In vivo delivery to immune cells

Abstract

The present disclosure provides a method of generating modified immune cells comprising a chimeric antigen receptor (CAR), the method comprising administering to a subject a composition comprising (a) one or more nucleic acid molecules, at least in part, encoding the CAR, and (b) a delivery vehicle, wherein after administration of the composition, the one or more nucleic acid molecules are translated in the immune cells to generate modified immune cells comprising the CAR in the subject, the modified immune cells comprising the CAR having target effector activity. Also provided herein is a composition comprising (a) one or more nucleic acid molecules, at least in part, encoding the CAR, and (b) a delivery vehicle comprising at least one targeting moiety.

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 / 287,725, filed December 9, 2021, which is incorporated 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 limitations faced remain to be addressed.

[0003] Thus, there is a need for the development of therapeutic compositions that target immune cells. Summary of the Invention

[0004] Cell therapy involving ex vivo modification of immune cells has attracted a great deal of attention from the scientific community over the past few years, including the first approval of a cell therapy for use in humans. Although the power of ex vivo modified immune cells is evident, there are several challenges in the generation, transportation and administration of such modified cells. Thus, the present disclosure encompasses the recognition that certain delivery vehicles are advantageous in facilitating the in vivo modification of immune cells to avoid the problems of ex vivo modification and subsequent patient administration. The teachings provided herein allow for the harnessing of the power of immune cells modified to target specific cells, tissues, or other undesirable targets while avoiding many of the current problems.

[0005] The disclosure encompasses, inter alia, methods of modifying immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) in a subject by delivering a composition comprising one or more nucleic acid molecules, at least in part, encoding a CAR, and a delivery vehicle.

[0006] In one aspect, the disclosure provides a method of generating a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising administering to a subject a composition comprising (a) one or more nucleic acid molecules at least in part encoding the CAR, and (b) a delivery vehicle, wherein after administration of the composition, the one or more nucleic acid molecules are translated in an immune cell to generate a modified immune cell comprising the CAR, wherein the immune cell is a stem cell, monocyte, macrophage, or dendritic cell in the subject, and wherein the modified immune cell comprising the CAR has target effector activity.

[0007] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a composition comprising (a) one or more nucleic acid molecules encoding, at least in part, a chimeric antigen receptor (CAR); and (b) a delivery vehicle, wherein after administration of the composition, the one or more nucleic acid molecules are translated in an immune cell to generate a modified immune cell comprising the CAR, wherein the immune cell is a stem cell, monocyte, macrophage, or dendritic cell in the subject, and wherein after administration, at least one sign or symptom of the disease or disorder is ameliorated in the subject.

[0008] In some embodiments, the composition is a pharmaceutical composition that includes a pharma- ceutically acceptable carrier.

[0009] In some embodiments, the one or more nucleic acid molecules comprise DNA or messenger RNA (mRNA). In some embodiments, the one or more nucleic acid molecules comprise modifications including modified nucleotides, modifications to the 5' untranslated region (UTR), modifications to the 3'UTR, a cap structure, a poly(A) tail, or a combination thereof. 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.

[0010] In some embodiments, the one or more nucleic acid molecules are purified nucleic acid molecules. In some embodiments, the purified nucleic acid molecules are generated by methods including silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin-based purification, polyA isolation, RNeasy, or a combination thereof.

[0011] In some embodiments, the one or more nucleic acid molecules are codon-optimized. In some embodiments, the one or more nucleic acid molecules are codon-optimized for expression in stem cells, monocytes, macrophages, or dendritic cells.

[0012] In some embodiments, the composition further comprises an additional payload. In some embodiments, the additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic polycytidylic acid (poly I:C), a TLR7 / 8 agonist, a CpG oligodeoxynucleotide, a NOD-like receptor (NLR) agonist, a RIG-I-like receptor (RLR) agonist, a C-type lectin receptor (CLR) agonist, a cytoplasmic DNA sensing, a cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) agonist, an interferon-inducible protein 16 (IFI16) agonist, a DEAD box helicase 41 (DDX41) agonist, an LRR-binding FLII interacting protein 1 (LRRFIP1) agonist, an absent in melanoma 2 (AIM2) agonist, an aryl hydrocarbon receptor (AhR) ligand, or a combination thereof.

[0013] In some embodiments, the one or more nucleic acid molecules and additional payload are encapsulated in a delivery vehicle. In some embodiments, the delivery vehicle is or comprises a liposome, a lipid nanoparticle, a polymer, an adeno-associated virus (AAV) vector, an adenoviral vector, a retroviral vector, or a combination thereof. In some embodiments, the liposome or lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or a combination thereof. In some embodiments, the retroviral vector comprises a lentiviral vector or a gamma retroviral vector. In some embodiments, the lentiviral vector is packaged with a Vpx protein. In some embodiments, the adenoviral vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, the Ad5 vector comprises an Ad5f35 adenoviral vector.

[0014] In some embodiments, the administering step is or includes intraarterial, subcutaneous, intravenous, intradermal, intratumoral, intralymph node, intramedullary, intramuscular, or intraperitoneal delivery.

[0015] In some embodiments, the targeted effector activity directed against a target cell is or includes phagocytosis, targeted cytotoxicity, antigen presentation, or cytokine secretion.

[0016] In some embodiments, at least a portion of the one or more nucleic acid molecules encode a peptide agent. In some embodiments, at least a portion of the one or more nucleic acid molecules encode a peptide agent that stimulates and / or polarizes a modified immune cell toward a proinflammatory phenotype. In some embodiments, the peptide agent is or comprises a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, an immune ligand, a dominant negative receptor, a switch receptor, a secreted antibody or fragment thereof, a transcription factor, an angiopoietin receptor, or a combination thereof.

[0017] In some embodiments, after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell to generate a modified immune cell that comprises a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, an immune ligand, a dominant negative receptor, a switch receptor, a secreted antibody or fragment thereof, a transcription factor, an angiopoietin receptor, or a combination thereof.

[0018] In some embodiments, the portion of the one or more nucleic acid molecules encoding a CAR and the portion of the one or more nucleic acid molecules encoding a peptide agent are present in the composition in a ratio of approximately 1000:1, 500:1, 250:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 1:250, 1:500, or 1:1000.

[0019] In some embodiments, a portion of the one or more nucleic acid molecules encoding the CAR is present in a first delivery vehicle, and a portion of the one or more nucleic acid molecules encoding the peptide agent is present in a second delivery vehicle. In some embodiments, the first delivery vehicle is or comprises a first population of lipid nanoparticles or liposomes, and the second delivery vehicle is or comprises a second population of lipid nanoparticles or liposomes.

[0020] In some embodiments, the portion of the one or more nucleic acid molecules encoding a CAR and the portion of the one or more nucleic acid molecules encoding a peptide agent are separated by a truncation peptide.

[0021] In some embodiments, the compositions are administered alone or in combination with one or more therapeutic agents, procedures, or modalities.

[0022] In some embodiments, the immune cells are mobilized prior to administering the composition to the subject. In some embodiments, the immune cells are mobilized by administering G-CSF, GM-CSF, FLT3-ligand, or plerixafor to the subject.

[0023] In another aspect, the disclosure provides a composition comprising (a) one or more nucleic acid molecules, at least a portion of which encodes a CAR, and (b) a delivery vehicle. In some embodiments, the delivery vehicle comprises at least one targeting moiety. In some embodiments, the targeting moiety facilitates passive targeting of the composition to a desired target. In some embodiments, the targeting moiety facilitates active targeting of the composition to a desired target. In some embodiments, the targeting moiety is or comprises an antibody or any fragment thereof. In some embodiments, the antibody or any fragment thereof binds to CD14, CD11b, CD163, CD206, CD33, CD209, or a combination thereof. In some embodiments, the targeting moiety is or comprises a small molecule. In some embodiments, the targeting moiety is or comprises a combination of specific lipids or hydrophobic elements.

