeptides within the class whose amino acid sequence and / or function are known. In some embodiments, such exemplary polypeptides are reference polypeptides of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (and in some embodiments with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with the reference polypeptide of the class (and in some embodiments, with all polypeptides in the class). For example, in some embodiments, a member polypeptide exhibits an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region that, in some embodiments, is or may contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and often up to 20 or more, amino acids, and in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide, hi some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0062] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a series of at least two amino acids linked together by a peptide bond). A protein can contain moieties other than amino acids (e.g., it can be a glycoprotein, proteoglycan, etc.) and / or can be otherwise processed or modified. Those of skill in the art will understand that a "protein" can be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may include, for example, two or more polypeptide chains linked by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known to those of skill in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0063] Signal Transduction Pathway: As used herein, the term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0064] Single-chain antibody: As used herein, the term "single-chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered stretch of amino acids. Various methods for producing single-chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0065] Specific binding: As used herein, the term "specific binding," with respect to an antigen-binding domain such as an antibody agent, refers to an antigen-binding domain or antibody agent that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antigen-binding domain or antibody agent that specifically binds to an antigen from one species may also bind to antigens from more than one species. However, such cross-species reactivity does not, in itself, alter the classification of the antigen-binding domain or antibody agent as specific. In another example, an antigen-binding domain or antibody agent that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, alter the classification of the antigen-binding domain or antibody agent as specific. In some cases, the terms "specific binding" or "specifically bind" can be used in reference to the interaction of an antigen-binding domain or antibody agent, protein, or peptide with a second chemical species and may mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antigen-binding domain or antibody agent recognizes and binds to a specific protein structure rather than the entire protein. If an antigen-binding domain or antibody agent is specific for epitope "A," then in a reaction involving labeled "A" and an antigen-binding domain or antibody agent, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0066] Stimulation: As used herein, the term "stimulation" refers to a primary response induced by a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex) binding to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signaling via the Fc receptor mechanism or a synthetic CAR. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structure. As used herein, the term "stimulatory molecule" refers to a molecule on a monocyte, macrophage, or dendritic cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. In some embodiments, the stimulatory molecule comprises an FcR extracellular domain comprising a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, FcαRI (CD89), or CD40 domain. In some embodiments, the stimulatory molecule comprises a TLR extracellular domain comprising a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, macrophage, dendritic cell, B cell, etc.) or tumor cell, specifically binds to a cognate binding partner (referred to herein as a "stimulatory molecule") on a monocyte, macrophage, or dendritic cell, thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, Toll-like receptor (TLR) ligands, anti-toll-like receptor antibodies, agonists, and antibodies against monocyte / macrophage receptors. In addition, cytokines such as interferon-gamma are potent stimulators of macrophages.

[0067] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog). In some embodiments, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is afflicted with a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is predisposed to a disease, disorder, or condition; in some embodiments, a predisposed subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or a reference population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit certain symptoms (e.g., clinical symptoms of the disease) or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or therapy is to be administered.

[0068] Substantially purified: As used herein, the term "substantially purified," e.g., when applied to cells, refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous cell population. In other instances, the term simply refers to cells that have been separated from cells with which they are naturally associated in their native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0069] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that is the subject of the provided methods, systems, and / or compositions, e.g., a cell, tissue, organ, or site in the body that is in need of treatment or that is preferentially bound by, for example, an antibody (or fragment thereof) or a CAR.

[0070] Target site: As used herein, the term "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0071] T cell receptor: As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, TCRs are composed of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains: a variable domain and a constant domain. In some embodiments, TCRs can be modified on any cell containing a TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0072] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic benefit is achieved by suppressing, ameliorating, or eradicating a disease state.

[0073] Transfected: As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0074] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to the partial or complete alleviation, amelioration, delay in onset, inhibition, prevention, mitigation, and / or reduction in incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who does not exhibit signs or characteristics of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment can be administered to a subject who exhibits only early or mild signs or characteristics of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, or condition. In some embodiments, treatment can include administering to an immune cell (e.g., a monocyte, macrophage, or dendritic cell) or contacting an immune cell with a modulator of a pathway activated by in vitro transcribed mRNA.

[0075] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissues. In some embodiments, a tumor can include precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a sign of cancer. In some embodiments, a tumor can be a dispersed tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0076] Vector: As used herein, the term "vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0077] Throughout this disclosure, various aspects of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0078] Detailed Description immune cells The present disclosure provides, inter alia, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) comprising at least one chimeric antigen receptor (CAR) described herein. Thus, in some embodiments, the immune cells comprising at least one CAR comprise (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein).

[0079] In some embodiments, the immune cell populations described herein comprise monocytes, macrophages, dendritic cells, and / or their precursors. In some embodiments, the population of immune cells comprises a purified population of monocytes, macrophages, or dendritic cells, or a cell line.

[0080] In some embodiments, immune cells are activated, e.g., the immune cells exhibit increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, e.g., compared to inactive cells. In some embodiments, activated immune cells exhibit altered gene expression, e.g., induced expression of one, two, three, four, five, six, or seven of the following pro-inflammatory genes (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to inactive cells. In certain embodiments, activated immune cells undergo cell division. In some embodiments, target effector activity of immune cells is enhanced by inhibition of CD47 and / or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating immune cells with anti-CD47 or anti-SIRPα antibodies, or by any method known to those of skill in the art.

[0081] In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. The immune cells may be autologous or provided from an allogeneic or universal donor. Cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to those of skill in the art, such as Ficoll separation. In some embodiments, cells from a subject's circulating blood are obtained by apheresis or leukapheresis. Cells collected by apheresis can be washed to remove the plasma fraction and resuspended in various buffers (e.g., phosphate-buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g., monocytes) involves plastic adherence. In some embodiments, differentiation of the enriched immune cells (e.g., monocytes) involves stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukopheresis composition (e.g., Leukopak), is used for enrichment. In some embodiments, the leukopheresis composition (e.g., Leukopak) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g., monocytes) is followed by mobilization with GM-CSF. In certain embodiments, selection of immune cells (e.g., monocytes) involves CD14-positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, immune cell precursors (e.g., precursors of macrophages, monocytes, or dendritic cells) are used in the compositions and methods described herein. Immune cell precursors can be differentiated into immune cells in vivo or ex vivo. Non-limiting examples of immune progenitor cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof.For example, induced pluripotent stem cells can be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSCs) can be derived from normal human tissues such as peripheral blood, fibroblasts, skin, keratinocytes, and renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs can differentiate into myeloid lineages (e.g., monocytes, macrophages, dendritic cells, or their precursors).

[0082] Immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be isolated from peripheral blood, e.g., by lysing red blood cells and depleting lymphocytes and red blood cells, e.g., by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. Specific subpopulations of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20. In some embodiments, enrichment of immune cell populations, e.g., by negative selection, can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. As non-limiting examples, cell selection can also include negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

[0083] During the isolation of a desired population of immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein by positive or negative selection, the concentration and surface (e.g., particles such as beads) of the immune cells can be varied. To ensure maximum contact area between the cells and the beads, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together.

[0084] In some embodiments, prior to administration, immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein (e.g., comprising a CAR described herein) are treated with a pro-inflammatory agent. In some embodiments, treatment with a pro-inflammatory agent increases the anti-tumor activity of immune cells described herein. In some embodiments, treatment with a pro-inflammatory agent promotes an M1 phenotype in immune cells described herein (e.g., a switch from an M2 phenotype to an M1 phenotype). In some embodiments, the pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L). In some embodiments, the pro-inflammatory agent includes or is a 41BB ligand agonist (e.g., 4-1BB).

[0085] In some embodiments, the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein (e.g., comprising a CAR described herein) are administered to a subject in combination with a pro-inflammatory agent. In some embodiments, the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein (e.g., comprising a CAR described herein) are administered to a subject substantially simultaneously with, before, or after the pro-inflammatory agent. In some embodiments, administration with a pro-inflammatory agent increases the anti-tumor activity of the immune cells described herein. In some embodiments, administration with a pro-inflammatory agent promotes an M1 phenotype in the immune cells described herein (e.g., a switch from an M2 phenotype to an M1 phenotype). In some embodiments, the pro-inflammatory agent includes or is a CD40 agonist (e.g., CD40L). In some embodiments, the pro-inflammatory agent includes or is a 41BB ligand agonist (e.g., 4-1BB).

[0086] macrophages Macrophages are immune cells specialized for the detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and are capable of at least three distinct antitumor functions: phagocytosis of dead or dying cells, microorganisms, cancer cells, cellular debris, or other foreign bodies; cytotoxicity against tumor cells; and presentation of tumor antigens to orchestrate adaptive antitumor immune responses.

[0087] Accumulating evidence suggests that macrophages are abundant in the tumor microenvironment of numerous cancers and can adopt many phenotypes, collectively referred to as tumor-associated macrophages (TAMs). The immunosuppressive nature of the tumor microenvironment typically results in more M2-like TAMs, which further contributes to the general suppression of antitumor immune responses. However, recent studies have confirmed that TAMs can be "reprogrammed" via proinflammatory signals and that a switch from an M2 phenotype to a more M1 phenotype is associated with a productive antitumor immune response. Engineering macrophages that induce endogenous TAMs to switch to M1 cells and are unable to subvert to M2 would significantly improve antitumor immunotherapy, representing a significant advance in the field.

[0088] In some embodiments, the macrophages comprise or are undifferentiated or M0 macrophages. In certain embodiments, the macrophages comprise or express one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Upon exposure to various stimuli, M0 macrophages are induced to polarize into several distinct populations that can be identified by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0089] In some embodiments, the macrophages include or are polarized macrophages. Under classical conditions of activation, M0 macrophages can be exposed to proinflammatory signals, such as LPS, IFNγ, or GM-CSF, and polarize into M1 macrophages. Generally, M1 macrophages are associated with proinflammatory immune responses, such as Th1 and Th17 T cell responses. Exposure to other stimuli can polarize macrophages into various "alternatively activated" or M2 macrophage populations.

[0090] In some embodiments, the macrophages comprise or are M1 macrophages. In some embodiments, the macrophages express one or more markers of M1 macrophages (e.g., CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, an IFIT family member, or an ISG family member (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18).

[0091] In some embodiments, macrophages comprising or expressing at least one CAR described herein produce relatively higher levels of one or more inflammatory cytokines (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of IL-1, TNF, IL-12, IL-18, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-6, IL-8, or IL33) or chemokines (e.g., IL-1, TNF, IL-12, IL-18, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-6, IL-8, or IL-33) compared to, for example, macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein stimulate an immune response and / or inflammation, e.g., as compared to macrophages that do not comprise a CAR described herein. In some embodiments, the macrophages comprise or are M2 macrophages (e.g., M2a, M2b, M2c, and M2d macrophages). M2a macrophages can be induced by IL-4, IL-13, and / or fungal infection. M2b macrophages can be induced by IL-1R ligands, immune complexes, and / or LPS. M2c macrophages can be induced by IL-10 and / or TGFβ. M2d macrophages can be induced by IL-6 and / or adenosine. In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce an immune response in a subject, for example, compared to macrophages that do not comprise a CAR described herein. In some embodiments, the macrophages express one or more markers of M2 macrophages (e.g., one, two, or three of CD206, CD163, or CD209).In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL-10 or TGFβ), e.g., compared to macrophages that do not comprise a CAR described herein.

[0092] In some embodiments, macrophages comprise at least one upregulated M1 marker and / or at least one downregulated M2 marker. In some embodiments, at least one M1 marker (e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members, and / or ISG family members) is upregulated in macrophages. In some embodiments, at least one M2 marker (e.g., CD206, CD163, and / or CD209) is downregulated in macrophages.

[0093] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased phagocytosis, e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumoricidal cytokines (e.g., TNFα)), e.g., compared to macrophages that do not comprise a CAR described herein.

[0094] In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one or both of increased expression of favorable genes (e.g., CD80, CD86, MHC-I, MHC-II, CD40, 41BBL, TNF, IFN-α, IFN-β, IFN-γ, IL2, IL12, IL6, IL8, IL1b, and / or CXCL12) or decreased expression of unfavorable genes (e.g., CD163, CD206, TGFβ, IL10, and / or IL4), e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit increased production of ROS, e.g., compared to macrophages that do not comprise a CAR described herein.In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit metabolic reprogramming (e.g., interferon signaling pathway, TH1 pathway, PTEN signaling, PI3K signaling, MTOR signaling, TLR signaling, CD40 signaling, 41BB signaling, 41BBL signaling, macrophage maturation signaling, dendritic cell maturation signaling, CD3 zeta signaling, FcRγ signaling, CD64 signaling, CD32a signaling, CD32c signaling, CD16a signaling, TLR1 signaling, TLR2 signaling, TLR3 signaling, TLR4 signaling, TLR5 signaling, TLR6 signaling, TLR7 signaling, TLR8 signaling, TLR9 signaling, ALK signaling, AXL signaling, DDR2 signaling, EGFR signaling, EphA1 signaling, INSR signaling, cMET signaling, MUSK signaling, null signaling, PDGFR signaling, PTK7 signaling, RET signaling, ROR1 signaling, ROS1 signaling, RYK signaling, TIE2 signaling, TRK signaling, VEGFR signaling, CD40 signaling, CD19 signaling, CD20 signaling, 41BB signaling, CD28 signaling, OX40 signaling, GITR signaling, TREM-1 signaling, TREM-2 signaling, DAP12 signaling, MR signaling, ICOS signaling, MyD88 signaling, V / I / LxYxxL / V signaling, SIRPα signaling, CD45 signaling, Siglec-10 signaling, PD1 signaling, SHP-1 signaling, SHP-2 signaling, KIR-2DL signaling, KIR-3DL signaling, NKG2A signaling, CD170 signaling, CD33 signaling, BTLA signaling, CD32b signaling, SIRPβ signaling, CD22 signaling, PIR-B signaling, and / or LILRB1 signaling).In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit an induction of cell survival mechanisms, e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit an induction of cell death mechanisms, e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., compared to macrophages that do not comprise a CAR described herein: increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity), or increased proliferation. In some embodiments, macrophages comprising or expressing at least one CAR described herein exhibit one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased CAR expression levels, or reduced background activity of the CAR, e.g., compared to macrophages that do not comprise a CAR described herein.

[0095] In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce infection (e.g., of an infectious pathogen) in a subject, e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, the infectious pathogen comprises or is a virus, a protozoan (e.g., Trypanosoma, Malaria, or Toxoplasma), a bacterium (e.g., Mycobacterium, Salmonella, or Listeria), a fungus (e.g., Candida), or a combination thereof. In some embodiments, the virus includes a hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, or hepatitis E), a retrovirus, a human immunodeficiency virus (e.g., HIV1 or HIV2), a T-cell leukemia virus, a lymphotropic virus (e.g., HTLV1 or HTLV2), a herpes simplex virus (e.g., herpes simplex virus type 1 or 2), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, a coronavirus (e.g., severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), Ebola virus, West Nile virus, or a variant or combination thereof.