[0024] The drawings are for illustration purposes only and not for limitation. [Brief description of the drawings]

[0025] [Figure 1] 1 shows exemplary fusion proteins including IFN-β and IFNAR1 / 2, and IL10 and IL10RA / B.

[0026] [Diagram 2] 1 shows an exemplary fusion protein design including a signal peptide, a cytokine, a linker, and a cytokine receptor, where the cytokine is bound to the cytokine receptor.

[0027] [Figure 3A] An exemplary method of the disclosure is shown, which includes gene transfer of a construct encoding a fusion protein into immune cells (e.g., macrophages), dimerization of the fusion protein with an endogenous receptor, and polarization of the macrophages into a pro-inflammatory (i.e., M1) state. [Figure 3B]An exemplary method of the disclosure is shown, which includes gene transfer of a construct encoding a fusion protein (and optionally a CAR) into immune cells (e.g., macrophages), dimerization of the fusion protein with an endogenous receptor, and polarization of the macrophages into a pro-inflammatory (i.e., M1) state.

[0028] [Figure 4A] An exemplary method of the disclosure is shown, which includes gene transfer of a construct encoding a fusion protein into immune cells (e.g., macrophages), dimerization of the fusion protein with an endogenous receptor, and polarization of macrophages into an anti-inflammatory (i.e., M2) state. [Figure 4B] An exemplary method of the disclosure is shown, which includes gene transfer of a construct encoding a fusion protein (and optionally a CAR) into immune cells (e.g., macrophages), dimerization of the fusion protein with an endogenous receptor, and polarization of the macrophage into an anti-inflammatory (i.e., M2) state. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, and other terms, are set forth throughout the specification. Publications and other reference materials referred to herein are incorporated by reference in their entirety to describe the background of the invention and to provide further details regarding its practice.

[0030] 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.

[0031] 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, 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%, 1% in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except when such a number exceeds 100% of the possible values).

[0032] 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 functions. Activation may also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, gene expression changes, antigen processing and presentation, metabolic changes, and other functions.

[0033] Agent: As used herein, the term "agent" (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. Agents 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. An agent can bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on the target or target cell. An agent can diffuse or be transported into the cell and act within the cell.

[0034] Antibody: As used herein, the term "antibody" refers to a polypeptide that contains sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As known in the art, an intact antibody, as produced in nature, is a tetrameric agent of about 150 kD, containing two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 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 about 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 (located at the base tip of the Y). A short region, known as a "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 join the two heavy chain polypeptides together in an intact antibody. Each light chain is composed 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 is composed of two heavy-light chain dimers in which the heavy and light chains are linked together by one disulfide bond and two other disulfide bonds connect the heavy chain hinge regions together to form a tetramer. Naturally produced antibodies are also 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 to provide 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 one hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, such as 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, an antibody produced and / or utilized in accordance with the present invention (e.g., as a component of a CAR) comprises a glycosylated Fc domain, e.g., an Fc domain with modified or engineered glycosylation. In some embodiments, any polypeptide, or complex of polypeptides, that includes sufficient immunoglobulin domain sequences 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., produced by an animal in response 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 mouse, 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, in appropriate embodiments (unless otherwise stated or clear from the context), can refer 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.).

[0035] 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 include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include 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 of the constructs or formats known or developed in the art for utilizing the structural and functional features 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 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 a polypeptide whose amino acid sequence includes one or more structural elements recognized by those of skill in the art as a complementarity determining region (CDR), or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those of skill in the art as a complementarity determining region (CDR).In some embodiments, an antibody agent is a polypeptide that includes 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 includes a polypeptide that includes 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 1-5 amino acid substitutions compared to the reference CDR. 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 CDR. 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 CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDR is deleted, added, or substituted when compared to the reference CDR, but the included CDR has 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 1-5 amino acids in the included CDR are deleted, added, or substituted when compared to the reference CDR, but the included CDR has 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 in the included CDR is substituted when compared to the reference CDR, but the included CDR has 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 1-5 amino acids within the included CDRs are deleted, added, or substituted as compared to the reference CDR, but the included CDRs have 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 includes structural elements recognized by those of skill in the art as an immunoglobulin variable domain, or includes a polypeptide whose amino acid sequence includes structural elements recognized by those of skill 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 includes structural elements recognized by those of skill in the art as an immunoglobulin variable domain, and / or does not include a polypeptide whose amino acid sequence includes structural elements recognized by those of skill in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent can be or include a molecule or composition that does not include immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule that includes at least one antigen binding domain).

[0036] 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.

[0037] 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.

[0038] 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 conformation.

[0039] 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 that specifies the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0040] 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 appreciate 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 appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen" as the term is used herein. Furthermore, those skilled in the art will appreciate 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 are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0041] Antitumor effect: As used herein, the term "antitumor 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 a cancerous condition. An "antitumor effect" may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in preventing the development of an initial tumor.

[0042] Autologous: As used herein, the term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0043] Allogeneic: As used herein, the term "allogeneic" refers to any material (eg, a population of cells) derived from a different animal of the same species.

[0044] Xenogenic: As used herein, the term "xenogenic" refers to any material (e.g., a population of cells) derived from an animal of a different species.

[0045] 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 through the bloodstream or lymphatic system to other parts of the body. 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, etc.

[0046] Conservative sequence modifications: As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies conforming to 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 the ability to bind antigen using the functional assays described herein.

[0047] Costimulatory Ligand: As used herein, the term "costimulatory ligand" refers to a molecule on an antigen presenting cell (e.g., APC, dendritic cell, 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 the Toll ligand receptor, 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.

[0048] Cytotoxic: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging cells. In some embodiments, the cytotoxicity of the modified cells is improved, e.g., the cytolytic activity of macrophages is increased.

[0049] 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.

[0050] 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 macrophages is to engulf and digest cellular debris, foreign bodies, microorganisms, cancer cells, and other unhealthy cells through phagocytosis.

[0051] 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, either having 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 codes for 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, the nucleotide sequence of which is identical to the mRNA sequence and which is usually shown in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0052] Endogenous: As used herein, "endogenous" refers to any substance that originates or is produced within a particular organism, cell, tissue or system.

[0053] 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.

[0054] Expansion: As used herein, the term "expansion" refers to an increase in number, such as in an increase in the number of cells, e.g., monocytes, macrophages, and / or dendritic cells. In some embodiments, expansion can occur in vivo.

[0055] 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 a 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.

[0056] Expression vector: As used herein, the term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and 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).

[0057] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that includes a distinct portion of a whole, but lacks one or more portions found in the whole structure. In some embodiments, a fragment consists of such a distinct portion. In some embodiments, a fragment consists of or includes 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 monomeric 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 monomeric units (e.g., residues) found in the whole nucleotide. The whole substance or entity may in some embodiments be referred to as the "parent" of the whole.

[0058] 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., containing 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 the comparison of sequences to determine the degree of homology, including, for example, by allowing gaps of a specified length in one sequence to another sequence when considering which residues in different sequences "correspond" to each other. Calculation of the percentage 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 in 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. The nucleotides at corresponding nucleotide positions are then 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, and 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 percentage of homology between the 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.

[0059] Identity: As used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if each position of the 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 positions that are matched or identical, e.g., 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, and if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0060] 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.