[0096] In some embodiments, macrophages comprising or expressing at least one CAR described herein reduce the formation of at least one protein aggregate via phagocytosis and / or degrade existing aggregates in a subject (e.g., a subject with a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, an amyloidosis, or a combination thereof), e.g., compared to macrophages that do not comprise a CAR described herein. In some embodiments, the neurodegenerative disease is selected from the group consisting of tauopathies, synucleopathies, presenile dementia, senile dementia, Alzheimer's disease, progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine diseases, trinucleotide repeat diseases, and prion diseases. In some embodiments, the inflammatory disease is selected from the group consisting of systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn's disease, chronic infections, hereditary periodic fevers, malignancies, systemic vasculitis, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, immunodeficiency, Muckle-Wells (MWS) disease, and familial Mediterranean fever (FMF). In some embodiments, the amyloidosis is selected from the group consisting of primary amyloidosis (AL), secondary amyloidosis (AA), familial amyloidosis (ATTR), beta2 microglobulin amyloidosis, localized amyloidosis, heavy chain amyloidosis (AH), light chain amyloidosis (AL), primary systemic amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, apolipoprotein C2 amyloidosis, apolipoprotein C3 amyloidosis, corneal lactoferrin amyloidosis, transthyretin-related amyloidosis, dialysis amyloidosis, fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and lysozyme amyloidosis.In some embodiments, the cardiovascular disease is selected from the group consisting of atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, and heart failure. In some embodiments, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, bone marrow fibrosis, and skin fibrosis.

[0097] Monocytes Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen and generally do not proliferate in a steady state. Monocytes vary greatly in size, ranging from approximately 10 to 30 μm in diameter. The nucleus-to-cytoplasm ratio of monocytes can range from approximately 2:1 to approximately 1:1. Monocytes typically contain chemokine receptors and pathogen recognition receptors that mediate migration from the blood to tissues, such as during infection. Monocytes produce proinflammatory cytokines, internalize cells and / or toxic molecules, and can differentiate into dendritic cells or macrophages.

[0098] In some embodiments, the monocytes comprise or express one or more phenotypic markers. Examples of phenotypic markers for human monocytic cells include CD9, CD11b, CD11c, CDw12, CD13, CD14, CD15, CDw17, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD116, CD119, CDwl2lb, CDw123, CD127, CDw128, CDw131, CD147, CD155, CD156a, CD157, and CD162. Examples of phenotypic markers for mouse monocytic cells include, but are not limited to, CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD2132, CDw210, CD226, CD281, CD282, CD284, and CD286. Examples of phenotypic markers for mouse monocytic cells include, but are not limited to, CD11a, CD11b, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdw131, CD281, CD282, CD284, CD286, F4 / 80, and CD49b. In certain embodiments, monocytes comprise one, two, or three of CD11b, CD14, or CD16. In certain embodiments, the monocytes comprise CD14+ CD16- monocytes, CD14+ CD16+ monocytes, or CD14- CD16+ monocytes.

[0099] In some embodiments, monocytes are differentiated into macrophages. In some embodiments, monocytes are differentiated into dendritic cells (DCs). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages is induced by macrophage colony-stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with IL-4.

[0100] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased phagocytosis, e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit improved survival, e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into macrophages (e.g., M1 or M2 macrophages), e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit enhanced differentiation into DCs (e.g., resident or migratory DCs and / or in lymphoid and non-lymphoid tissues), e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to monocytes that do not comprise a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumoricidal cytokines (e.g., TNFα)), e.g., compared to monocytes that do not comprise a CAR described herein.

[0101] In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., compared to monocytes not comprising a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit increased production of ROS, e.g., compared to monocytes not comprising a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit metabolic reprogramming, e.g., compared to monocytes not comprising a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, e.g., compared to monocytes not comprising a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, e.g., compared to monocytes not comprising a CAR described herein. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., compared to monocytes that do not comprise a CAR described herein: increased resistance to phagocytic checkpoints; increased expression of chemokine receptors to aid trafficking; increased expression of chemokines to recruit other immune cells; increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity); or increased proliferation. In some embodiments, monocytes comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR expression, improved stability of the CAR on the cell surface, increased CAR expression levels, or reduced background activity of the CAR, compared to monocytes that do not comprise a CAR described herein.

[0102] dendritic cells Dendritic cells (DCs) are specialized bone marrow-derived antigen-presenting cells that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells can be found in both lymphoid and non-lymphoid organs and are generally thought to originate from either the lymphoid or myeloid lineages.

[0103] In some embodiments, DCs comprise or express one or more phenotypic markers. Exemplary phenotypic markers of DCs include, but are not limited to, CD11c, CD83, CD1a, CD1c, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286, and in part, CD206, CD207, CD208, and CD209.

[0104] Immature DCs may be characterized by a high capacity for antigen capture but a relatively low capacity for T cell stimulation. Inflammatory mediators promote the maturation of DCs. When DCs reach a mature stage, their properties change dramatically compared to immature DCs, such as a decreased capacity for antigen capture and / or an increased capacity for T cell stimulation. In some embodiments, the DCs include or are immature DCs. In other embodiments, the DCs include or are mature DCs.

[0105] Without wishing to be bound by theory, it is believed that by modifying DC cells to contain or express at least one CAR described herein, the mature DCs can simultaneously exhibit increased antigen capture capacity and increased T cell stimulation, e.g., compared to DCs that do not contain a CAR described herein. In some embodiments, DCs that contain or express at least one CAR described herein mediate tumor antigen presentation, e.g., increase tumor antigen presentation, compared to DCs that do not contain a CAR described herein. In some embodiments, DCs that contain or express at least one CAR described herein mediate tumor T cell stimulation, e.g., increase T cell stimulation, compared to DCs that do not contain a CAR described herein.

[0106] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to DCs that do not comprise a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased phagocytosis, e.g., compared to DCs that do not comprise a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis), increased antigen processing, increased antigen cross-presentation, increased T cell sensitization, and / or T cell stimulation, e.g., compared to DCs that do not comprise a CAR described herein.

[0107] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one or both of increased expression of preferred genes or decreased expression of undesired genes, e.g., compared to DCs not comprising a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit increased production of ROS, e.g., compared to DCs not comprising a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit metabolic reprogramming, e.g., compared to DCs not comprising a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit induction of cell survival mechanisms, e.g., compared to DCs not comprising a CAR described herein.

[0108] In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit induction of cell death mechanisms, e.g., compared to DCs that do not comprise a CAR described herein. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one, two, three, four, or five of the following, e.g., compared to DCs that do not comprise a CAR described herein: increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity), or increased proliferation. In some embodiments, DCs comprising or expressing at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR expression, improved stability of the CAR on the cell surface, increased CAR expression levels, or reduced background activity of the CAR, e.g., compared to DCs that do not comprise a CAR described herein.

[0109] Methods for modifying immune cells In some embodiments, the present disclosure provides a method of modifying immune cells, the method comprising: (a) modifying a nucleic acid encoding a chimeric antigen receptor (CAR), (b) purifying the nucleic acid, and (c) delivering the nucleic acid to an immune cell, wherein the immune cell comprises a macrophage, monocyte, or dendritic cell, and the modified immune cell comprises the CAR. In some embodiments, the present disclosure provides a method of modifying immune cells, the method comprising: (a) modifying a messenger RNA (mRNA) encoding a chimeric antigen receptor (CAR), (b) purifying the mRNA, and (c) delivering the mRNA to an immune cell, wherein the immune cell comprises a macrophage, monocyte, or dendritic cell, and the modified immune cell comprises the CAR.

[0110] In some embodiments, the present disclosure provides a method comprising delivering modified mRNA to an immune cell, wherein the mRNA comprises a CAR. In some embodiments, the provided method further comprises, optionally prior to the delivering step, treating the immune cell with an RNaseL inhibitor. In some embodiments, the provided method further comprises culturing the immune cell with a cytokine or immunostimulatory recombinant protein (e.g., IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB receptor, TLR agonist, beta-glucan, IL-4, IL-13, IL-10, TGF-β, glucocorticoid, immune complex, or a combination thereof). In some embodiments, the cytokine comprises IFN-β.

[0111] Nucleic acid modification In some embodiments of the present disclosure, the nucleic acid construct is or comprises mRNA. In some embodiments, mRNA according to the present disclosure can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to increase stability. Modification of mRNA can include, for example, modifications of nucleotides of the RNA. Thus, modified mRNA according to the present disclosure can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, modifying the mRNA includes including modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, and / or a poly(A) tail in the mRNA.In some embodiments, the mRNA may contain purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl- Cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-furan and 5-methyl-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.The preparation of such analogs is known to those skilled in the art from, for example, U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0112] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding CAR) can comprise RNA backbone modifications. Typically, backbone modifications are modifications in which the backbone phosphate of the nucleotide contained in RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methyl phosphonate, methyl phosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium group, etc., which include replacing phosphodiester bonds with other anionic groups, cationic groups, or neutral groups.

[0113] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a CAR) may contain a sugar modification. Exemplary sugar modifications are chemical modifications of the sugar of the nucleotide contained therein, such as 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotide, 2'-deoxy Sugar modifications include, but are not limited to, sugar modifications selected from the group consisting of 2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracytidine 5'-triphosphate, 2'-arauidine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0114] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding CARs) can contain modifications of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, and 6-azacytidine 5'-triphosphate. , 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or xanthosine 5'-triphosphate. In some embodiments, the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof.

[0115] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminal (5') end and a "tail" to the C-terminal (3') end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0116] Thus, in some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a CAR) comprises a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to generate a 5' triphosphate linkage; and the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A) and G(5')ppp(5')G). In some embodiments, the cap comprises a Cap0 structure. The Cap0 structure lacks 2'-O-methyl residues on the ribose attached to bases 1 and 2. In some embodiments, the cap comprises an AGCap1 structure. The AGCap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap comprises a Cap2 structure. The Cap2 structure has 2'-O-methyl residues attached to both bases 2 and 3. In some embodiments, the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, the modified mRNA of the disclosure comprises m6AGCap1 and modified nucleotides including pseudouridine (PsU).

[0117] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a CAR) comprises a 3' poly(A) tail structure. The poly(A) tail at the 3' end of the mRNA typically comprises about 10-400 adenosine nucleotides (e.g., about 100-400 adenosine nucleotides, about 10-200 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-100 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA comprises a 3' poly(C) tail structure. A suitable poly(C) tail at the 3' end of an mRNA typically contains about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly(C) tail may be in addition to or in place of the poly(A) tail.

[0118] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a CAR) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron-responsive element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length.

[0119] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 or more nucleotides in length.

[0120] nucleic acid delivery The present disclosure provides, inter alia, methods for modifying immune cells (e.g., monocytes, macrophages, or dendritic cells), the method comprising delivering to the immune cells a nucleic acid construct comprising one or more nucleic acid sequences encoding a chimeric antigen receptor (CAR) or a fragment thereof. The method may comprise delivering to the immune cells (e.g., monocytes, macrophages, or dendritic cells) a nucleic acid construct comprising one or more nucleic acid sequences encoding (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein), such that the immune cells comprise a CAR comprising (a)-(c). In some embodiments, the nucleic acid construct comprising the one or more nucleic acid sequences further encodes one, two, or three of: (d) an extracellular leader domain (e.g., an extracellular leader domain described herein), (e) an extracellular hinge domain (e.g., an extracellular hinge domain described herein), or (f) an intracellular costimulatory domain (e.g., an intracellular costimulatory domain described herein).

[0121] Nucleic acid constructs comprising one or more nucleic acid sequences encoding at least one CAR described herein can be introduced into immune cells (e.g., monocytes, macrophages, or dendritic cells) by physical, chemical, or biological methods. In some embodiments, one or more physical, chemical, or biological methods of nucleic acid delivery described herein can be used to introduce one or more nucleic acid sequences encoding at least one CAR described herein and one or more nucleic acid sequences that do not encode a CAR. Physical methods for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) can include electroporation, calcium phosphate precipitation, lipofection, Viromer-mediated transfection, particle bombardment, microinjection, mechanotransduction (e.g., squeeze-type technology), or a combination thereof. Nucleic acid constructs can be introduced into immune cells using commercially available methods, such as electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.), Gene Pulser II® (BioRad, Denver, Colo.), Multiporator® (Eppendort, Hamburg, Germany)), Maxcyte STX (Maxcyte), Maxcyte VLX (Maxcyte), Maxcyte GT (Maxcyte), CliniMacs electroporator (Miltenyi Biotec), or Neon transfection system (Thermo Fisher).Nucleic acid constructs can also be introduced into immune cells using mRNA transfection, e.g., cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001), which is incorporated herein by reference in its entirety).

[0122] Biological methods for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become widely used to insert genes into mammalian cells (e.g., human cells). Viral vectors can also be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses (e.g., Adf535), or adeno-associated viruses (see, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, which are incorporated herein by reference in their entireties). Retroviral vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer, allowing for long-term and stable integration of the transgene and its propagation in daughter cells. In some embodiments, lentiviral vectors are packaged with Vpx proteins (e.g., as described in International Publication No. WO 2017 / 044487, which is incorporated herein by reference in its entirety). In some embodiments, Vpx comprises a virion-associated protein (e.g., an accessory protein for viral replication). In some embodiments, the Vpx protein is encoded by human immunodeficiency virus type 2 (HIV-2). In some embodiments, the Vpx protein is encoded by simian immunodeficiency virus (SIV). In some embodiments, an immune cell (e.g., monocyte, macrophage, or dendritic cell) described herein is transfected with a lentiviral vector packaged with a Vpx protein. In some embodiments, Vpx inhibits at least one antiviral factor in an immune cell (e.g., monocyte, macrophage, or dendritic cell) described herein. In some embodiments, a lentiviral vector packaged with a Vpx protein exhibits increased transfection efficiency of an immune cell (e.g., monocyte, macrophage, or dendritic cell) described herein, e.g., compared to a lentiviral vector not packaged with a Vpx protein.In some embodiments, immune cells (e.g., monocytes, macrophages, or dendritic cells) described herein are electroporated and / or transfected with at least one Vpx mRNA prior to transfection with a viral vector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)). Chemical means for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) include colloidal dispersion systems, polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).