[0061] Immune cell: As used herein, the term "immune cell" refers to a cell involved in an immune response, such as promoting or suppressing 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, and stem cells that can differentiate into one or more of these cell types. The source of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0062] 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 and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0063] Immunoglobulin: As used herein, the term "immunoglobulin" or "Ig" refers 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. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in most control primary immune responses. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but 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.

[0064] 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.

[0065] Modification: As used herein, the term "modification" 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 a nucleic acid.

[0066] 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 a 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.

[0067] Moiety: One of skill in the art will understand that a "moiety" is a defined chemical group or entity that has a particular structure and / or activity (e.g., targeting of modified immune cells to a desired target) as described herein.

[0068] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single stranded and double stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid may comprise one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or a backbone comprising one or more peptide bonds, e.g., as in "peptide nucleic acids". In some embodiments, a nucleic acid comprises one or more or all naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises 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 compared to those of the natural residues (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). 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., by replication in a recombinant cell or system, in vivo or in vitro, or by 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.

[0069] 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 the expression of the latter. 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 when 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.

[0070] 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 disease 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 hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0071] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The 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 of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., as well as by synthetic means.

[0072] 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 engineered amino acid sequence, in that it is artificially designed and / or created. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., modifications 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, the polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be added 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 thus 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 (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 (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 which, in some embodiments, may be or 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 contiguous 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 its parent polypeptide.

[0073] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a series of at least two amino acids linked together by peptide bonds). A protein can include moieties other than amino acids (e.g., it can be a glycoprotein, a 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, a protein 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.

[0074] 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 the transmission of 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.

[0075] Single chain antibody: As used herein, the term "single chain antibody" refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region through an engineered amino acid stretch. Various methods for producing single chain antibodies are known, including those described in U.S. Patent 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.

[0076] Specific binding: As used herein, the term "specific binding" 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, with respect to an antigen-binding domain, such as an antibody agent. For example, an antigen-binding domain or antibody agent that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such cross-species reactivity does not in itself change 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 change the classification of the antigen-binding domain or antibody agent as specific. In some cases, the term "specific binding" or "specifically binding" may be used in relation to the interaction of an antigen-binding domain or antibody agent, a protein, or a 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 particular 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 a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0077] Stimulation: As used herein, the term "stimulation" refers to a primary response induced by a stimulatory molecule (e.g., FcR complex, TLR complex, or TCR / CD3 complex) binding, for example, to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the Fc receptor mechanism or synthetic CAR. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures. As used herein, the term "stimulatory molecule" refers to a molecule of 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 (herein referred to 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 for macrophages.

[0078] 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 susceptible to a disease, disorder, or condition, and in some embodiments, a susceptible 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 a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., a clinical symptom of a disease) or characteristic of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptom or characteristic of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, a subject is an individual to whom and / or to whom a diagnosis and / or therapy is to be administered.

[0079] 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 are separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous cell population. In other instances, the term simply refers to cells that are separated from the 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.

[0080] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that is the subject of the methods, systems, and / or compositions provided, e.g., a cell, tissue, organ, or site in the body that is in need of treatment or that is preferentially bound, e.g., by an antibody (or fragment thereof) or a CAR.

[0081] Target site: As used herein, the term "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0082] 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 the presentation of antigen. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of heterodimers of alpha (α) and beta (β) chains, although in some cells the TCR is composed of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms that 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 that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0083] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0084] 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.

[0085] Treat: As used herein, the terms "treat", "treatment", or "treating" refer to 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 subjects who do not exhibit signs or characteristics of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to subjects who exhibit only early or mild signs or characteristics of a disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to subjects who exhibit 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 the in vitro transcribed mRNA.

[0086] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may 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 may be a dispersed or liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0087] Vector: As used herein, the term "vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to introduce the isolated nucleic acid into 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. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0088] Throughout this disclosure, various aspects of the invention can 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 interpreted as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, as well as the 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, as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0089] Detailed Description The present disclosure provides, inter alia, methods and compositions for promoting the in vivo modification of immune cells (e.g., stem cells, monocytes, macrophages, and / or dendritic cells) in a subject. In some embodiments, the modified immune cells are generated through the use of one or more delivery vehicles, which may include one or more targeting moieties.

[0090] Methods for in vivo immune cell modification The disclosure provides, inter alia, methods of generating modified immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) in a subject, the methods comprising administering to the subject a composition described herein comprising (a) one or more nucleic acid molecules encoding, at least in part, a chimeric antigen receptor (CAR), a fusion protein, and / or a peptide agent, and (b) a delivery vehicle. Thus, in some embodiments, after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) to generate a modified immune cell comprising a CAR, a fusion protein, and / or a peptide agent. In some embodiments, the modified immune cell comprising a CAR, a fusion protein, and / or a peptide agent has target effector activity.

[0091] The disclosure also provides, inter alia, a method of treating a disease or disorder in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a composition described herein comprising (a) one or more nucleic acid molecules, at least in part, encoding a CAR, a fusion protein, and / or a peptide agent, and (b) a delivery vehicle. Thus, in some embodiments, after administration of the composition, the one or more nucleic acid molecules are translated in an immune cell (e.g., a stem cell, a macrophage, a monocyte, or a dendritic cell) to generate a modified immune cell comprising a CAR, a fusion protein, and / or a peptide agent. In some embodiments, at least one sign or symptom of the disease or disorder is ameliorated in the subject after administration of a composition described herein.

[0092] Delivery Vehicle One or more nucleic acid molecules comprising one or more nucleic acid sequences encoding at least one CAR and / or peptide agent described herein can be introduced into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) by physical, chemical, or biological methods (each a "delivery vehicle" as used herein). Physical methods for introducing the nucleic acid constructs described herein into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) can include electroporation, lipofection, particle bombardment, or a combination thereof. One or more nucleic acid molecules can also be introduced into immune cells using mRNA transfection, such as cationic liposome-mediated transfection, lipofection, polymer encapsulation, or 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).

[0093] Biological methods for introducing one or more nucleic acid molecules described herein into immune cells (e.g., stem cells, macrophages, monocytes, 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., Ad5f35), or adeno-associated viruses (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362, which are incorporated herein by reference in their entirety). 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. WO2017 / 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 described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell) is transfected with a lentiviral vector packaged with a Vpx protein. In some embodiments, Vpx inhibits at least one antiviral factor of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell). In some embodiments, a lentiviral vector packaged with a Vpx protein exhibits increased transfection efficiency of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., compared to a lentiviral vector not packaged with a Vpx protein.In some embodiments, immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) are electroporated or transfected or both 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)).

[0094] Chemical means for introducing the nucleic acid constructs described herein into immune cells (e.g., stem cells, macrophages, monocytes, 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 (e.g., lipid nanoparticles), liposomes, and lipofectamine-nucleic acid complexes).

[0095] An exemplary type of delivery vehicle for delivering the nucleic acid constructs described herein is a lipid-based system. One or more nucleic acid molecules described herein may be encapsulated in the aqueous interior of a liposome, interspersed within a lipid bilayer, attached to a liposome via a linking molecule, attached to a lipid nanoparticle (LNP) via a linking molecule, entrapped in a liposome, entrapped in an LNP, complexed with a liposome, complexed with an LNP, dispersed in a solution or suspension containing lipid, mixed with lipid, complexed with a micelle, or otherwise associated with lipid. The lipids used in the methods described herein may be naturally occurring lipids 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 Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. In some embodiments, the lipid-based system may include one or more lipids that facilitate targeting of the composition to a desired cell type(s) (e.g., stem cells, monocytes, macrophages, or dendritic cells). In some embodiments, the delivery vehicle allows the composition to be selectively taken up (e.g., endocytosed, phagocytosed) by immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) compared to a composition that does not include a delivery vehicle.

[0096] targeting part In some embodiments, the delivery vehicle may include one or more targeting moieties. In some embodiments, the targeting moiety can facilitate passive targeting of the composition to a desired target. In some embodiments, the targeting moiety can facilitate active targeting of the composition to a desired target.

[0097] In some embodiments, the targeting moiety may be or include one or more of an antibody (e.g., monoclonal, polyclonal, synthetic, human, humanized, non-human antibody) or any fragment thereof, such as an scFv, aptamer, darpin, centyrin, naturally occurring or synthetic receptor, affibody, or other artificial protein recognition molecule, that binds, for example, to one or more of CD14, CD11b, CD163, CD206, CD33, CD209. In some embodiments, the targeting moiety may be or include a small molecule.

[0098] In some embodiments, the targeting moiety may be or include, for example, a specific lipid or hydrophobic element combination that is present on or forms the outer surface of a liposome or lipid nanoparticle (e.g., for targeting to a specific cell type(s)).

[0099] nucleic acid molecule In some embodiments of the present disclosure, the one or more nucleic acid molecules are or include mRNA. In some embodiments, the mRNA according to the present disclosure can be synthesized as unmodified or modified mRNA. Typically, the mRNA is modified to increase stability. Modification of the mRNA can include, for example, modification of the nucleotides of the RNA. Thus, the 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 in the mRNA modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, and / or a poly(A) tail.

[0100] In some embodiments, the mRNA of the present disclosure (e.g., mRNA encoding a CAR, a fusion protein, and / or a peptide agent) may include an RNA backbone modification. Typically, a backbone modification is a modification in which the backbone phosphate of the nucleotide contained in the 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 includes replacing phosphodiester bonds with other anionic, cationic, or neutral groups.

[0101] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a CAR, a fusion protein, and / or a peptide agent) may include a sugar modification. Exemplary sugar modifications are chemical modifications of the sugar of the nucleotides involved, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, 2'-deoxy -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 azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0102] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a CAR, a fusion protein, and / or a peptide agent) can include a modification of the base of a nucleotide (base modification). A modified nucleotide that includes a base modification is also referred to as a base-modified nucleotide.

[0103] 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.

[0104] Thus, in some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a CAR, a fusion protein, and / or a peptide agent) 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 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 a 2'-O-methyl residue 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, modified mRNAs of the disclosure comprise modified nucleotides including m6AGCap1 and pseudouridine (PsU).

[0105] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a CAR, a fusion protein, and / or a peptide agent) 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 10-200 cytosine nucleotides (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly(C) tail may be in addition to or may be a substitute for the poly(A) tail.

[0106] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a CAR, a fusion protein, and / or a peptide agent) comprises a 5' and / or a 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 response element. In some embodiments, the 5' untranslated region can be between about 50-500 nucleotides in length.

[0107] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the location of the mRNA in the 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.

[0108] Administration of additional payloads In some embodiments, the method of the present disclosure includes one or more steps of administering an additional payload to a subject to regulate immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) during the process of modifying the immune cells. In some embodiments, the composition may include one or more additional payloads. In some embodiments, the composition may include one or more additional payloads in the same delivery vehicle as the one or more nucleic acid molecules. In some embodiments, the composition may include one or more additional payloads in a delivery vehicle different from that used with the one or more nucleic acid molecules.

[0109] In some embodiments, the additional payload may be or include a modulator of a pathway activated by the 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 proinflammatory 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 (RNaseL), and overexpression and inhibition of self-amplifying mRNA replication are relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0110] 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 RNaseL, RNase T2 or RNase1 inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL inhibitor. In some embodiments, the RNaseL inhibitor comprises sunitinib. In some embodiments, the RNaseL inhibitor comprises ABCE1.

[0111] In some embodiments, administering an RNaseL inhibitor to a subject increases mRNA stability in the modified immune cells compared to mRNA stability in modified immune cells of the same type (e.g., stem cells, macrophages, monocytes, or dendritic cells) of a subject that has not been administered an RNaseL inhibitor. In some embodiments, administering an RNaseL inhibitor to a subject increases CAR expression in the modified immune cells compared to CAR expression in modified immune cells of the same type (e.g., stem cells, macrophages, monocytes, or dendritic cells) of a subject that has not been administered an RNaseL inhibitor. In some embodiments, administering an RNaseL inhibitor to a subject increases effector activity in modified immune cells compared to effector activity in modified immune cells of the same type (e.g., stem cells, macrophages, monocytes, or dendritic cells) of a subject that has not been administered an RNaseL inhibitor.

[0112] In some embodiments of the present disclosure, the step of administering an additional payload to the subject occurs before the step of administering a composition comprising the mRNA to the subject.

[0113] In some embodiments, the disclosed methods include administering to the subject a cytokine or immune stimulatory recombinant protein. In some embodiments, the cytokine is 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), TNF-β, CD154, lymphotoxin beta (LT-β), A proliferation-inducing ligand (APRIL), CD70, CD153, glucocorticoid 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 peroxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand ligand (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.

[0114] In some embodiments of the present disclosure, the step of administering to the subject a cytokine or immune stimulatory recombinant protein is performed after the step of administering to the subject a composition comprising mRNA.

[0115] In some embodiments, administration of the cytokine or immunostimulatory recombinant protein to a subject increases the survival rate of modified immune cells in the subject as compared to the same type of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administration of the cytokine or immunostimulatory recombinant protein to a subject increases protein (e.g., CAR, fusion protein, and / or peptide agent) expression in modified immune cells in the subject as compared to the same type of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administration of the cytokine or immunostimulatory recombinant protein to a subject extends the longevity of protein (e.g., CAR, fusion protein, and / or peptide agent) expression in modified immune cells in the subject as compared to the same type of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administration of a cytokine or immunostimulatory recombinant protein to a subject increases the effector activity of a modified immune cell in the subject compared to the same type of modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein. In some embodiments, administration of a cytokine or immunostimulatory recombinant protein to a subject increases the pro-inflammatory (M1) polarization of a modified immune cell in the subject compared to the same type of modified immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) in a subject that has not been administered the cytokine or immunostimulatory recombinant protein.

[0116] Assay A variety of assays can be performed to confirm the presence of one or more nucleic acid molecules described herein in immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) taken from a subject. For example, such assays include molecular biology 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.

[0117] A variety of assays can be performed to determine various characteristics of the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells), including, but not limited to, immune cell viability, nucleic acid expression, nucleic acid longevity, protein (e.g., CAR, fusion protein, and / or peptide agent) expression, protein (e.g., CAR, fusion protein, and / or peptide agent) longevity, effector activity, and proinflammatory (M1) polarization. For example, such assays include flow cytometry, quantitative PCR, and in vitro functional assays such as cytokine / chemokine secretion, phagocytosis, and specific lysis assays of tumor cells of interest.

[0118] Treatment methods The present disclosure provides, inter alia, a method of treating a disease or disorder in a subject (e.g., a disease or disorder described herein), comprising delivering a pharmaceutical composition described herein. In some embodiments, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder. The pharmaceutical composition 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.

[0119] 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). The pharmaceutical compositions described herein can be administered to a subject alone or in combination with one or more therapeutic agents, procedures, or modalities according to the dosing regimens described herein or as a physician, using sound medical judgment, may determine to be advantageous.

[0120] The pharmaceutical compositions 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.

[0121] Methods are provided for treating (e.g., reducing, inhibiting, or delaying the progression of) cancer or tumor in a subject using the pharmaceutical compositions described herein. The subject may have an adult or childhood form of cancer. The cancer may be in early, mid, or late stage, or may be metastatic cancer. The cancer may include, but is not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, or myeloma, such as multiple myeloma), or metastatic lesions. 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, reproductive 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).