[0123] In some embodiments, the system for delivering the nucleic acid constructs described herein is a lipid-based system. The nucleic acid constructs described herein may be encapsulated in the aqueous interior of liposomes, interspersed within lipid bilayers, attached to liposomes via linking molecules, entrapped in liposomes, complexed with liposomes, dispersed in a solution or suspension containing lipids, mixed with lipids, complexed with micelles, or otherwise associated with lipids. The lipids used in the methods described herein may be naturally occurring or synthetic lipids. Lipids may also be obtained from commercial sources. For example, dimyristyl phosphatidylcholine can be obtained from Sigma (St. Louis, MO), dicetyl phosphate can be obtained from K&K Laboratories (Plainview, NY), cholesterol can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol can be obtained from Avanti. It can be obtained from Polar Lipids, Inc. (Birmingham, Ala.) Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C.

[0124] Nucleic acid purification In some embodiments, the methods of the present disclosure include purifying a nucleic acid (e.g., an mRNA encoding a CAR). In some embodiments, purifying a nucleic acid (e.g., an mRNA encoding a CAR) includes the use of any standard purification method known in the art. In some embodiments, purifying a nucleic acid (e.g., an mRNA encoding a CAR) includes silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin-based purification, polyA isolation, RNeasy, or a combination thereof. In some embodiments, purifying a nucleic acid (e.g., an mRNA encoding a CAR) includes silica membrane purification. In some embodiments, purifying a nucleic acid (e.g., an mRNA encoding a CAR) includes high performance liquid chromatography (HPLC).

[0125] Treatment and culture of immune cells during modification In some embodiments, the methods of the present disclosure include one or more steps of treating immune cells (e.g., monocytes, macrophages, or dendritic cells) during the process of modifying the immune cells.

[0126] In some embodiments, the disclosed methods include treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with a modulator of a pathway activated by in vitro-transcribed mRNA. The in vitro-transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7, TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2'-5'-oligoadenylate synthase (OAS)). Signaling through these different pathways leads to inflammation associated with activation of type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-12, and a cascade of transcriptional programs. Collectively, these create a pro-inflammatory microenvironment poised to induce specific immune responses. Furthermore, downstream effects such as slowing down translation via eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, promoting RNA degradation by ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication are relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0127] In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase L, RNase T2, or RNase 1 inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase L inhibitor. In some embodiments, the RNase L inhibitor comprises sunitinib. In some embodiments, the RNase L inhibitor comprises ABCE1.

[0128] In some embodiments, treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with an RNaseL inhibitor increases mRNA stability in the modified immune cells compared to mRNA stability in modified immune cells of the same type that have not been treated with an RNaseL inhibitor. In some embodiments, treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with an RNaseL inhibitor increases CAR expression in the modified immune cells compared to CAR expression in modified immune cells of the same type that have not been treated with an RNaseL inhibitor. In some embodiments, treating immune cells (e.g., monocytes, macrophages, or dendritic cells) with an RNaseL inhibitor increases effector activity in the modified immune cells compared to effector activity in modified immune cells of the same type that have not been treated with an RNaseL inhibitor.

[0129] In some embodiments of the present disclosure, the step of treating the immune cells (eg, monocytes, macrophages, or dendritic cells) occurs before the step of delivering the mRNA to the immune cells.

[0130] In some embodiments, the methods of the present disclosure include culturing immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein. In some embodiments, the cytokine is selected from the group consisting of IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB, CD19 agonist, TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, ​​TLR-7, TLR-8, or TLR-9), TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3), glucocorticoid, immune complex, interleukin- 1 alpha (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), tumor necrosis factor (TNF-β), CD154, lymphotoxin beta (LT-β), and A-proliferation-inducing ligand (APRIL) ), CD70, CD153, glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L (CD252), TALL-1 (tumor necrosis factor ligand superfamily member 13B-TNFSF13B), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), TNF-related activation-inducing cytokine (TRANCE), erythropoietin (Epo), thyroid peroxisome proliferator-activated receptor (TPR) Tyridase precursor (Tpo), FLT-3L, stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), nucleotide-binding oligomerization domain-containing protein (NOD) ligands (e.g., NOD1, NOD2, or NOD1 / 2 agonists), RIG-I-like receptor (RLR) ligands (e.g., 5'ppp-dsRNA, 3p-hpRNA, Poly(I:C), or Poly(dA:dT)),C-type lectin receptor (CLR) ligands (e.g., curdlan, β-glucan, HKCA, laminarin, pustulan, scleroglucan, dispersible WGP, soluble WGP, zymosan, zymosan degraded, furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB-HS15, or TDM), cyclic dinucleotide sensor ligands (e.g., C-Gas agonists or stimulator of interferon genes (STING) ligands), inflammasome inducers (e.g., alum, ATP, CPPD crystals, hemozoin, MSU crystals, nanoSiO2, nigericin, or TDB), aryl hydrocarbon (AhR) ligands (e.g., FICZ, indirubin, ITE, or L-kynurenine), alpha protein kinase 1 (ALPK1) ligands, multi-PRR ligands, NFKB / NFAT activators (e.g., concanavalin A A), ionomycin, PHA-P, or PMA), or a combination thereof. In some embodiments, the cytokine comprises IFN-β.

[0131] In some embodiments of the present disclosure, the step of culturing immune cells (eg, monocytes, macrophages, or dendritic cells) is performed after the step of delivering mRNA to the immune cells.

[0132] In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the viability of the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases protein (e.g., CAR) expression in the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the longevity of protein (e.g., CAR) expression in the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the effector activity of the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., monocytes, macrophages, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases M1 polarization of the modified immune cells compared to the same type of modified immune cells that were not cultured with the cytokine or immunostimulatory recombinant protein.

[0133] modified immune cells In some embodiments, the modified immune cells are produced by the methods of the present disclosure.

[0134] In some embodiments, the modified immune cells exhibit increased viability compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% increase in viability compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR.

[0135] In some embodiments, the modified immune cells exhibit increased expression of the mRNA encoding the CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR. In some embodiments, the modified immune cells exhibit at least a 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000% increase in expression of the mRNA encoding the CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR. In some embodiments, the modified immune cells exhibit at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold increase in expression of the mRNA encoding the CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR.

[0136] In some embodiments, the modified immune cells exhibit increased CAR expression compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit at least a 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000% increase in CAR expression compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold increase in CAR expression compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR.

[0137] In some embodiments, the modified immune cells exhibit an increased longevity of the mRNA encoding the CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR. In some embodiments, the modified immune cells exhibit an increased longevity of the mRNA encoding the CAR by at least 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, or 1 month compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR.

[0138] In some embodiments, the modified immune cells exhibit an increased longevity of the CAR compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR. In some embodiments, the modified immune cells exhibit an increased longevity of the CAR of at least 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, or 1 month compared to modified immune cells of the same type comprising an unmodified mRNA encoding the CAR.

[0139] In some embodiments, the modified immune cells exhibit increased effector activity compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the modified immune cells exhibit a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% increase in effector activity compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the increased effector activity comprises increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation.

[0140] In some embodiments, the modified immune cells exhibit increased M1 polarization compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the increased M1 polarization comprises increased levels of M1 markers including CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, an IFIT family member, or an ISG family member. In some embodiments, the modified immune cells exhibit a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% increase in M1 polarization compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR.

[0141] In some embodiments, the modified immune cells exhibit decreased M2 polarization compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR. In some embodiments, the decreased M2 polarization comprises a decrease in the levels of an M2 marker, including CD206, CD163, or CD209. In some embodiments, the modified immune cells exhibit a 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% decrease in M2 polarization compared to modified immune cells of the same type comprising an unmodified mRNA encoding a CAR.

[0142] Assay Various assays can be performed to confirm the presence of the nucleic acid constructs described herein in immune cells (e.g., monocytes, macrophages, or dendritic cells). For example, such assays include molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR, as well as biochemical assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting). Other assays of the present disclosure include, for example, fluorescence-activated cell sorting (FACS), immunofluorescence microscopy, MSD cytokine analysis, mass spectrometry (MS), RNA-Seq, and functional assays.

[0143] Various assays can be performed to determine various characteristics of the modified immune cells (e.g., monocytes, macrophages, or dendritic cells), including, but not limited to, immune cell viability, nucleic acid (e.g., mRNA) expression, nucleic acid (e.g., mRNA) longevity, protein (e.g., CAR) expression, protein (e.g., CAR) longevity, effector activity, and M1 polarization.

[0144] All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0145] Chimeric antigen receptor (CAR) As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically target the cells and / or bind to an antigen. CARs can be used as a therapy, e.g., by adoptive cell transfer. For example, in some embodiments, monocytes, macrophages, and / or dendritic cells are removed from a patient (e.g., from blood, tumor, or ascites) and modified to express a receptor specific for a particular form of antigen. In some embodiments, the CAR is specifically expressed against an antigen, e.g., a tumor-associated antigen. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0146] In some embodiments, modified immune cells, e.g., modified macrophages, monocytes, or dendritic cells, are generated by expressing a CAR therein. In some embodiments, the immune cells comprise a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and the immune cells comprise macrophages, monocytes, or dendritic cells.

[0147] In some embodiments, the CAR may further comprise one or more extracellular leader domains, one or more extracellular hinge domains, and one or more intracellular costimulatory domains.

[0148] In some embodiments, the CAR comprises a spacer domain or hinge (e.g., a CD8 or CD28 hinge domain) between the extracellular domain and the transmembrane domain. In some embodiments, the CAR comprises a spacer domain or hinge between the intracellular domain and the transmembrane domain. As used herein, the term "spacer domain" or "hinge" refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular or intracellular domain of a polypeptide chain. In some embodiments, the spacer domain or hinge may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, a short oligopeptide or polypeptide linker, preferably between 2-10 amino acids in length, may form the link between the transmembrane and intracellular domains of the CAR. Examples of linkers include a glycine-serine doublet.

[0149] In some embodiments, an immune cell comprising a CAR may comprise one or more regulatory systems, including, but not limited to, a safety switch (e.g., an on switch and an off switch, a suicide switch), a logic gate, e.g., an AND gate (e.g., two or more CARs, each lacking one or more signaling domains such that activation of both / all CARs is required for activation or function of the intact immune cell (such as a macrophage, monocyte, or dendritic cell)), an OR gate (e.g., two or more CARs, each having an intracellular domain, e.g., CD3ζ and a costimulatory domain), and / or a NOT gate (e.g., two or more CARs, one of which includes an inhibitory domain that antagonizes the function of the other CAR(s)).

[0150] The present disclosure also provides an immune cell comprising a nucleic acid sequence (e.g., an isolated nucleic acid sequence) encoding a CAR, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an extracellular domain, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid sequence encoding an intracellular domain, and wherein the cell is a monocyte, macrophage, or dendritic cell that expresses the CAR.

[0151] In some embodiments, the CAR comprises an extracellular domain operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, for expression in an immune cell. In some embodiments, the nucleic acid encoding the extracellular domain is operably linked to the nucleic acid encoding the transmembrane domain, which is operably linked to the nucleic acid encoding the intracellular domain.

[0152] In some embodiments, the effector activity of an immune cell comprising a CAR is directed against a target cell that contains an antigen that specifically binds to the antigen-binding domain of the CAR. In some embodiments, the targeted effector activity directed against the target cell is or includes phagocytosis, targeted cytotoxicity, antigen presentation, or cytokine secretion.

[0153] In some embodiments, a CAR described herein comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain) that inhibits anti-phagocytic signaling in an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). In some embodiments, a CAR described herein improves the effector activity of an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell), e.g., by enhancing inhibition of CD47 and / or SIRPα activity. In some embodiments, a CAR described herein binds to CD47 and acts as a dominant-negative receptor, inhibiting SIRPα activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds to SIRPα comprises, e.g., an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction between CD47 and SIRPα. In some embodiments, a CAR is or comprises a phagocytosis logic gate.

[0154] In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR described herein) comprises or expresses at least one variant or fragment of SIRPα (e.g., dominant-negative SIRPα or a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), PD1 (e.g., dominant-negative PD1 or HAC-I), anti-PD1 scFv (e.g., E27 or durvalumab), Siglec-10, Siglec-9, Siglec-11, and / or SHP-1. In some embodiments, the variant or fragment comprises a mutant intracellular domain. In some embodiments, the variant or fragment does not comprise or express at least one intracellular domain (e.g., the immune cell comprises or expresses an anti-CD47 scFv, a CD8 hinge domain, and a CD8 transmembrane). In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR described herein) comprises a dominant negative receptor, e.g., that blocks an inhibitory checkpoint.

[0155] In some embodiments, the CARs described herein further comprise a truncated peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide) and at least one second CAR comprising at least one inhibitory domain of anti-phagocytic signaling. In some embodiments, the at least one second CAR comprises SIRPα (e.g., a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), or a CD47-binding extracellular domain or a fragment thereof. In some embodiments, the at least one second CAR comprises a SIRPα transmembrane domain or a fragment thereof. In certain embodiments, the second CAR further comprises a hinge domain (e.g., a CD8 hinge domain). In certain embodiments, at least one second CAR comprises (i) a leader sequence (e.g., a CD8 leader), ii) an extracellular domain (e.g., a SIRPα, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain), and ii) a transmembrane domain (e.g., a SIRPα transmembrane domain). In some embodiments, the CARs described herein further comprise a truncated peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD20 or a fragment thereof, CD19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and / or a fluorescent protein).

[0156] In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR described herein) comprises or expresses one or more phosphatase dead domains (e.g., phosphatase dead Shp1, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and / or phosphatase dead SHIP-1 domain) and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain). In some embodiments, a CAR described herein further comprises a truncated peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide) and one or more phosphatase dead domains (e.g., phosphatase dead Shp1, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and / or phosphatase dead SHIP-1 domain) and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain).

[0157] Extracellular domain The present disclosure provides chimeric antigen receptors (CARs) comprising an extracellular domain. In some embodiments, the extracellular domain comprises an Fc receptor (FcR) extracellular domain. In some embodiments, the extracellular domain comprises a toll-like receptor (TLR) extracellular domain. In some embodiments, the extracellular domain comprises a leader domain. In some embodiments, the extracellular domain comprises an antigen-binding domain. In some embodiments, the extracellular domain comprises a hinge domain. In some embodiments, the extracellular domain comprises one or more of an FcR extracellular domain, a TLR extracellular domain, a leader domain, an antigen-binding domain, and a hinge domain. In some embodiments, the extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0158] FcR extracellular domain In some embodiments, the FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain (or portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, the FcR extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0159] TLR extracellular domains In some embodiments, the TLR ectodomain comprises a full-length TLR ectodomain. In some embodiments, the TLR ectodomain comprises a portion of a full-length TLR ectodomain. In some embodiments, the TLR ectodomain (or portion thereof) is or comprises a human TLR ectodomain. In some embodiments, the TLR ectodomain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0160] Leader Domain In some embodiments, the CAR comprises one or more extracellular leader domains. In some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence encoding an extracellular leader domain, but the extracellular leader domain is cleaved from the CAR before the CAR is expressed in an immune cell. In some embodiments, the extracellular leader domain is or comprises a human extracellular leader domain. In some embodiments, the extracellular leader domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain comprises a CD8 extracellular leader domain. In some embodiments, the extracellular leader domain comprises a leader domain derived from a stimulatory or costimulatory domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88 domain).

[0161] antigen-binding domain In some embodiments, the CAR comprises an antigen-binding domain that binds to, for example, an antigen on a target cell. In some embodiments, the CAR comprises an antigen-binding domain that binds to an antigen associated with a viral infection, a bacterial infection, a parasitic infection, an autoimmune disease, and / or a cancer cell. In some embodiments, the antigen-binding domain recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state.

[0162] In some embodiments, the antigen binding domain binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B-cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), protease-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21 (testisin or PRSS21), vascular endothelial growth factor receptor 2 (V EGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, beta9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), and claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8),Melanoma antigen recognized by T cell 1 (MelanA or MART1), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Proteins) Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mut) hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), or immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, the tumor antigen isIn certain embodiments, the tumor antigen comprises ERBB2 (Her2 / neu). In certain embodiments, the tumor antigen comprises PSMA. In certain embodiments, the tumor antigen comprises mesothelin.

[0163] In some embodiments, the antigen-binding domain binds to a misfolded protein antigen or a protein in a protein aggregate, e.g., a protein specific to a disease / disorder of interest. In some embodiments, the disease / disorder is a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease / disorder, or amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or transthyretin). In some embodiments, the neurodegenerative disease / disorder is a tauopathy, amyloid nucleopathy, presenile dementia, senile dementia, Alzheimer's disease (mediated by beta-amyloid protein aggregates), Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine diseases, trinucleotide repeat diseases, familial British dementia, fatal familial insomnia, Gerstmann-Sträussler-Scheinker syndrome, hereditary cerebral hemorrhage with amyloidosis (Icelandic type) (HCHW). AI), sporadic fatal insomnia (sFI), variably protease-sensitive prion disease (VPSPr), familial Danish dementia, and prion diseases (such as Creutzfeldt-Jakob disease, CJD, and variant Creutzfeldt-Jakob disease (vCJD)).

[0164] In some embodiments, the antigen-binding domain comprises any domain that binds to an antigen. In some embodiments, the antigen-binding domain is or comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, or any fragment thereof, such as an scFv. In some embodiments, the antigen-binding domain is or comprises an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule. In some embodiments, the antigen-binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, the antigen-binding domain is derived, in whole or in part, from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR comprises a human antibody, a humanized antibody, or a fragment thereof (e.g., an scFv). In some embodiments, the antigen-binding domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the antigen-binding domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0165] In some embodiments, the CAR comprises one or more antigen-binding domains. In some embodiments, the CAR comprises two or more antigen-binding domains. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the immune cells comprise two or more different CARs comprising one or more antigen-binding domains. In some embodiments, immune cells comprising bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of two antigens. In some embodiments, the immune cells comprise bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains, where the CARs alone are insufficient to mediate activation of the modified cell, but together are synergistic and provide distinct signals that stimulate activation of the modified cell. In some embodiments, such a configuration may be referred to as an "AND" logic gate.

[0166] In some embodiments, immune cells containing bispecific CARs and / or two or more different CARs containing one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of one antigen and the absence of a second, normal protein antigen before cellular activity is stimulated. In some embodiments, such a configuration may be referred to as a "NOT" logic gate. In contrast to AND gates, NOT-gate CAR-modified cells are activated by binding to a single antigen. However, binding of a second receptor to the second antigen functions to neutralize the activation signal perpetuated through the CAR. Typically, such inhibitory receptors target antigens that are abundantly expressed in normal tissues but absent in tumor tissues.

[0167] Hinge domain In some embodiments, the CAR comprises one or more extracellular hinge domains. In some embodiments, the extracellular hinge domain is or comprises a human extracellular hinge domain. In some embodiments, the extracellular hinge domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular hinge domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the one or more extracellular hinge domains comprise a CD8a extracellular hinge domain or an IgG4 or CD28 extracellular hinge domain. In some embodiments, the extracellular hinge domain is selected based on, for example, optimal size (e.g., allowing for exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and / or homodimerization to tumor antigens. Optimize the physicochemical parameters of CAR, such as the lack of dimerization.

[0168] Transmembrane domain In some embodiments, the CAR comprises a transmembrane domain, e.g., connecting the extracellular domain to the intracellular domain. In some embodiments, the transmembrane domain is naturally associated with one or more other domain(s) of the CAR. In some embodiments, to minimize interaction with other members of the receptor complex, the transmembrane domain can be modified to avoid binding of other surface membrane proteins to the transmembrane domain. In some embodiments, the transmembrane domain can be derived from either naturally occurring or synthetic sources. In some embodiments, the transmembrane domain is derived from a naturally occurring membrane-associated or transmembrane protein. In some embodiments, the transmembrane domain is or comprises a human transmembrane domain. In some embodiments, the transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the transmembrane domain is selected from the group consisting of CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD 28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3 zeta, FcRγ, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPβ, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain.

[0169] FcR transmembrane domain In some embodiments, the FcR transmembrane domain comprises a full-length FcR transmembrane domain. In some embodiments, the FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, the FcR transmembrane domain is or comprises a human FcR transmembrane domain or a portion thereof. In some embodiments, the FcR transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR transmembrane domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0170] TLR transmembrane domains In some embodiments, the TLR transmembrane domain comprises a full-length TLR transmembrane domain. In some embodiments, the TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, the TLR transmembrane domain is or comprises a human TLR transmembrane domain or a portion thereof. In some embodiments, the TLR transmembrane domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0171] Intracellular domain In some embodiments, a CAR comprises one or more intracellular domains. In some embodiments, the intracellular domain is or comprises a human intracellular domain or portion thereof. In some embodiments, the intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the intracellular domain and / or other cytoplasmic domains of a CAR are involved in activation of the cell (e.g., immune cell) in which the CAR is expressed. In some embodiments, the intracellular domain of a CAR is involved in signal activation and / or transduction in the immune cell comprising the CAR.

[0172] In some embodiments, the intracellular domain of the CAR comprises at least one domain involved in signal activation and / or transduction. In some embodiments, the intracellular domain is or comprises at least one of a costimulatory molecule and a signaling domain. In some embodiments, the intracellular domain of the CAR comprises dual signaling domains. In some embodiments, the intracellular domain of the CAR comprises three or more signaling domains.

[0173] In some embodiments, the intracellular domain comprises a cytoplasmic portion of a surface receptor. In some embodiments, the intracellular domain comprises a costimulatory molecule. In some embodiments, the intracellular domain comprises a molecule that acts to initiate signaling in an immune cell.

[0174] In some embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules, for example, at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0175] FcR intracellular domain In some embodiments, the FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, the FcR intracellular domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, the FcR intracellular domain is or comprises a human FcR intracellular domain or a portion thereof. In some embodiments, the FcR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR intracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0176] TLR intracellular domain In some embodiments, the TLR intracellular domain comprises a full-length TLR intracellular domain. In some embodiments, the TLR intracellular domain comprises a portion of a full-length TLR intracellular domain. In some embodiments, the TLR intracellular domain is or comprises a human TLR intracellular domain or a portion thereof. In some embodiments, the TLR intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR intracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0177] Signaling domains In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is or comprises a human intracellular signaling domain or a portion thereof. In some embodiments, the signaling domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the signaling domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0178] In some embodiments, the one or more intracellular signaling domains are selected from the group consisting of CD3-zeta, FcR γ, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL , DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD 40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPβ, CD22, PIR-B, LILRB1, Syk, 41BB ligand (41BBL, TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, or one or more cytokine receptor signaling domains (e.g., IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, Dectin-1, or ICOSL intracellular signaling domain).

[0179] In some embodiments, the intracellular domain of the CAR comprises dual signaling domains, e.g., 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, Dectin-1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, in any combination with any of the signaling domains listed in the paragraph above.

[0180] Costimulatory domain As used herein, a "costimulatory molecule" or "costimulatory domain" refers to a molecule in an immune cell that is used to enhance or attenuate initial stimulation. For example, pathogen-associated pattern recognition receptors such as TLRs or the CD47 / SIRPα axis are molecules on immune cells that enhance or attenuate initial stimulation, respectively. In some embodiments, the costimulatory domain is selected from the group consisting of TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, and LIG. HT, NKG2C, B7-H3, CD83-specific binding ligand, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, I TGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), Costimulatory molecules include CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0181] In some embodiments, the costimulatory domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the costimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0182] As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal, such as activation of a CAR on an immune cell, results in activation of the immune cell.

[0183] Cleaved peptide As used herein, a cleavage peptide refers to a peptide that can induce cleavage of a recombinant protein within a cell. In some embodiments, the cleavage peptide is a 2A peptide. In some embodiments, the cleavage peptide is or comprises a P2A, F2A, E2A, or T2A peptide. In some embodiments, the nucleic acids described herein comprise one or more nucleic acid sequences encoding one or more cleavage peptides. In some embodiments, the nucleic acid comprising the nucleic acid sequence encoding the cleavage peptide also comprises one or more nucleic acid sequences encoding one or more intracellular domains and one or more nucleic acid sequences comprising one or more peptide agents, wherein translation of the nucleic acid results in a protein comprising one or more intracellular domains separated from the one or more peptide agents by the cleavage peptides. In some embodiments, a first promoter is operably linked to one or more nucleic acids encoding a CAR, and a second promoter is operably linked to one or more nucleic acids encoding a peptide agent. In some embodiments, the nucleic acid sequence comprising a CAR and optionally one or more peptide agents further comprises an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA, facilitating initiation of translation.

[0184] Peptide drugs As used herein, a peptide agent refers to a peptide that is co-expressed with a CAR in an immune cell. In some embodiments, the peptide agent is co-expressed with a CAR to ensure stoichiometric balance and optimal signaling of the CAR. In some embodiments, a peptide agent forms a homodimer with the same peptide agent. In some embodiments, a peptide agent forms a heterodimer with a different peptide agent. In some embodiments, a nucleic acid described herein comprises one or more nucleic acid sequences encoding one or more peptide agents. In some embodiments, a peptide agent is or comprises an FcR gamma chain. In some embodiments, a peptide agent comprises any peptide, protein, receptor, secreted antibody or fragment thereof (e.g., scFv, Fab, Fab', F(ab')2, Fc, or nanobody). In some embodiments, the peptide agent comprises one or more cytokines (e.g., one or more of IL-1, IL-2, IL-6, IL-8, TNF-α, IFNα, IFNβ, IFN-γ, GMCSF, or MCSF), CD40-L, dominant negative SIRPα, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC10, or dominant negative LILRB.

[0185] Fc receptor (FcR) In some embodiments, the CAR comprises one or more antigen-binding domains and an FcR extracellular domain, and / or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and / or the intracellular domain of the CAR comprises an FcR intracellular domain. In some embodiments, the CAR comprises, from N-terminus to C-terminus, one or more extracellular binding domains, an FcR extracellular domain, an FcR transmembrane domain, and an FcR intracellular domain. In some embodiments, one or more of the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain are or comprise human FcR domains. In some embodiments, the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together comprise a full-length FcR. In some embodiments, the FcR extracellular domain, the FcR transmembrane domain, and the FcR intracellular domain together comprise a portion of a full-length FcR. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, the FcR intracellular domain comprises a portion of a full-length FcR intracellular domain.

[0186] Toll-like receptors (TLRs) In some embodiments, the CAR comprises one or more antigen binding domains and a Toll-like receptor (TLR) extracellular domain, and / or the transmembrane domain of the CAR comprises a TLR transmembrane domain, and / or the intracellular domain of the CAR comprises a TLR intracellular domain. In some embodiments, the CAR comprises, from N-terminus to C-terminus, one or more extracellular binding domains, a TLR extracellular domain, a TLR transmembrane domain, and a TLR intracellular domain. In some embodiments, one or more of the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain are or comprise human TLR domains. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a full-length TLR. In some embodiments, the TLR extracellular domain, the TLR transmembrane domain, and the TLR intracellular domain together comprise a portion of a full-length TLR. In some embodiments, the TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, the TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, the TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.

[0187] Nucleic Acid Constructs The present disclosure provides, inter alia, a nucleic acid molecule encoding at least one CAR or fragment thereof described herein. An immune cell can comprise a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) encoding at least one CAR described herein. In some embodiments, the nucleic acid molecule encoding at least one CAR comprises (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein).

[0188] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns to the extent that a nucleotide sequence encoding a protein, depending on its type, may contain intron(s). "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (e.g., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand can be used as a template for transcription of the gene or cDNA and can encode the protein or other product of that gene or cDNA.

[0189] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be adjacent to each other and, where necessary to join two protein-coding regions, in the same reading frame.

[0190] The nucleic acid molecule encoding at least one CAR or fragment thereof described herein can be a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, the nucleic acid molecule comprises or is a messenger RNA (mRNA) transcript encoding at least one CAR or fragment thereof described herein. In some embodiments, the nucleic acid molecule comprises or is a DNA construct encoding at least one CAR or fragment thereof described herein.

[0191] In some embodiments, all or a fragment of a CAR described herein is encoded by a codon-optimized nucleic acid molecule, e.g., for expression in a cell (e.g., a mammalian cell). Various codon optimization methods are known in the art, e.g., as disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148, each of which is incorporated herein by reference in its entirety.

[0192] Expression of the nucleic acids described herein can be achieved by operably linking a nucleic acid encoding a CAR polypeptide or fragment thereof to a promoter in an expression vector. Exemplary promoters (e.g., constitutive promoters) include, but are not limited to, the elongation factor-1α (EF-1α) promoter, the immediate-early cytomegalovirus (CMV) promoter, the ubiquitin C promoter, the phosphoglycerokinase (PGK) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the Moloney murine leukemia virus (MoMuLV) promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, or the creatine kinase promoter. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. The vector can also contain additional promoter elements, such as enhancers, to control the frequency of transcription initiation.

[0193] In some embodiments, the vector comprising the nucleic acid molecule encoding at least one CAR or a fragment thereof described herein comprises or is a viral vector. Viral vector technology is well known and described in the art (e.g., Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, or retroviral vectors (e.g., lentiviral vectors or gamma retroviral vectors). In some embodiments, the vector comprises a lentiviral vector (e.g., as described in U.S. Patent No. 9,149,519 or International Publication No. WO2017 / 044487, each of which is incorporated herein by reference in its entirety).