[0122] 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 tissue, 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 cell carcinoma (e.g., renal cell carcinoma), cancer), 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 or metastatic colorectal cancer, e.g., microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair deficient repair proficient, or mismatch repair deficient colon 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 tumors (NETs) (e.g., atypical pulmonary carcinoid tumors)), lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders), lymphomas (e.g., T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma), myelomas (e.g., multiple myeloma), or leukemias (e.g., myeloid leukemia or lymphocytic leukemia).

[0123] In some embodiments, the cancer is a brain tumor, e.g., a glioblastoma, a gliosarcoma, or a recurrent brain tumor. In some embodiments, the cancer is a pancreatic cancer, e.g., advanced pancreatic cancer. In some embodiments, the cancer is a skin cancer, e.g., a melanoma (e.g., stage II-IV melanoma, HLA-A2 positive melanoma, unresectable melanoma, or metastatic melanoma), or a Merkel cell carcinoma. In some embodiments, the cancer is a kidney cancer, e.g., a renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer is a 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.

[0124] 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.

[0125] The pharmaceutical compositions described herein can be used to enhance or modulate immune response in a subject. In one embodiment, the pharmaceutical compositions described herein enhance, stimulate, or increase 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 immunocompromised or at risk. For example, the subject is undergoing or has undergone chemotherapy and / or radiation therapy.

[0126] 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 of the liver, 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.

[0127] 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 described herein are administered by injection or infusion. The pharmaceutical compositions described herein can be administered to a patient intraarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodal, intramedullary, 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 directly injected into the subject at the site of inflammation, the site of local disease, lymph nodes, organs, tumors, or the site of infection.

[0128] composition The present disclosure provides, inter alia, compositions comprising one or more nucleic acid molecules and a delivery vehicle (e.g., a delivery vehicle modified with one or more targeting moieties). In some embodiments, the compositions of the present disclosure comprise one or more nucleic acid molecules, at least in part, encoding a CAR, and a delivery vehicle. In some embodiments, the compositions of the present disclosure comprise one or more nucleic acid molecules, at least in part, encoding a fusion protein, and a delivery vehicle. In some embodiments, the compositions of the present disclosure comprise one or more nucleic acid molecules, at least in part, encoding a peptide agent, and a delivery vehicle. In some embodiments, the compositions of the present disclosure comprise one or more nucleic acid molecules, at least in part, encoding a CAR and at least in part, encoding a fusion protein, and a delivery vehicle. In some embodiments, the compositions of the present disclosure comprise one or more nucleic acid molecules, at least in part, encoding a CAR and at least in part, encoding a peptide agent, and a delivery vehicle. In some embodiments, the compositions of the present disclosure comprise one or more nucleic acid molecules, at least in part, encoding a fusion protein and at least in part, encoding a peptide agent, and a delivery vehicle. In some embodiments, a composition of the disclosure comprises one or more nucleic acid molecules, at least in part, encoding a CAR, at least in part encoding a fusion protein, and at least in part encoding a peptide agent, and a delivery vehicle.

[0129] In some embodiments, compositions of the disclosure comprise one or more nucleic acid molecules at least partially encoding a CAR, a fusion protein, and / or a peptide agent, where the portions of the one or more nucleic acid molecules encoding a CAR, a fusion protein, and / or a peptide agent are all present in a single nucleic acid molecule. In some embodiments, compositions of the disclosure comprise one or more nucleic acid molecules at least partially encoding a CAR, a fusion protein, and / or a peptide agent, where the portions of the one or more nucleic acid molecules encoding a CAR, a fusion protein, and / or a peptide agent are all present in separate nucleic acid molecules.

[0130] In some embodiments, the delivery vehicle comprises or is a liposome, lipid nanoparticle, polymer, adeno-associated virus (AAV) vector, adenovirus vector, retrovirus vector, or a combination thereof. In some embodiments, the liposome or lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or a combination thereof. In some embodiments, the retrovirus vector comprises a lentivirus vector or a gamma retrovirus vector. In some embodiments, the lentivirus vector is packaged with Vpx protein. In some embodiments, the adenovirus vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, the Ad5 vector comprises an Ad5f35 adenovirus vector.

[0131] In some embodiments, the compositions of the present disclosure include an additional payload. In some embodiments, the additional payload is a pathogen recognition receptor agonist (e.g., a TLR ligand (e.g., TLR1, 2, 3, 4, 5, 6, 7, 8, 9, or 13); polyinosinic polycytidylic acid (poly I:C); a TLR7 / 8 agonist (e.g., a TLR7 / 8 agonist that is a single-stranded RNA or base analog that activates TLR7 / 8); a CpG oligodeoxynucleotide (ODN) (e.g., class A, B, or C CpG ODNs; NOD-like receptor (NLR) agonists (e.g., NOD1, NOD2, or NOD1 / 2 agonists); RIG-I-like receptor (RLR) agonists (e.g., RIG-I agonists, MDA-5 agonists, double-stranded RNA, 5' triphosphate hairpin RNA, 5' triphosphate dsRNA, or Poly(dA:dT)); C-type lectin receptor (CLR) agonists (e.g., Dectin-1 agonists, Mincle agonists, DC-SIGN agonists, Dectin-2 agonists, beta-glucan, curdlan, HKCA, laminarin, pustulan, scleroglucan, WGP, zymosan, fluf furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB-HS15, or TDM); cytoplasmic DNA sensing, cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) agonists; interferon-inducible protein 16 (IFI16) agonists; DEAD-box helicase 41 (DDX41) agonists; LRR-binding FLII-interacting protein 1 (LRRFIP1) agonists; absent in melanoma 2 (AIM2) agonists (e.g., dsDNA-EC, G3-YSD, HSV-60, ISd, ODN TTAGGG, PolydA:dT, VACV70, diABZI, 2'3'-cGAMP, 3'3'cGAMP, c-di-AMP, c-di-GMP, cAIMP, 2'2'cGAMP, 2'3'cGAM(PS)2(R p / Sp), fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'c-di-AM(PS)2(Rp,Rp)VacciGrade, fluorinated c-diGMP, 2'3'-c-diGMP, or DMXAA);an aryl hydrocarbon receptor (AhR) ligand; or a combination thereof;

[0132] In some embodiments, the one or more nucleic acid molecules and the additional payload are encapsulated in a delivery vehicle. In some embodiments, the one or more nucleic acid molecules and the additional payload are encapsulated in the same delivery vehicle. In some embodiments, the one or more nucleic acid molecules and the additional payload are encapsulated in separate delivery vehicles.

[0133] nucleic acid molecule The present disclosure provides, inter alia, a nucleic acid molecule encoding at least one CAR, fusion protein, and / or peptide agent, or fragment thereof, as described herein. In some embodiments, the present disclosure provides a nucleic acid molecule encoding at least one CAR, or fragment thereof, as described herein. In some embodiments, the present disclosure provides a nucleic acid molecule encoding at least one fusion protein, or fragment thereof, as described herein. In some embodiments, the present disclosure provides a nucleic acid molecule encoding at least one peptide agent, or fragment thereof, as described herein. An immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) may comprise one or more nucleic acid molecules (e.g., one or more exogenous nucleic acid molecules) encoding at least one protein (e.g., a CAR, fusion protein, or peptide agent) as described herein.

[0134] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns to the extent that a nucleotide sequence encoding a protein may contain intron(s) depending on the type. "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, either with 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 the 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 a protein or other product of that gene or cDNA.