[0194] In some embodiments, the viral vector comprises an adenoviral vector. Adenoviruses are a large family of viruses that contain double-stranded DNA. Adenoviruses replicate in the nucleus of host cells, using the host's cellular machinery to synthesize viral RNA, DNA, and proteins. Adenoviruses are known in the art to infect both replicating and non-replicating cells, accommodate large transgenes, and encode proteins without integrating into the host cell genome. In some embodiments, the adenoviral vector comprises an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector).

[0195] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, for example, Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., PNASUSA, 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699-708 (2012)). Methods for producing and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0196] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof. Generally, any AAV serotype can be used to deliver at least one CAR described herein. In some embodiments, the AAV serotype has tropism for a specific tissue.

[0197] In some embodiments, the CRISPR / Cas9 system facilitates high-level, precise genome editing using adeno-associated virus (AAV) vectors, which have recently been shown to serve as donor template DNA during homologous recombination (HR).

[0198] In some embodiments, the vector comprises a gammaretroviral vector (e.g., as described in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun;3(6):677-713, which is incorporated herein by reference in its entirety). Exemplary gammaretroviral vectors include murine leukemia virus (MLV), spleen-limited focus-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0199] In some embodiments, the vector comprises two or more nucleic acid sequences encoding a CAR, e.g., at least one CAR described herein, and a second CAR, e.g., a different CAR described herein. In some embodiments, the two or more nucleic acid sequences encoding the CAR and the second CAR are encoded by a single nucleic acid molecule, e.g., in the same frame as a single polypeptide chain. In some embodiments, the two or more CARs are separated by one or more cleavage peptide sites (e.g., autocleavage sites or substrates for intracellular proteases). In certain embodiments, the cleavage peptide comprises a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, or a variant thereof.

[0200] In some embodiments, the vector comprises at least one nucleic acid sequence encoding a CAR, e.g., at least one CAR described herein, and at least one nucleic acid encoding at least one gene that co-expresses the CAR, e.g., a cytokine described herein (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, and / or IL-1) or a stimulatory ligand described herein (e.g., CD7, B7-1 (CD80)), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD40L, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and / or B7-H3 ligand).

[0201] Pharmaceutical Composition The present disclosure provides, inter alia, pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0202] In some embodiments, pharmaceutical compositions of the present disclosure include compositions comprising one or more modified mRNAs, the modified mRNAs comprising modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, a polyA tail, or a combination thereof, and one or more RNase L inhibitors. In some embodiments, the pharmaceutical composition comprises one or more modified mRNAs comprising an AGCap1 or m6AGCap1 cap structure. In some embodiments, the pharmaceutical composition comprises one or more modified mRNAs comprising modified nucleotides including pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), or N1-methyl-pseudouridine (N1mPsU). In some embodiments, the pharmaceutical composition comprises sunitinib. In some embodiments, the pharmaceutical composition comprises ABCE1. In some embodiments, the pharmaceutical composition comprises macrophages transfected with an mRNA comprising m6-AGCap1 and a PsU modification, wherein the mRNA encodes a CAR, and the macrophages are cultured with IFN-β, wherein culturing the macrophages with IFN-β enhances CAR expression, CAR persistence, CAR macrophage function, M1 phenotype, resistance to M2 inducers, or a combination thereof, compared to macrophages transfected with the same mRNA but not cultured with IFN-β.

[0203] Where a "therapeutically effective amount," "immunologically effective amount," "anti-immune response effective amount," or "immune response inhibiting effective amount" is indicated, the exact amount of a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be determined by a physician, taking into account individual differences in the age, weight, immune response, and condition of the patient (subject).

[0204] Pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein may include buffers such as neutral buffered saline or phosphate buffered saline (PBS), carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol, proteins, polypeptides, or amino acids (e.g., glycine), antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), serum, and preservatives such as cryoprotectants. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, e.g., free of detectable levels of contaminants (e.g., endotoxins).

[0205] The pharmaceutical compositions described herein can be administered in a manner appropriate for the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration will depend on factors such as the condition of the patient and the type and severity of the patient's disease, disorder, or condition, but the appropriate dosage can be determined by clinical trials.

[0206] The pharmaceutical compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0207] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous infusion or injection. In some embodiments, the pharmaceutical compositions disclosed herein are formulated for intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be formulated for administration using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, which is incorporated herein by reference in its entirety).

[0208] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumor, and intrasternal injection and infusion.

[0209] The pharmaceutical compositions comprising the immune cells described herein may be administered in an amount of about 10 4 ~about 10 9 cells / kg body weight (e.g., approximately 10 5 ~about 10 6 In some embodiments, the dose of immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein is at least about 1 x 10 cells / kg body weight, including all integer values ​​within these ranges. 6 , about 1.1×10 6 , about 2×10 6 , about 3.6×10 6 , about 5×10 6 , 1×10 7 , about 1.8×10 7 , about 2×10 7 , about 5×10 7 , about 1×10 8 , about 2×10 8 , about 5×10 8 , about 1×10 9, about 2×10 9 , or about 5 × 10 9 The pharmaceutical compositions described herein may also be administered multiple times at a particular dosage. The optimal dosage and treatment regime for a particular patient can be readily determined by one of skill in the art by monitoring the patient for symptoms of a disease, disorder, or condition and adjusting the treatment accordingly.

[0210] It may be desirable to administer a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein to a subject, followed by redrawing blood (or performing apheresis), activating the collected immune cells, and reinfusing the activated immune cells into the subject. This process can be performed multiple times, for example, every few weeks. Immune cells (e.g., macrophages, monocytes, or dendritic cells) can be activated from a blood draw of about 10 cc to about 400 cc. In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are activated from a blood draw of about 20 cc, about 30 cc, about 40 cc, about 50 cc, about 60 cc, about 70 cc, about 80 cc, about 90 cc, or about 100 cc. Without being bound by theory, methods involving multiple blood draws and reinfusions described herein can be selected for certain immune cell populations. In some embodiments, a pharmaceutical composition comprising an immune cell (e.g., a macrophage, monocyte, or dendritic cell) described herein is administered in combination with (e.g., before, simultaneously with, or after) a second therapy. For example, the second therapy may include an antiviral therapy (e.g., cidofovir, interleukin-2, cytarabine (ARA-C), or natalizumab), chimeric antigen receptor-T cell (CAR-T) therapy, T cell receptor (TCR)-T cell therapy, chemotherapy, radiation, an immunosuppressant (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, FK506 antibody, or glucocorticoid), an antagonist (e.g., a PD-1 antagonist, a PD-L1 antagonist), or a combination thereof. The therapeutic agent includes, but is not limited to, one or more of an anti-CD52 antibody (e.g., alemtuzumab), an anti-CD3 antibody, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, or irradiation.

[0211] In some embodiments, pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein are administered in combination with (e.g., before, concurrently with, or after) bone marrow transplantation or lymphocyte depletion therapy using chemotherapeutic agents (e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or Rituxan). In certain embodiments, the subject undergoes standard of care with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of a pharmaceutical composition comprising immune cells described herein. The pharmaceutical compositions described herein can be administered before or after surgery.

[0212] The dosage of any of the aforementioned therapies administered to a subject varies depending on the disease, disorder, or condition being treated and on the specific subject. Scaling of dosages for human administration can be performed according to art-recognized practices. For example, the dosage of alemtuzumab is generally about 1 mg to about 100 mg for an adult, usually administered daily for a period of about 1 day to about 30 days, e.g., a daily dose of about 1 mg to about 10 mg per day (e.g., as described in U.S. Patent No. 6,120,766, the entire contents of which are incorporated herein by reference).

[0213] Treatment method The present disclosure provides, inter alia, methods of treating a disease or disorder (e.g., a disease or disorder described herein) in a subject, the method comprising delivering a pharmaceutical composition comprising an immune cell (e.g., a macrophage, monocyte, or dendritic cell) described herein. In some embodiments, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having the disease or disorder. The pharmaceutical compositions described herein may be for use in the manufacture of a medicament for treating a disease or disorder in a subject or for stimulating an immune response in a subject.

[0214] The subject treated with the methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having or at risk of having a disease or disorder described herein). In some embodiments, the immune cells (e.g., macrophages, monocytes, or dendritic cells) can be autologous, allogeneic, or xenogeneic with respect to the subject. The pharmaceutical compositions described herein can be administered to a subject alone or in combination with one or more therapeutic agents, procedures, or treatment regimens according to the dosing regimens described herein.

[0215] Pharmaceutical compositions comprising the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be used to treat or prevent tumor or cancer-related diseases, neurodegenerative diseases or disorders, inflammatory diseases or disorders, cardiovascular diseases or disorders, fibrotic diseases or disorders, diseases associated with amyloidosis, and combinations thereof.

[0216] Methods are provided for treating (e.g., reducing, inhibiting, or delaying the progression of) cancer or tumor in a subject using pharmaceutical compositions comprising the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein. The subject may have an adult or childhood form of cancer. The cancer may be in early, intermediate, or late stage, or may be metastatic. The cancer may include, but is not limited to, a solid tumor, a blood cancer (e.g., leukemia, lymphoma, or myeloma, such as multiple myeloma), or a metastatic lesion. Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas and carcinomas, e.g., adenocarcinomas, including those affecting the lung, breast, ovary, lymphatic system, gastrointestinal (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma, e.g., cutaneous melanoma), pancreas, and bone (e.g., chordoma).

[0217] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., non-small cell lung cancer (NSCLC) of squamous and / or non-squamous histology, or NSCLC adenocarcinoma), or small cell lung cancer (SCLC)), skin cancer (e.g., Merkel cell carcinoma or melanoma (e.g., advanced melanoma)), ovarian cancer, mesothelioma, bladder cancer, soft tissue sarcoma (e.g., hemangiopericytoma (HPC)), bone cancer (osteosarcoma), kidney cancer (e.g., renal carcinoma (e.g., renal cell carcinoma)), or small cell lung cancer (SCLC)). cystic carcinoma), liver cancer (e.g., hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of the estrogen receptor, progesterone receptor, or Her2 / neu, e.g., triple-negative breast cancer), colorectal cancer (e.g., recurrent colorectal cancer or metastatic colorectal cancer, e.g., microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair proficiency, etc.), repair proficient, or mismatch repair deficient colorectal cancer), nasopharyngeal cancer, duodenal cancer, endometrial cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (e.g., anaplastic thyroid carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), neuroendocrine tumor (NET) (e.g., atypical pulmonary carcinoid tumor)), lymphoproliferative disorder (e.g., post-transplant lymphoproliferative disorder), lymphoma (e.g., T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma), myeloma (e.g., multiple myeloma), or leukemia (e.g., myeloid leukemia or lymphocytic leukemia).

[0218] In some embodiments, the cancer is a brain tumor, e.g., glioblastoma, gliosarcoma, or recurrent brain tumor. In some embodiments, the cancer is pancreatic cancer, e.g., advanced pancreatic cancer. In some embodiments, the cancer is skin cancer, e.g., melanoma (e.g., stage II-IV melanoma, HLA-A2-positive melanoma, unresectable melanoma, or metastatic melanoma), or Merkel cell carcinoma. In some embodiments, the cancer is kidney cancer, e.g., renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer is breast cancer, e.g., metastatic breast cancer or stage IV breast cancer, e.g., triple-negative breast cancer (TNBC). In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is anal canal cancer (e.g., squamous cell carcinoma of the anal canal). In some embodiments, the cancer is cervical cancer (e.g., squamous cell carcinoma of the cervix). In some embodiments, the cancer is gastric cancer (e.g., Epstein-Barr virus (EBV)-positive gastric cancer, or gastric or gastroesophageal junction cancer). In some embodiments, the cancer is head and neck cancer (e.g., HPV-positive and -negative squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the cancer is nasopharyngeal carcinoma (NPC). In some embodiments, the cancer is colorectal cancer, e.g., recurrent colorectal cancer, metastatic colorectal cancer, e.g., microsatellite-unstable colorectal cancer, microsatellite-stable colorectal cancer, mismatch repair-proficient colorectal cancer, or mismatch repair-deficient colorectal cancer.

[0219] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the cancer is a lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory HL or DLBCL). In some embodiments, the cancer is a myeloma, such as multiple myeloma.

[0220] Pharmaceutical compositions comprising the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein can be used to enhance or modulate an immune response in a subject. In one embodiment, the pharmaceutical compositions described herein enhance, stimulate, or increase an immune response in a subject (e.g., a subject having or at risk of a disease or disorder described herein). In certain embodiments, the subject is or is at risk of being immunocompromised. For example, the subject is undergoing or has undergone chemotherapy and / or radiation therapy.

[0221] In some embodiments, the subject has or is at risk of developing an inflammatory disorder (e.g., a chronic or acute inflammatory disorder). In some embodiments, the subject has or is at risk of developing an autoimmune disease or disorder. Exemplary autoimmune diseases that can be treated with the methods described herein include, but are not limited to, Alzheimer's disease, asthma (e.g., bronchial asthma), allergies (e.g., atopic allergies), acquired immune deficiency syndrome (AIDS), atherosclerosis, Behcet's disease, celiac disease, cardiomyopathy, Crohn's disease, cirrhosis, diabetes, diabetic retinopathy, eczema, fibromyalgia, fibromyositis, glomerulonephritis, graft-versus-host disease (GVHD), Guillain-Barré syndrome, hemolytic anemia, multiple sclerosis, myasthenia gravis, osteoarthritis, polychondritis, psoriasis, rheumatoid arthritis, sepsis, stroke, vasculitis, ventilator-induced lung injury, transplant rejection, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, ulcerative colitis, uveitis, vitiligo, or Wegener's granulomatosis.

[0222] Administration of the pharmaceutical compositions described herein can be by any convenient method (e.g., injection, ingestion, infusion, inhalation, implantation, or implantation). In some embodiments, the pharmaceutical compositions disclosed herein are administered by injection or infusion. The pharmaceutical compositions described herein can be administered to a patient intraarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramuscularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, the pharmaceutical compositions described herein are administered by intravenous infusion or injection. In some embodiments, the pharmaceutical compositions described herein are administered by intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be injected directly into the subject at the site of inflammation, the site of local disease, a lymph node, an organ, a tumor, or the site of infection.

[0223] The present disclosure is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way. [Example]

[0224] The following examples are provided to illustrate to one of ordinary skill in the art how to make and use the methods and compositions described herein and are not intended to limit the scope of the disclosure.