[0135] The nucleic acid molecule encoding at least one protein (e.g., a CAR, a fusion protein, or a peptide agent) described herein, or a fragment thereof, 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 protein (e.g., a CAR, a fusion protein, or a peptide agent) described herein, or a fragment thereof. In some embodiments, the nucleic acid molecule comprises or is a DNA construct encoding at least one protein (e.g., a CAR, a fusion protein, or a peptide agent) described herein, or a fragment thereof.

[0136] In some embodiments comprising nucleic acid molecules encoding a CAR and a peptide agent, the portion of the one or more nucleic acid molecules encoding the CAR and the portion of the one or more nucleic acid molecules encoding the peptide agent are present in the composition in a ratio of approximately 1000:1, 500:1, 250:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 1:250, 1:500, or 1:1000.

[0137] In some embodiments, a nucleic acid molecule encoding at least one protein (e.g., a CAR, a fusion protein, or a peptide agent) or a fragment thereof described herein comprises a modification. In some embodiments, the modification comprises a modified nucleotide, a modification to the 5' untranslated region (UTR), a modification to the 3'UTR, a cap structure, a poly(A) tail, or a combination thereof. 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.

[0138] In some embodiments, the nucleic acid molecule encoding at least one protein (e.g., a CAR, a fusion protein, or a peptide agent) or fragment thereof described herein comprises a purified nucleic acid molecule. In some embodiments, the purified nucleic acid molecule is produced by a method including 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, the purified nucleic acid molecule is produced by standard methods of mRNA purification known in the art.

[0139] In some embodiments, all or a fragment of a protein (e.g., a CAR, a fusion protein, or a peptide agent) described herein is encoded by a codon-optimized nucleic acid molecule, for example, for expression in a cell (e.g., a mammalian cell). In some embodiments, one or more nucleic acid molecules of the present disclosure are codon-optimized for expression in stem cells, monocytes, macrophages, or dendritic cells. Various codon optimization methods are known in the art, for example, 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.

[0140] Expression of the nucleic acid described herein can be achieved by operably linking the nucleic acid encoding the protein (e.g., CAR, fusion protein, or peptide agent) or a fragment thereof to a promoter in an expression vector. Exemplary promoters (e.g., constitutive promoters) include, but are not limited to, elongation factor-1 alpha promoter (EF-1 alpha) promoter, immediate early cytomegalovirus (CMV) promoter, ubiquitin C promoter, phosphoglycerokinase (PGK) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, or creatine kinase promoter. Examples of inducible promoters include, but are not limited to, metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter. The vector can also contain additional promoter elements, such as enhancers, to control the frequency of transcription initiation.

[0141] In some embodiments, the vector comprising the nucleic acid molecule encoding at least one protein (e.g., CAR, fusion protein, or peptide agent) or fragment thereof described herein comprises or is a viral vector. Viral vector technology is well known and described in the art (e.g., described in 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., described in U.S. Pat. No. 9,149,519 or International Publication No. WO2017 / 044487, each of which is incorporated herein by reference in its entirety).

[0142] In some embodiments, the viral vector comprises an adenovirus 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 affect both replicating and non-replicating cells, to accommodate large transgenes, and to encode proteins without integrating into the host cell genome. In some embodiments, the adenovirus vector comprises an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenovirus vector, e.g., a helper-dependent Ad5F35 adenovirus vector).

[0143] In some embodiments, the viral vector is an adeno-associated viral (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 generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0144] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, and variants thereof. In general, any AAV serotype can be used to introduce at least one protein (e.g., CAR, fusion protein, or peptide agent) or fragment thereof described herein. In some embodiments, the AAV serotype has tropism for a particular tissue.

[0145] 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 function as donor template DNA during homologous recombination (HR).

[0146] 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.

[0147] In some embodiments, compositions of the disclosure include two or more nucleic acid sequences encoding a protein, e.g., a CAR, a fusion protein, and / or a peptide agent. In some embodiments, a vector includes two or more nucleic acid sequences encoding a protein, e.g., a CAR, a fusion protein, and / or a peptide agent. In some embodiments, two or more nucleic acid sequences encoding a protein (e.g., a CAR, a fusion protein, and / or a peptide agent) are encoded by a single nucleic acid molecule, e.g., in the same frame as a single polypeptide chain. In some embodiments, two or more proteins (e.g., a CAR, a fusion protein, and / or a peptide agent) are separated by one or more cleavage peptide sites (e.g., autocleavage sites or substrates for intracellular proteases). In certain embodiments, the cleavage peptides include 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 variants thereof.

[0148] In some embodiments, the vector comprises at least one nucleic acid sequence encoding a protein, (e.g., a CAR, a fusion protein, and / or a peptide agent), and at least one nucleic acid encoding at least one gene that is co-expressed with a second protein, 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 a B7-H3 ligand).

[0149] Pharmaceutical Compositions The present disclosure provides, inter alia, pharmaceutical compositions comprising the compositions described herein (e.g., one or more nucleic acid molecules and a delivery vehicle) in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients.

[0150] 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 the pharmaceutical compositions 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).

[0151] The pharmaceutical compositions 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), preservatives such as serum and cryoprotectants. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, e.g., have no detectable levels of contaminants (e.g., endotoxins).

[0152] The pharmaceutical compositions described herein can be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, disorder, or condition, although appropriate dosages can be determined by clinical trials.

[0153] The pharmaceutical compositions described herein may be in various 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. A preferred composition may be an injectable or infusible solution. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0154] 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).

[0155] 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.

[0156] The pharmaceutical compositions described herein may also be administered multiple times at a particular dosage. Optimal dosages and treatment regimes 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 treatment accordingly.

[0157] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously with, or after) a second therapy. For example, the second therapy can 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, mycophenolic acid, FK506 antibody, or glucocorticoid), an antagonist (e.g., PD-1 antagonist, PD-L1 antagonist, or PD-L2 antagonist), or an anti-cancer drug (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate ... methotrexate, methotre In some embodiments, the second therapy may include, but is not limited to, an immune cell mobilizing treatment of the present disclosure (e.g., G-CSF, GM-CSF, FLT3-ligand, or plerixafor).

[0158] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, concurrently with, or after) bone marrow transplantation or lymphodepletion 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 transplant, the subject receives an infusion of a pharmaceutical composition described herein. The pharmaceutical compositions described herein can be administered before or after surgery.

[0159] The dosage of any of the aforementioned therapies administered to a subject varies depending on the disease, disorder, or condition being treated and based on the particular subject. Scaling of dosages for administration to humans can be performed according to art-accepted practices. For example, the dosage of alemtuzumab is generally about 1 mg to about 100 mg for an adult, and is 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, which is incorporated herein by reference in its entirety).

[0160] Chimeric antigen receptors (CARs) As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor that is engineered to be expressed on immune effector cells and specifically targets the cells and / or binds to an 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.

[0161] In some embodiments, the modified immune cell, e.g., a modified stem cell, macrophage, monocyte, or dendritic cell, is generated by expressing a CAR therein. In some embodiments, the immune cell comprises a CAR that comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the immune cell comprises a stem cell, macrophage, monocyte, or dendritic cell.

[0162] In some embodiments, the CAR may further comprise one or more of one or more extracellular leader domains, one or more extracellular hinge domains, and one or more intracellular costimulatory domains.

[0163] In some embodiments, the CAR comprises a spacer domain or hinge between the extracellular and transmembrane domains. In some embodiments, the CAR comprises a spacer domain or hinge between the intracellular and transmembrane domains. 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 glycine-serine doublets.