[0225] method Differentiation of monocytes into macrophages In an exemplary method of the present disclosure, leukoplaks derived from apheresis of normal donors were subjected to elutriation using the Elutra Cell Separation System (Terumo BCT) or cell separation using CliniMACS Prodigy (Miltenyi Biotec) or CliniMACS Plus (Miltenyi Biotec) to deplete red blood cells, platelets, lymphocytes, and granulocytes. Monocytes were enriched using elutriation or positively selected using MACS CD14+ Select (Miltenyi Biotec) according to the manufacturer's instructions. Purity and viability of pre- and post-selection (positive and negative fractions) were tested using flow cytometry. Selected CD14+ monocytes were differentiated into macrophages for up to 7 days. Differentiated macrophages were harvested on days 5–7 and either cryopreserved in freezing medium or used untreated for experiments. In some cases, cells were utilized after selection in the monocytic state.

[0226] Electroporation In an exemplary method of the present disclosure, untreated or thawed human macrophages were thawed and cultured overnight at 37°C one day before electroporation. Macrophages were collected and washed with PBS. Viable cells were counted using an NC200. Neon Transfection For macrophages transfected using the Neon Transfection System, 50 nM to 300 nM of mRNA per 1e+6 macrophages was mixed in electroporation buffer on ice. Macrophages were electroporated using the Neon Transfection System or the Maxcyte Electroporation System. After electroporation, cells were kept on ice for 10 minutes. Cells were collected, counted, and cultured in macrophage culture medium for further use. Cells were then transferred to plates and incubated at 37°C for 15 minutes for recovery. For further use, cells were plated in 6-well plates containing macrophage culture medium.

[0227] Fluorescence-activated cell sorting (FACS) Cell viability was assessed using the Live / Dead Fixable Aqua Dead Cell Staining Kit (Thermo) or similar reagents, and also using NC-200 (Chemometec).

[0228] Primary human macrophages were tested for CAR-HER2 expression using a two-step staining protocol: primary staining with human HER2 / ERBB2 protein-His tag (Sino Biological Inc., 10004-H08H-100), followed by secondary staining with Human TruStain FcX (Biolegend, 422302) and anti-His tag APC (R&D Systems, IC050A). TruStain FcX (Biolegend, 422302) was used for FACS staining of monocytes, macrophages, or monocytic cell lines expressing Fc receptors. Macrophage purity was tested using the following panel: anti-CD11b PE (Biolegend, 301306), anti-CD14 BV711 (Biolegend, 301838), anti-CD3 FITC (eBioscience, 11-0038-42), anti-CD19 PE-CY7 (eBioscience, 25-0198-42), anti-CD66b PerCP-CY5.5 (Biolegend, 305108), anti-CD56 BV605 (Biolegend, 318334), and the Live / Dead Fixable Aqua (L / D aqua) Dead Cell Staining Kit (Thermo Fisher Scientific, L34957). The same panel was used to test monocyte purity after CD14 MACS selection prior to seeding for differentiation. M1 / M2 markers for primary human macrophages were measured using the following panel: anti-CD11B PE (Biolegend, 301306), anti-CD80 BV605 (Biolegend, 305225), anti-CD86 BV711 (Biolegend, 305440), anti-CD206 BV421 (Biolegend, 321126), anti-CD163 APC-CY7 (Biolegend, 333622), anti-HLA-DR BV785 (Biolegend, 307642), anti-HLA ABC PE / CY7 (Biolegend, 311430), and Live / Dead Fixable Aqua. Detected using a Dead Cell Staining Kit.

[0229] Functional assays Co-culture of target cancer cells and CAR macrophages Target cells, such as HER2+ breast cancer cells (CRL2351) or HER2+ ovarian cancer cells (Sartorious #4475) stably expressing NucLight Green GFP, were used. Additional cell lines expressing the cognate antigen targeted by the CAR were evaluated, as described below. CAR macrophages were cocultured with target cells at the indicated ratios for the indicated time periods. To quantify the in vitro antitumor effect, the relative number of target cancer cells was measured using an Incucyte live imaging microscope (Sartorius) that monitors the green fluorescence intensity of culture wells approximately every hour. The antitumor activity of CAR macrophages was compared with control macrophages or target cells alone. GraphPad Data were graphed and statistical analysis was performed using Prism.

[0230] Treatment of CAR macrophages with interferon (IFN) After transfection with CAR mRNA, macrophages were cultured in macrophage culture medium containing 10–300 ng / mL recombinant human IFN-α, IFN-β, or IFN-γ (Peprotech) for 4–24 hours, as indicated. After the indicated time, the IFN-containing medium was washed out. IFN-sensitized CAR macrophages were used for further analysis.

[0231] Treatment of CAR macrophages with pro-inflammatory factors After transfection of CAR mRNA, 2e+6 macrophages were cultured in macrophage culture medium containing various concentrations of pro-inflammatory mediators. Exemplary pro-inflammatory mediators include: TLR1 / 2 agonists (e.g., Pam3CSK4), TLR3 agonists (e.g., Poly(I:C)), TLR4 agonists (e.g., LPS-EK standard (lipopolysaccharide from Escherichia coli K12)), TLR5 agonists (e.g., FLA-ST standard (flagellin from S. typhimurium)), TLR6 / 2 agonists (e.g., FSL-1), TLR7 agonists (e.g., imiquimod), TLR8 agonists (e.g., ssRNA40 / LyoVec), TLR9 agonists (e.g., CpG oligonucleotides), IFN-γ 20 ng / mL, TNF-α for 4-48 hours. 20 ng / mL β-glucan, recombinant human CD40 ligand, recombinant human 41BB ligand, recombinant human 41BB receptor, TNFα, IL6, IL12, STING agonist, cGAS agonist, and other pro-inflammatory mediators (cytokines, agonists, antibodies, small molecules, peptides). After the indicated times, the medium was washed away, and the sensitized CAR macrophages were used for further analysis.

[0232] Assessment of macrophage phenotype (M1 / M2) M1 / M2 markers of primary human macrophages were detected with the following panel: anti-CD11B PE (Biolegend, 301306), anti-CD80 BV605 (Biolegend, 305225), anti-CD86 BV711 (Biolegend, 305440), anti-CD206 BV421 (Biolegend, 321126), anti-CD163 APC-CY7 (Biolegend, 333622), anti-HLA-DR BV785 (Biolegend, 307642), anti-HLA ABC PE / CY7 (Biolegend, 311430), and Live / Dead Fixable Aqua Dead Cell Staining Kit. Cytokine production (IL12, IFN-γ, TNF-α, IL6, IL8, IL1b, MCP-1, IL10, IL4, IL13, and other human cytokines) was assessed in supernatants collected from macrophages (control or CAR) treated as described below using an MSD instrument (Meso Scale Discovery) according to the manufacturer's recommendations. In some cases, macrophages were cocultured with antigen-bearing target cells at the indicated effector-to-target ratios.

[0233] Detection of mCherry expression The transfection efficiency, persistence of expression, and intensity of expression of the fluorescent reporter gene mCherry in human monocytes or macrophages were assessed using real-time live microscopy on an Incucyte (Sartorius) for the indicated time periods.

[0234] Treatment of macrophages with sunitinib Macrophages were treated with 0-10 μM sunitinib (an RNAse-L inhibitor) for 2 hours before mRNA electroporation or transfection. The expression level and persistence of the encoding transgene were assessed using the methods described above.

[0235] Real-time PCR Real-time PCR of the encoding transgene (e.g., CAR) was performed using standard methods. Briefly, RNA was isolated using the Ambion RiboPure RNA Purification Kit (AM1924, Thermo Fisher Scientific) and reverse-transcribed using iScript RT Supermix for RT-qPCR (1708841, Bio-Rad). For quantitative PCR, template cDNA, primers, Taqman Gene Expression primers / probes, and Taqman Gene Expression Master Mix (4369016, Applied Biosystems) were used according to the manufacturer's instructions.

[0236] Determining the duration of CAR expression Flow cytometry In an exemplary method of the present disclosure, 50,000 CAR macrophages were plated in a 96-well plate. To measure anti-HER2 CAR expression, His-tagged recombinant HER2 protein was added to the cells in a buffer, such as PBS supplemented with BSA, and incubated. The cells were then spun down at 300 × g for 5 minutes, and the supernatant was removed. Fc receptors were then blocked using an Fc blocking solution, such as human TruStain FcX (BioLegend, catalog 422302), in PBS for 5 minutes. Following Fc blocking, staining for cell viability and other surface markers can be performed. For example, cell viability can be determined using the LIVE / DEAD™ Fixable Aqua Dead Cell Staining Kit (Invitrogen, catalog L34957). Additionally, an anti-His antibody, such as a His-tagged APC-conjugated antibody (R&D Systems, catalog IC050A), was added. CAR expression was determined by flow cytometry, first by gating on single-cell populations, then by selecting live cells, and finally by measuring the APC fluorescence of the cells. CAR-expressing cells were brighter in the APC channel than control cells not exposed to anti-His antibodies. The brightness of CAR-positive cells determined the degree of expression, and repeated measurements over time allowed us to track CAR expression over time.

[0237] Immunofluorescence (IF) microscopy In an exemplary method of the present disclosure, cells were cultured on glass slides as appropriate. The medium was then removed, and the cells were washed three times with PBS. Next, the cells were fixed using 4% paraformaldehyde or methanol. The incubation time in the fixative depended on the identity of the fixative. After the appropriate time, the fixative was removed, and the cells were again washed three times with PBS. If intracellular staining was required, the cells were incubated with 1% Triton X-100 in PBS (Thermo Fisher Scientific, catalog BP151-100) and washed three times with PBS. A blocking solution, such as BSA in PBS, was then added to the cells for 60 minutes to block. The blocking solution was then removed, and the cells were washed. A fluorescent dye-conjugated antibody was diluted according to the manufacturer's instructions and allowed to bind to the cells overnight. The antibody solution was then removed, and the cells were washed. Finally, a mounting solution, such as ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen, catalog P36966), was applied to the cells, and a coverslip was placed on top. This was allowed to dry for 24 hours before imaging, which was performed via fluorescence microscopy: cells expressing CAR were brighter than cells not expressing CAR in the appropriate channel for the fluorophore.

[0238] RT-PCR In an exemplary method of the present disclosure, macrophages were lysed and RNA was collected using a one-step RT-PCR kit (SuperScript™ III Platinum™ One-Step qRT-PCR Kit, Invitrogen catalog 11732-020) according to the manufacturer's instructions. Primers specific for CAR were used in the assay. Macrophages containing CAR mRNA showed a signal in the RT-PCR assay, whereas untransduced macrophages showed no signal.

[0239] Determining CAR effector cell function In an exemplary method of the present disclosure, a CAR transgene was introduced into monocytes or macrophages via electroporation / transfection with nucleic acids (e.g., DNA, mRNA, or chemically modified mRNA) or via viral transduction with lentivirus, adenovirus, or alternative viral vectors. CAR expression was confirmed using antigen-specific staining by flow cytometry, real-time PCR, or fluorescence microscopy. These techniques can also be used to determine the intensity and kinetics of CAR expression. CAR constructs expressed on the surface of macrophages were tested for activity in tumor phagocytosis and / or tumor killing assays against target-positive cell lines. Constructs that triggered phagocytosis and / or killing of target cells were tested for cytokine secretion, chemokine secretion, immune cell recruitment capacity, phenotypic change (i.e., autopolarization to M1 / M2), and T cell stimulation / antigen presentation function.

[0240] Phagocytosis assay Flow phagocytosis assay (cells) In an exemplary method of the present disclosure, target-positive and target-negative tumor cells were labeled with the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, catalog C34554) according to the manufacturer's instructions, or the cells were engineered to express a fluorescent protein (e.g., GFP). CAR-expressing and control macrophages were then plated into a U-bottom 96-well plate at a macrophage:tumor cell ratio of 1:1 and cultured for 4 hours. At the end of the incubation, cells were removed from the wells and stained for flow cytometry. The panel included a viability dye and a macrophage-specific marker, e.g., CD11b. When live cells were gated, CD11b / CFSE double-positive cells were identified as macrophages that had presumably phagocytosed the cells. When CAR macrophages were cultured with target-positive tumor cells, the percentage of double-positive cells should be increased compared to macrophages without CAR. The specificity of target-based phagocytosis was tested using control macrophages cultured with cells negative for the target.

[0241] Flow phagocytosis assay (beads) In an exemplary method of the present disclosure, polystyrene beads were functionalized with a CAR target or an unrelated protein. These beads were then labeled with pHrodo™ Red, SE (Invitrogen, catalog P36600), a pH-responsive dye. Upon acidification, the dye's fluorescence level increased. The beads were then cultured with CAR macrophages and untransduced macrophages. After a period of time, the macrophages were removed and stained for flow cytometry using a viability dye and a macrophage-specific marker, e.g., CD11b. After gating on live cells, cells that were CD11b / pHrodo double-positive were considered macrophages that had presumably phagocytosed the beads. When CAR macrophages were cultured with target-positive beads, the percentage of double-positive cells should increase compared to macrophages without CAR. The specificity of target-based phagocytosis was tested using control macrophages cultured with target-negative beads.

[0242] Incucyte(cell) In an exemplary method of the present disclosure, target-positive tumor cells expressing a fluorescent protein, such as GFP, were cultured with CAR macrophages and non-transduced macrophages in a 96-well plate. The ratio between effector macrophages and target tumor cells was varied from 10:1E:T to 1:10E:T, in addition to a target cell-only control of 0:1E:T. The number of macrophages was kept constant at 10e3 macrophages per well. The change in fluorescence over time was measured every 4 hours to determine the amount of tumor cell killing that occurred during the culture. Furthermore, image analysis techniques were used to determine the location of macrophages in the culture and the number of macrophages that phagocytosed tumor cells.

[0243] Incucyte (beads) In an exemplary method of the present disclosure, pHrodo-functionalized beads carrying the protein target of CAR were added to both CAR macrophages and non-transduced macrophages in wells of a 96-well plate. Macrophages were plated at a concentration of 20e3 per well, and beads were added at a 5:1 bead-to-macrophage ratio. pHrodo fluorescence was measured every 30 minutes for 5 hours. The ratio of the increase in fluorescence between the initial time point and the 1-hour time point was used to determine the amount of phagocytosis that had occurred. CAR macrophages were expected to show a greater change in fluorescence than non-transduced controls.

[0244] Mouse model Xenograft mouse model In an exemplary method of the present disclosure, NSGS (NSG-SGM3) mice were challenged intraperitoneally with SKOV3, a HER2+ human ovarian cancer, to model peritoneal carcinomatosis. Mice were treated with human CAR macrophages generated by mRNA electroporation, with or without IFN-β sensitization (or a negative control). Tumor burden was monitored via bioluminescence imaging.

[0245] Syngeneic Mouse Models BALB-C immunocompetent mice were implanted subcutaneously with CT26-HER2, a model of HER2+ colon cancer. Once tumors were implanted, mice were treated with CAR macrophages generated by mRNA electroporation, with or without IFN-β sensitization (or a negative control). Tumor burden was monitored by caliper measurement of tumor mass.

[0246] Example 1: Effect of mRNA modification on expression and viability in primary human macrophages Donor macrophages (HC153444 and / or HC156308) were differentiated from CD14+ monocytes and electroporated or transfected with mCherry mRNA. mCherry mRNA contained variant cap structures, including AGCap1, m6AGCap1, or anti-reverse cap analog (ARCA), and modified nucleotides, including pseudouridine (PsU), 5-methoxyuridine (5mU), or 5-methylcytidine / pseudouridine (5meC PsU). mRNA expression and cell viability were detected by FACS on days 1 or 15 after transfection.

[0247] Although mCherry mRNA variants did not affect macrophage viability when electroporated, the viability of mRNA-transfected macrophages ranged from 36% to 70% depending on the variant (Figure 1A). The percentage of mCherry-expressing cells was nearly 100% in electroporated cells and 70-90% in transfected cells (data not shown). The mean mCherry fluorescence intensity (representing the number of mCherry proteins expressed per cell) depended on the mRNA modification and transfection method (Figure 1B). AGCap1 produced greater mCherry intensity in electroporated cells, whereas PsU modification and HPLC purification produced greater mCherry intensity in transfected cells.

[0248] As shown in Figure 1C, the persistence of mCherry RNA depended on the mRNA modification and mRNA delivery method used. mCherry mRNA persisted longer in transfected macrophages compared to electroporated macrophages. AGCap1-containing mRNA persisted better than m6AGCap1 mRNA in electroporated cells. HPLC purification had a greater effect on transfected cells than on electroporated cells.

[0249] Example 2: Effect of CAR mRNA modification on macrophage viability Donor macrophages (HC153444) were differentiated from CD14+ monocytes and transfected with mRNA encoding a CAR (CAR mRNA) using electroporation or transfection. The mRNA encoding a CAR containing the HER2 extracellular domain and the CD3 zeta intracellular domain was used. CAR expression and cell viability were detected by FACS on day 1.

[0250] As shown in Figure 2A, the exemplary viability of macrophages electroporated with mRNA was >80% for all mRNA modifications evaluated. Macrophages transfected with Viromer mRNA showed >90% viability for most CAR-expressing macrophages and naive macrophages, whereas macrophages transfected with mRNA containing m6AGCap1 and PsU modifications and mRNA containing 5moU modifications had a 65% viability. Furthermore, as shown in Figure 2B, in electroporated macrophages, CAR mRNA containing m6AGCap1 and PsU modifications induced the highest CAR expression. In macrophages transfected with mRNA containing m6AGCap1, N1mPsU, and / or PsU modifications induced the highest CAR expression. As shown in Figure 2C, optimization of the mRNA 5'Cap to m6AGCap1 and modification of uracil to pseudouracil (PsU) increased CAR expression in human macrophages by approximately threefold.

[0251] Example 3: Effect of CAR mRNA modification on macrophage function Human macrophages were differentiated from CD14+ monocytes and electroporated with mRNA encoding a HER2 CAR (CAR mRNA). The CAR was injected into macrophages at a 5:1 effector (CAR macrophage) to target (cancer cell) ratio. Two days after mRNA transfection, fluorescently labeled HER2+ breast cancer cells (CRL2351) were co-cultured with CAR macrophages.

[0252] As shown in Figure 3A, macrophages transfected with HER2 CAR mRNA containing the m6AGCap1PsU modification exhibited potent killing activity comparable to that of macrophages transduced with Ad5f35. Macrophages transfected with HER2 CAR mRNA containing the m6AGCap1PsU modification exhibited the highest target cell killing activity compared to macrophages transfected with HER2 CAR mRNA containing the 5moU modification (Figures 3B-3D). These results also indicate that macrophages transfected with optimized mRNA modifications exhibit higher antitumor activity, even at a lower effector (CAR-M) to target (cancer cell) ratio.

[0253] Example 4: M1 polarization enhances mRNA persistence and macrophage / CAR-macrophage function HER2 CAR mRNA containing m6AGCap1 and PsU modifications was electroporated into human macrophages. Cells were cultured for up to 48 hours with cytokines that induce an M1 phenotype, such as IFN-alpha, IFN-beta, IFN-gamma, IFN-gamma plus lipopolysaccharide (LPS), TNF-alpha, IL-6, or STING ligand (STING-L). Afterwards, cytokines were washed away and fresh medium was added. CAR expression and M1 marker expression were measured on days 2 and 7 post-transfection. Two days after macrophage transfection, fluorescently labeled HER2+ breast cancer cells (CRL2351) were co-cultured with HER2 CAR macrophages. Cancer cell growth was monitored via fluorescence every 4 hours using an Incucyte live imaging microscope. The effector (CAR macrophage) to target (cancer cell) ratio was 5:1.

[0254] As shown in Figure 4A, the tested interferon cytokines did not result in a decrease in the viability of CAR-transfected macrophages on day 2. Surprisingly, macrophages treated with IFN-β showed higher CAR expression than macrophages treated with control medium, IFN-α, or IFN-γ (Figure 4B). As shown in Figure 4C, treating macrophages with interferon cytokines did not result in a decrease in the viability of CAR-transfected macrophages on day 7. Surprisingly, macrophages treated with IFN-β showed higher CAR expression than macrophages treated with control medium, IFN-α, or IFN-γ, indicating that IFN-β improves the expression duration of mRNA-encoding transgenes such as CAR in human macrophages (Figure 4D). Furthermore, treatment of mRNA-transfected CAR macrophages with IFN-α, IFN-β, or IFN-γ induced an M1 phenotype (based on CD86 expression, Figure 4E) and reduced M2 markers (based on CD163 expression, Figure 4F). IFN-β induced the strongest M1 phenotype of the interferons evaluated, which persisted for at least 7 days after treatment (Figure 4E).

[0255] Example 5: Effect of IFN treatment on CAR expression, CAR macrophage function, M1 phenotype markers and cytokine production To determine whether interferon treatment differentially affected macrophages transfected with mRNA containing different modifications, five different mRNA modifications were also tested. HER2 CAR mRNA containing different mRNA modifications was electroporated into human macrophages. CAR expression and M1 markers were detected by flow cytometry (Attune) four days after electroporation. Next, day 4 CAR macrophage cells were co-cultured with Nuc-Light-labeled HER2+ breast cancer cell line (CRL2351) at a 5:1 effector (CAR macrophage) to target (cancer cell) ratio. Cancer cell growth was monitored via fluorescence every four hours using an Incucyte live imaging microscope.

[0256] As shown in Figure 5A, macrophages transfected with all evaluated chemicals, with or without IFN-β treatment, exhibited high viability. Macrophages transfected with AGCap1 or m6AGCap1 and PsU- or N1mPsU-modified CAR mRNA exhibited the highest levels of expression, with IFN-β slightly improving the percentage of cells expressing CAR for all modifications at day 4 (Figure 5B). However, IFN-β treatment significantly increased the number of CARs expressed per cell based on CAR MFI for all evaluated mRNA modifications, with m6AGCap1 / PsU-comodified mRNA exhibiting the highest expression (Figure 5C).

[0257] To further evaluate the effect of IFN-β on CAR persistence, human macrophages electroporated with m6AGCap1 / PsU mRNA encoding the HER2 CAR were evaluated on days 2 and 7. IFN-β treatment significantly improved CAR expression rates on day 7 compared to CAR macrophages not treated with IFN-β (Figure 6).

[0258] To examine the effect of IFN-β treatment on improving CAR expression and to optimize the IFN-β concentration, macrophages transfected with M6AGCap1 / PsU-modified mRNA were treated with 0, 3, 10, 30, or 100 ng / mL IFN-β for 4 hours, and viability, CAR percentage, and CAR expression were measured on days 4 and 7 after electroporation. MFI was assessed. An IFN-β dose-dependent effect on CAR expression by human macrophages was observed ( FIG. 7A ). As described in Example 4, treatment of CAR macrophages with IFN-β induced an M1 phenotype, so further experiments were performed to determine whether the effect was dose-dependent. As shown in FIG. 7B , induction of M1 markers CD80, CD86, and HLA-DR was IFN-β dose-dependent.

[0259] Considering that macrophage phenotypes are thought to be plastic and that immunosuppressive cytokines such as IL-10 are known to induce an M2 phenotype, we evaluated the effects of IL-10 treatment on IFN-β-treated or untreated HER2 CAR mRNA-transfected macrophages. IFN-β-treated CAR macrophages resisted the effects of IL-10 and did not express the M2 marker CD163. Instead, they maintained expression of the M1 marker CD86 for 48 hours (Figure 8A) and 7 days after treatment with IL-10 (Figure 8B). IFN-β-sensitized CAR macrophages also resisted other M2 inducers.

[0260] To evaluate the antitumor function of macrophages transfected with mRNA encoding a HER2 CAR, with or without IFN-β sensitization, untransduced (UTD) or CAR macrophages were sensitized with 0, 3, 10, 30, or 100 ng / mL IFN-β for 4 or 20 hours. These effector cells were then cocultured with the HER2+ breast cancer cell line CRL2351-GFP at an effector-to-target ratio of 3:1 or 1.5:1, and antitumor activity was measured based on GFP expression using an Incucyte live imaging microscope. IFN-β sensitization improved the ability of CAR macrophages to kill cancer cells (Figure 9A). To evaluate whether IFN-β treatment improved the antitumor activity of CAR macrophages with mRNA containing the unique modification, we evaluated the antitumor activity of macrophages electroporated with mRNA containing the unique modification, with or without IFN-β treatment. IFN-β treatment improved the antitumor activity of all CAR macrophages except those transfected with 5moU mRNA (Figure 9B). To assess whether the improved antitumor activity of mRNA-transfected CAR macrophages was universal to all interferons or only interferon beta, macrophages electroporated with M6AGCap1 / PsU mRNA were treated with IFN-alpha, beta, or gamma and their cancer cell-killing ability was assessed. IFN-β-treated CAR macrophages had a greater effect than IFN-α or IFN-γ, demonstrating the highest cancer cell killing (Figure 9C).

[0261] To assess whether interferon treatment of macrophages improves other antitumor functions, we evaluated cytokine secretion in interferon-treated or untreated mRNA-transfected HER2 CAR macrophages after co-culture with HER2+ breast cancer cells. 150 nM HER2 CAR mRNA containing m6AGCap1 and PsU modifications was electroporated into human macrophages. HER2+ breast cancer cells (CRL2351) were transfected with CAR mRNA and then co-cultured with CAR macrophages at a 3:1 effector (CAR macrophages) to target (cancer cells) ratio. Supernatants were collected 48 hours after co-culture of cancer cells and macrophages, and Meso was analyzed. Cytokine levels were measured using a Scale Discovery (MSD) instrument. As shown in Figure 10, treatment of macrophages with IFN-α, IFN-β, or IFN-γ increased the secretion of cytokines IL-6, IL-8, and TNFα from macrophages.

[0262] Additional studies were conducted to determine whether treatment with interferon could further improve CAR mRNA persistence in macrophages and extend the functionality of CAR-macrophages. 300 nM CAR mRNA containing m6AGCap1 and PsU modifications was electroporated into human macrophages. Cells were cultured with 20 ng / mL IFN for 24 hours, after which the cells were washed to remove the cytokine. CAR expression was detected by flow cytometry (Attune) 2 days after transfection. Next, the cells were cocultured with a Nuc-Light-labeled HER2+ breast cancer cell line (CRL2351) at an effector-to-target ratio of 3:1. Cancer cell proliferation was monitored via fluorescence using an Incucyte live imaging microscope. CAR expression and M1 markers in macrophages were detected by flow cytometry (Attune) 7 days after transfection.

[0263] As shown in Figure 11A, 2 days after transfection with CAR mRNA, the viability and CAR expression of CAR macrophages were very high, except for macrophages treated with IFN-γ. Furthermore, as shown in Figures 11B and 11C, IFN treatment enhanced the target cell killing activity of CAR macrophages. Figure 11C shows target cell killing after 72 hours of coculture of cancer cells and macrophages. IFN treatment also affected macrophage viability, CAR expression, M1 marker expression, and CAR macrophage function. As shown in Figure 12A, treatment of transfected macrophages with IFN-β increased cell viability, HER2 CAR expression, and the expression of M1 markers CD80, CD86, and HLA-DR at 7 days compared with macrophages not treated with interferon. Figure 12A shows that although all macrophages showed high viability at 7 days, IFN-β-treated macrophages had significantly higher CAR expression levels. Figure 12B shows that of all CAR macrophages tested in the cancer cell killing assay 7 days after electroporation, IFN-β-treated macrophages exhibited the highest level of cancer killing (greatest reduction in tumor growth). Seven days after electroporation, macrophages were co-cultured with target cancer cells for 72 hours. As shown in Figure 12C, all interferons improved the cancer cell killing activity of CAR macrophages compared to CAR macrophages not treated with interferon.

[0264] Example 6: Transfected macrophages are sensitive to IFNγ Human macrophages were transfected with mCherry mRNA containing m6AGCap1 and PsU or N1mPsU modifications and then cultured with different doses of IFN-γ for 1 day. mCherry expression was monitored using an Incucyte live imaging microscope. As shown in Figure 13A, when macrophages were transfected with the mRNA, IFN-γ reduced mCherry mRNA expression.

[0265] In vitro transcribed mRNA has previously been shown to be recognized by various endosomal innate immune receptors (e.g., Toll-like receptor (TLR) 3, TLR7, and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2'-5'-oligoadenylate synthase (OAS)). Signaling through these distinct pathways leads to inflammation associated with activation of type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-12, and a cascade of transcriptional programs. Collectively, these create a proinflammatory microenvironment poised to induce specific immune responses. Furthermore, downstream effects such as reduced translation via eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, enhanced RNA degradation by ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication are all relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0266] Activation of IFN-γ through the TLR pathway can activate 2'-5'-oligoadenylate synthase (OAS) to generate 2'-5'-oligoadenylate (2-5A), which in turn can activate RNase L, leading to RNA degradation and apoptosis.

[0267] Example 7: Effects of RNaseL inhibitors, sunitinib and ABCE1 on CAR macrophages To determine whether RNase L inhibitors could rescue IFN-γ-induced instability of transfected mRNA, human macrophages were treated with 1 μM sunitinib (RNase L inhibitor) 2 hours before transfection of mCherry mRNA containing m6AGCap1 and PsU modifications. The transfected cells were then cultured with different doses of IFN-γ for 1 day. mCherry expression was monitored using an Incucyte live imaging microscope. As shown in Figure 14A, sunitinib rescued IFN-γ-induced degradation of the mRNA.