[0164] In some embodiments, an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR may comprise one or more control systems including, but not limited to, a safety switch (e.g., an on switch and an off switch, a suicide switch), a logic gate, such as an AND gate (e.g., two or more CARs, each of which lacks one or more signaling domains such that activation of both / all CARs is required for activation or function of the complete immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell), an OR gate (e.g., two or more CARs, each of which has an intracellular domain such as CD3ζ and a costimulatory domain), and / or a NOT gate (e.g., two or more CARs, one of which contains an inhibitory domain that antagonizes the function of the other CAR(s).

[0165] The disclosure also provides an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic 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 the cell is a stem cell, macrophage, monocyte, or dendritic cell that expresses a CAR.

[0166] 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 a nucleic acid encoding a transmembrane domain, and the nucleic acid encoding the transmembrane domain is operably linked to a nucleic acid encoding the intracellular domain.

[0167] In some embodiments, the effector activity of an immune cell comprising a CAR is directed against a target cell that comprises 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.

[0168] 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 stem cell, a macrophage, a monocyte, or a dendritic cell). In some embodiments, a CAR described herein improves effector activity of an immune cell described herein (e.g., a stem cell, a macrophage, a monocyte, or a dendritic cell), e.g., by enhancing inhibition of CD47 and / or SIRPα activity. In some embodiments, a CAR described herein binds, e.g., CD47 and acts as a dominant negative receptor to inhibit SIRPα activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds 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.

[0169] In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR, fusion protein, and / or peptide agent 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 mutated 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 fusion protein described herein and / or a CAR) comprises a dominant negative receptor, e.g., that blocks an inhibitory checkpoint.

[0170] In some embodiments, the CAR described herein further comprises a truncated peptide (e.g., a P2A, F2A, E2A and / or T2A peptide) and a second CAR comprising at least one inhibitory domain of anti-phagocytic signaling. In some embodiments, the 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 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, the 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 CAR described herein further comprises 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).

[0171] In some embodiments, an immune cell described herein (e.g., comprising or expressing a CAR, fusion protein, and / or peptide agent 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 domains) and / or constitutively active kinase domains (e.g., a constitutively active LYN domain). In some embodiments, the CARs described herein further comprise a truncated peptide (e.g., a P2A, F2A, E2A and / or T2A peptide) and one or more phosphatase dead domains (e.g., a phosphatase dead Shp1, a phosphatase dead 72-5ptase (INPP5E), a phosphatase dead Shp2, and / or a phosphatase dead SHIP-1 domain) and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain).

[0172] Extracellular domain The present disclosure provides a chimeric antigen receptor (CAR) 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).

[0173] 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 a 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.

[0174] TLR extracellular domains In some embodiments, the TLR extracellular domain comprises a full-length TLR extracellular domain. In some embodiments, the TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, the TLR extracellular domain (or a portion thereof) is or comprises a human TLR extracellular domain. In some embodiments, the TLR 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 TLR 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 TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0175] 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., 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).

[0176] Antigen-binding domain In some embodiments, the CAR comprises an antigen binding domain that binds, for example, to 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 a target cell associated with a particular disease state.

[0177] 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, CD2, CD5, CD7, CD147, 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 proliferation Growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific fetal 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, beta type, 9 (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 (TEM7) R), 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 cell 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, surviving, 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 breakpoints, 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 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 anchoring 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 papilloma virus E6 (HPV E6), human papilloma virus 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), immunoglobulin lambda-like polypeptide 1 (IGLL1),delta-like ligand 3 (DLL3); delta-like canonical notch ligand 4 (DLL4), TROP2, cMET, alpha fetoprotein (AFP), AXL, roundabout homolog 1 (ROBO1), CS1, or aspartate beta-hydroxylase (ASPH). In 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.

[0178] 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 an amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or transthyretin). In some embodiments, the neurodegenerative disease / disorder is a tauopathy, an 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's 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, variant Creutzfeldt-Jakob disease (vCJD)).

[0179] 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).

[0180] 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 cell comprises two or more different CARs comprising one or more antigen binding domains. In some embodiments, immune cells comprising bispecific CARs and / or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and / or on-target extra-tissue effects by requiring two antigens to be present. In some embodiments, the immune cell comprises bispecific CARs and / or comprising 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 separate signals that stimulate activation of the modified cell. In some embodiments, such a configuration may be referred to as an "AND" logic gate.

[0181] In some embodiments, immune cells that contain bispecific CARs and / or two or more different CARs that contain one or more antigen binding domains can reduce off-target and / or on-target extra-tissue effects by requiring the presence of one antigen and the absence of a second, normal protein antigen before the activity of the cell is stimulated. In some embodiments, such a configuration may be referred to as a "NOT" logic gate. In contrast to an AND gate, a NOT gate CAR-modified cell is activated by binding to a single antigen. However, binding of a second receptor to a second antigen functions to nullify the activation signal that is perpetuated through the CAR. Typically, such inhibitory receptors target antigens that are abundantly expressed in normal tissues but not present in tumor tissues.

[0182] 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., modified immune cells 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., modified immune cells 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 optimizes the physicochemical parameters of the CAR, such as, for example, optimal size (e.g., allowing for the exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and / or lack of homodimerization, for tumor antigens. In some embodiments, the hinge domain is or comprises a CD8a or CD28 domain.

[0183] 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 interactions 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., modified immune cells provided herein). In some embodiments, the transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., modified immune cells 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 In some embodiments, the transmembrane domain is or comprises a CD8a or CD28 domain.

[0184] 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.

[0185] 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.

[0186] Intracellular domain In some embodiments, the CAR comprises one or more intracellular domains. In some embodiments, the intracellular domain is or comprises a human intracellular domain or a portion thereof. In some embodiments, the intracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., modified immune cells provided herein). In some embodiments, the intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., modified immune cells provided herein). In some embodiments, the intracellular domain and / or other cytoplasmic domains of the CAR participate in activation of the cell (e.g., immune cell) in which the CAR is expressed. In some embodiments, the intracellular domain of the CAR participates in signal activation and / or transduction in the immune cell that contains the CAR.

[0187] 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.

[0188] 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.

[0189] In some embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules, such as at least one signaling domain from CD3, the Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0190] 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.

[0191] 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.

[0192] 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 can be a domain that is endogenous to a particular immune cell type (e.g., modified immune cells provided herein). In some embodiments, the signaling domain can be a domain that is not endogenous to a particular immune cell type (e.g., modified immune cells provided herein).

[0193] In some embodiments, the one or more intracellular signaling domains are selected from the group consisting of CD3-zeta, FcR gamma, 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, CD40, CD19, CD20, 41BB, CD28, O X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SH P-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, ICOSL intracellular signaling domain), FLT3, any other intracellular signaling domain described herein, any derivative, variant, or fragment thereof, any synthetic sequence of intracellular signaling domains having the same functionality, and any combination thereof.

[0194] 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, FLT3, CD40, or any derivative, variant, or fragment thereof, in any combination with any of the signaling domains listed in the paragraph above.

[0195] 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, 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.

[0196] In some embodiments, a 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, a costimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0197] As used herein, a "costimulatory 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.

[0198] Cleavage peptide As used herein, a cleavage peptide refers to a peptide that can induce cleavage of a recombinant protein in a cell. In some embodiments, the cleavage peptide is a 2A peptide. In some embodiments, the cleavage peptide is or includes a P2A, F2A, E2A, or T2A peptide. In some embodiments, the nucleic acid described herein comprises 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, and 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 an mRNA to facilitate initiation of translation.