[0268] To evaluate whether RNAse L inhibition could improve the antitumor function of mRNA-transfected CAR macrophages, macrophages were transfected with mRNA containing the modifications, pretreated with sunitinib, and then evaluated as effector cells in a cancer cell killing assay. CAR macrophages pretreated with 1 nM sunitinib exhibited greater cancer cell killing than CAR macrophages not pretreated with sunitinib or untransfected control macrophages treated with or without sunitinib (Figure 14B). The improved cancer-killing ability of sunitinib-sensitized CAR macrophages in a 48-hour CRL2351 breast cancer cell killing assay is shown in Figure 14C.

[0269] The effect of another RNase L inhibitor (RLI or ABCE1) was also tested to further validate the concept. Human macrophages were co-transfected with mRNA encoding mCherry containing m6AGCap1 and PsU modifications and mRNA encoding ABCE1.

[0270] As shown in Figure 15, 48 hours after electroporation, ABCE1 co-expression significantly improved the expression of the mRNA-encoding transgene of interest. The viability of ABCE1-cotransfected macrophages was unaffected and remained high. ABCE1 co-transfection increased mCherry expression approximately two-fold, and pretreatment with sunitinib further enhanced this effect.

[0271] Example 8: Effects of genetically encoded RNaseL inhibitors on CAR macrophages To evaluate whether other genetically encoded RNaseL inhibitors could improve CAR expression, co-transfection of CAR mRNA containing the modification with mRNA encoding NS1 was evaluated. NS1 is an influenza A-derived gene that encodes the NS1A protein. As shown in Figure 16, co-transfection of CAR mRNA with ABCE1 or NS1 with CAR mRNA increased the level of CAR expression compared to co-transfection of CAR with the control gene mCherry. NS1 co-transfection resulted in higher CAR expression than ABCE1 co-transfection.

[0272] To assess the mechanism by which ABCE1 and / or NS1 affect CAR expression, the decay of electroporated mRNA encoding HER2 CAR containing modifications was assessed by RT-qPCR. The relative abundance of CAR mRNA in macrophages transfected with CAR plus mCherry mRNA, CAR plus ABCE1 mRNA, or CAR plus NS1 mRNA at 8 hours was compared to the abundance of CAR mRNA at 2 hours. As shown in Figure 17, the level of CAR mRNA cotransfected with mCherry (negative control) decreased by more than 50% at 6 hours, whereas the level of CAR mRNA cotransfected with ABCE1 mRNA or NS1 mRNA continued to increase.

[0273] Example 9: Sensitization of macrophages To generate Her2-zeta CAR-expressing macrophages, 90 x 10 primary human macrophages were cultured at 4°C for 12 h. 6The cells were suspended in EP buffer (MaxCyte) containing 300 nM mRNA (TriLink) at a concentration of 100 cells / mL. 100 μL of the cell mixture was added to an electroporation cassette (OC100x2, MaxCyte) and electroporated using experimental T cell 1 settings. The cells were removed from the cassette and plated in 3 mL of TexMACS medium (Miltenyi Biotech) containing 20% ​​FBS (Gibco) on an UpCell plate (Thermo Scientific) and incubated overnight at 37°C, 5% CO2.

[0274] Recombinant CD40 ligand (Peprotech), 4-1BB ligand (Enzo Life Sciences), and 4-1BB receptor (Peprotech) were resuspended in molecular-grade water to a stock concentration of 100 μg / mL. The stock solutions were then used to create working solutions ranging from 2 to 0.002 μg / mL in PBS. 100 μL of the working solution was added to wells of a 96-well plate and left at room temperature for 4 hours.

[0275] The plates were removed from the incubator and left at room temperature for 30 minutes. Cells were detached from the plates, counted using an NC-200 automated cell counter (Chemomtech), and resuspended in TexMACS medium containing 10% FBS. After washing the protein-coated plates twice with PBS, macrophages were added to a final volume of 100 μL of TexMACS medium containing 10% FBS. The plates were incubated at 37°C and 5% CO2 for 3 hours. After 3 hours, 10,000 CRL-2351 cells expressing nuclear GFP were added to each well. The final concentration of GM-CSF was 10 ng / mL in all wells. Cell lysis was detected using an Incucyte (Essen Bioscience). Tumor cell death was calculated by the integrated GFP intensity per well relative to time 0.

[0276] For cell surface protein detection, macrophages were plated in agonist molecule-coated wells in a final volume of 200 μL of TexMACS medium + 10% FBS + 10 ng / mL GM-CSF and incubated at 37°C and 5% CO for 3 days. Cells were incubated with 300 μL Accutase (Sigma) for 30 minutes and transferred to a 96-well round-bottom plate for staining. Cells were incubated in FACS buffer containing 20 μg / mL Her2-His for 20 minutes at room temperature, followed by incubation with Human TruStain FcX for 10 minutes at room temperature. Surface protein staining was performed using the following panel: CD80-FITC, CD86-PE, CD163-APC-Cy7, CD206-BV421, anti-His-APC, and Aqua Live / Dead aqueous solution. Detection of surface protein expression was completed using an Attune NxT flow cytometer (Thermo Fisher).

[0277] Treatment of CAR macrophages with CD40L significantly improved the tumor-killing ability of macrophages and CAR macrophages (Figure 18A). Priming with CD40L also induced an M1 phenotype in macrophages transfected with CAR mRNA (Figure 18B). Treatment with 4-1BB and 4-1BBL produced similar results, although less potently than CD40L (Figures 19A-19B and Figures 20A-B). These results indicate that pretreatment or priming of CAR macrophages with a CD40 agonist, such as CD40L, can improve efficacy, and that combination therapy involving CAR macrophages with a CD40 agonist can improve efficacy.

[0278] Example 10: Effect of mRNA modification on human monocytes Delivery of CAR mRNA containing modifications to human monocytes was also evaluated. Specifically, mRNA encoding a HER2 CAR containing M6AGCap1 and PsU modifications was electroporated into monocytes from five human donors. As shown in Figure 21, high CAR expression was achieved, both in terms of intensity and percentage.

[0279] Example 11: Efficacy of CAR macrophages generated by mRNA electroporation in a xenograft solid tumor mouse model To evaluate the efficacy of CAR-macrophages generated by mRNA electroporation in xenograft solid tumor mouse models, five groups were used: no treatment, mock treatment, mock treatment with IFN-β, mRNA CAR-macrophage (mRNA-CAR) treatment, and mRNA CAR-macrophage with IFN-β (mRNA-CAR+IFNb) treatment.

[0280] NSGS mice were injected with 6e5 SKOV3 cells (n=5 mice per group). Mice were subsequently treated with 8e6 macrophages by intraperitoneal injection on days 0, 4, and 8, as indicated by the arrows in Figure 22. Tumor burden was measured by bioluminescence imaging using an IVIS imaging system (Perkin Elmer). Mice were imaged every 2-3 days.

[0281] As shown in Figure 22, mice treated with mRNA CAR-macrophages or mRNA CAR-macrophages stimulated with IFN-β showed suppression of tumor growth compared to controls.

[0282] Example 12: Efficacy of CAR macrophages generated by mRNA electroporation in a syngeneic solid tumor mouse model To evaluate the efficacy of CAR-macrophages generated by mRNA electroporation in a syngeneic solid tumor mouse model, five groups were used: a non-treated group, a mock-treated group, a mock-treated group with IFN-β, an mRNA CAR-macrophage (mRNA CAR)-treated group, and an mRNA CAR-macrophage with IFN-β (mRNA CAR+IFNb)-treated group.

[0283] BALB / c mice were subcutaneously injected with 7.5 e 5 CT26-HER2 colon cancer cells (n = 12-13 mice per group). 12 days after tumor injection (mean tumor mass was 50 mm 3 On days 16 and 20, approximately 3e6 macrophages were injected intratumorally (approximately 3e6 macrophages per mouse per injection, indicated by arrows in Figure 23). Tumor volume was measured biweekly using calipers and estimated using the following formula: Tumor volume = length x width^2 / 2, where length represents the maximum diameter of the tumor and width represents the perpendicular tumor diameter.

[0284] As shown in Figure 23, nearly 80% of mice receiving IFN-β-treated mRNA CAR-macrophages rejected their tumors, while approximately 60% of mice receiving mock, IFN-β-treated mock, or mRNA CAR-macrophages rejected tumors. IFN-β-sensitized mRNA CAR-macrophages significantly suppressed tumor growth compared to negative controls. Particularly in the context of immunocompetent mouse models, IFN-β-sensitized mRNA CAR macrophages outperformed mRNA CAR macrophages or IFN-β-sensitized control macrophages, indicating a synergistic effect between CAR engineering and IFN-β sensitization.

[0285] equivalent It should be understood that various changes, modifications, and improvements to this disclosure will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and any invention described in this disclosure is more particularly described by the following claims.

[0286] Those of ordinary skill in the art will understand the typical basis for deviation or error attributable to values ​​obtained in the assays or other processes described herein. Publications, websites, and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are hereby incorporated by reference in their entireties. The present invention provides, for example, the following items. (Item 1) 1. A method for modifying immune cells, comprising the steps of: (a) modifying a messenger RNA (mRNA) encoding a chimeric antigen receptor (CAR); (b) purifying the mRNA; (c) delivering the mRNA to the immune cells; the immune cells include macrophages, monocytes, or dendritic cells; The method, wherein the modified immune cells comprise a CAR. (Item 2) 2. The method of claim 1, wherein the modifying step comprises including in the mRNA modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, and / or a poly(A) tail. (Item 3) 3. The method of claim 2, wherein the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). (Item 4) 4. The method of any one of items 1 to 3, wherein the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof. (Item 5) 5. The method according to any one of items 1 to 4, wherein the purifying step comprises silica membrane purification and / or high performance liquid chromatography (HPLC). (Item 6) 6. The method of any one of items 1 to 5, wherein the delivering step comprises transfection. (Item 7) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including AGCap1 and 5moU in the mRNA, the purifying step comprises silica membrane purification, and the delivering step comprises electroporation. (Item 8) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including AGCap1 and PsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises electroporation. (Item 9) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including AGCap1 and N1mPsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises electroporation. (Item 10) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including m6-AGCap1 and N1mPsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises electroporation. (Item 11) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including m6-AGCap1 and PsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises electroporation. (Item 12) The modifying step includes modifying the mRNA to include AGCap1 and PsU, the purifying step includes HPLC, and the delivering step includes transfection. 7. The method according to any one of items 1 to 6, comprising transfection. (Item 13) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including m6-AGCap1 and PsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises transfection. (Item 14) 7. The method of any one of items 1 to 6, wherein the modifying step comprises including m6-AGCap1 and N1mPsU in the mRNA, the purifying step comprises HPLC, and the delivering step comprises transfection. (Item 15) 7. The method according to any one of items 1 to 6, wherein the modifying step comprises including AGCap1 and 5moU in the mRNA. (Item 16) 7. The method according to any one of items 1 to 6, wherein the modifying step comprises including m6AGCap1 and 5moU in the mRNA. (Item 17) 17. The method according to any one of items 1 to 16, further comprising treating the immune cells with an RNaseL inhibitor. (Item 18) 18. The method of claim 17, wherein the RNaseL inhibitor comprises sunitinib. (Item 19) 18. The method of item 17, wherein the RNaseL inhibitor comprises ABCE1. (Item 20) Item 18. The method of item 17, wherein the processing step occurs before the delivering step. (Item 21) 21. The method according to any one of items 1 to 20, further comprising culturing the immune cells with a cytokine or an immunostimulatory recombinant protein. (Item 22) 22. The method of claim 21, wherein the cytokine comprises IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB receptor, TLR agonist, beta-glucan, IL-4, IL-13, IL-10, TGF-β, glucocorticoid, immune complex, or a combination thereof. (Item 23) 23. The method of claim 21 or 22, wherein the cytokine comprises IFN-β. (Item 24) 24. The method according to any one of items 21 to 23, wherein the culturing step is carried out after the delivering step. (Item 25) 25. The method of any one of items 1 to 24, wherein the modified immune cells express the CAR. (Item 26) 26. The method of paragraph 25, wherein CAR expression is increased compared to CAR expression in modified immune cells of the same type to which unmodified mRNA encoding the CAR was delivered. (Item 27) 26. The method of paragraph 25, wherein the modified immune cells exhibit increased effector activity compared to the effector activity in modified immune cells of the same type into which unmodified mRNA encoding the CAR has been delivered. (Item 28) A modified immune cell produced by the method according to any one of items 1 to 27. (Item 29) 29. The modified immune cell of paragraph 28, which exhibits increased survival compared to the same type of modified immune cell comprising an unmodified mRNA encoding the CAR. (Item 30) 30. The modified immune cell of paragraph 28 or paragraph 29, wherein the cell exhibits increased expression of the mRNA encoding the CAR compared to a modified immune cell of the same type comprising an unmodified mRNA encoding the CAR. (Item 31) 31. The modified immune cell of any one of items 28 to 30, which exhibits increased CAR expression compared to a modified immune cell of the same type comprising an unmodified mRNA encoding said CAR. (Item 32) 32. The modified immune cell of any one of items 28 to 31, wherein the modified immune cell exhibits an extended lifespan of the mRNA encoding the CAR compared to a modified immune cell of the same type comprising an unmodified mRNA encoding the CAR. (Item 33) 33. The modified immune cell of any one of items 28 to 32, wherein the modified immune cell exhibits extended lifespan of the CAR compared to a modified immune cell of the same type comprising an unmodified mRNA encoding the CAR. (Item 34) 34. The modified immune cell of any one of items 28 to 33, which exhibits increased effector activity compared to a modified immune cell of the same type comprising an unmodified mRNA encoding the CAR. (Item 35) 35. The modified immune cell of any one of items 28 to 34, which exhibits increased M1 polarization compared to a modified immune cell of the same type comprising an unmodified mRNA encoding said CAR. (Item 36) one or more modified mRNAs comprising modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, a polyA tail, or a combination thereof; and A composition comprising one or more RNaseL inhibitors. (Item 37) 37. The composition of claim 36, wherein the cap structure comprises AGCap1 or m6AGCap1. (Item 38) 38. The method of claim 36 or 37, wherein the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), or N1-methyl-pseudouridine (N1mPsU). (Item 39) 39. The method of any one of items 36 to 38, wherein the one or more RNaseL inhibitors comprise sunitinib. (Item 40) 39. The method of any one of items 36 to 38, wherein the one or more RNaseL inhibitors comprise ABCE1.

Claims

[Claim 1] The invention described in the present specification.

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