[0199] Peptide drugs In some embodiments, at least a portion of one or more nucleic acid molecules of the disclosure encodes a peptide agent. In some embodiments, a peptide agent refers to a protein or peptide that is co-expressed with a CAR in an immune cell. In some embodiments, the peptide agent primes and / or polarizes a modified immune cell of the disclosure to a proinflammatory phenotype. 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, the peptide agent forms a homodimer with an identical peptide agent. In some embodiments, the peptide agent forms a heterodimer with a different peptide agent. In some embodiments, the nucleic acid described herein comprises one or more nucleic acid sequences that encode one or more peptide agents. In some embodiments, the peptide agent is or comprises an FcR gamma chain.

[0200] In some embodiments, a peptide agent includes 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 or is a cytokine (e.g., IFNα, IFNβ, IFNγ, GM-CSF, M-CSF, IFNλ, TNFα, IL-1b, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-18, or IL-33), a cytokine receptor, a chemokine, a chemokine receptor (e.g., CCR2), an immune ligand (e.g., CD40L, 41BBL, OX40L, CD80, CD86, FTL3 ligand, or CLEC9A), a dominant negative receptor (e.g., dominant negative SIRPa), a switch receptor, a secretory antibody or fragment thereof (e.g., an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CD47 antibody, or an anti-SIRPα antibody), a transcription factor, an angiopoietin receptor (e.g., Tie2), or a combination thereof. In some embodiments, the cytokine receptor is a constitutively active cytokine receptor (e.g., constitutively active GMCSF-R, MCSF-R, or IFNbR). In some embodiments, the transcription factor activates an immune cell of the disclosure to an M1 phenotype. In some embodiments, the peptide agent comprises or is a dominant negative SIRPα, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC10, dominant negative LILRB, or combinations thereof. In some embodiments, the peptide agent comprises DNGR1.

[0201] Fc receptors (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. 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.

[0202] Fusion proteins The term "fusion protein," as used herein, refers to an artificial chimeric protein that includes a cytokine fused to at least one of its corresponding receptor subunits such that the cytokine can bind intramolecularly to its tethered receptor and induce downstream signaling.

[0203] In some embodiments, the fusion protein may include one or more of one or more cytokines, one or more linkers, and one or more cytokine receptors (see FIG. 1). In some embodiments, the fusion proteins of the present disclosure are membrane bound. In some embodiments, the fusion proteins of the present disclosure are not membrane bound.

[0204] In embodiments, the fusion protein of the present disclosure further comprises a signal peptide. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a signal peptide, a cytokine, a linker, and a cytokine receptor (see FIG. 2).

[0205] In some embodiments, the fusion protein includes a linker between the cytokine and the cytokine receptor. As used herein, the term "linker" refers to any oligopeptide or polypeptide that functions to link the cytokine to the cytokine receptor in a polypeptide chain in the fusion protein of the present disclosure. In some embodiments, the linker may include up to 300 amino acids, preferably 5-100 amino acids, and most preferably 5-30 amino acids.

[0206] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) containing the fusion protein may include one or more control systems, including, but not limited to, a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g., a cell-specific promoter, a cell state-specific promoter, a downstream promoter of CAR activation, a downstream promoter of an endogenous signaling pathway, or drug-inducible transcription), post-transcriptional control of the fusion protein mRNA (e.g., RNA-based inhibition by endogenous or recombinant miRNA), or post-translational control of the fusion protein structure or stability (e.g., a fusion protein whose intracellular domain conditionally associates with the complete structure by drug / light-induced association (allowing signaling) or dissociation (inhibiting signaling), or a fusion protein whose stability is regulated by a drug for induced stabilization (allowing signaling) or degradation (inhibiting signaling)). These control systems can be combined to create logic gates, e.g., AND gates (e.g., a fusion protein with a CAR-inducible promoter and cytosolic domain that associate in a drug-dependent manner and thus requires CAR activation and the presence of a small molecule), OR gates (e.g., a fusion protein under the control of a promoter that is transcriptionally activated after either CAR activation or the addition of a small molecule), and / or NOT gates (e.g., a fusion protein whose mRNA is degraded by an endogenous miRNA that is expressed in a native immune cell signaling state (such as an miRNA that is upregulated by a particular cytokine signaling pathway and thus the fusion protein is expressed only in the absence of this cytokine). In some embodiments, modified immune cells, e.g., modified stem cells, macrophages, monocytes, or dendritic cells, are generated by expressing the fusion protein therein. In some embodiments, the immune cells comprise a fusion protein comprising a cytokine, a linker, and a cytokine receptor, where the immune cells comprise stem cells, macrophages, monocytes, or dendritic cells, and the cytokine binds to the cytokine receptor.

[0207] In some embodiments, a fusion protein of the disclosure comprises a cytokine selected from Table 1 and a corresponding receptor 1 selected from the same row of Table 1. In some embodiments, a fusion protein of the disclosure comprises a cytokine selected from Table 1 and a corresponding receptor 2 / co-receptor selected from the same row of Table 1. [Table 1]

[0208] In some embodiments, a fusion protein of the present disclosure comprises interleukin 10 (IL-10), a linker, and an interleukin-10 receptor (IL10R).

[0209] In some embodiments, a fusion protein of the present disclosure comprises interferon beta (IFNβ), a linker, and an interferon-α / β receptor (IFNAR).

[0210] In some embodiments, the modified immune cell (e.g., a stem cell, a macrophage, a monocyte, or a dendritic cell) further comprises a chimeric antigen receptor (CAR) of the present disclosure.

[0211] The disclosure also provides an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a nucleic acid sequence (e.g., an isolated nucleic acid sequence) encoding a fusion protein, the nucleic acid sequence comprising a nucleic acid sequence encoding a cytokine, a nucleic acid sequence encoding a linker, and a nucleic acid sequence encoding a cytokine receptor, wherein the cell is a stem cell, macrophage, monocyte, or dendritic cell that expresses the fusion protein.

[0212] In some embodiments, the fusion protein comprises a cytokine operably linked to another domain of the fusion protein, such as a cytokine receptor, for expression in an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell).

[0213] In some embodiments, the fusion proteins of the disclosure are expressed on the surface of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells). In some embodiments, the fusion proteins of the disclosure induce a pro-inflammatory phenotype as indicated by cytokine production, gene expression changes, cell surface markers, and / or functional assays. In some embodiments, the fusion proteins of the disclosure induce an anti-inflammatory phenotype as indicated by cytokine production, gene expression changes, cell surface markers, and / or functional assays.

[0214] In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 9. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is identical to a sequence selected from Table 9.

[0215] In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 80% identical to a sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 85% identical to a sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 90% identical to a sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 95% identical to a sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 96% identical to a sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 97% identical to a sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 98% identical to a sequence selected from Table 10. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is at least 99% identical to a sequence selected from Table 10. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is identical to a sequence selected from Table 10.

[0216] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the disclosure maintain a proinflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the disclosure maintain a proinflammatory phenotype for at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after the immune cells are modified with a nucleic acid encoding the fusion protein. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the disclosure maintain a proinflammatory phenotype longer than immune cells induced by pretreatment with a soluble cytokine.

[0217] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the disclosure maintain an anti-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the disclosure maintain an anti-inflammatory phenotype for at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after the immune cells are modified with a nucleic acid encoding the fusion protein. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the disclosure maintain an anti-inflammatory phenotype longer than immune cells induced by pre-treatment with a soluble cytokine.

[0218] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the present disclosure maintain a proinflammatory phenotype and / or resist destruction when induced by anti-inflammatory cytokines. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose response curve of a proinflammatory marker by treating modified immune cells comprising the proinflammatory fusion protein with increasing concentrations of an anti-inflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose response curve of a proinflammatory marker by treating modified immune cells comprising the proinflammatory fusion protein with increasing concentrations of a proinflammatory cytokine (e.g., by quantifying the effect of soluble IFN-β on modified immune cells comprising the IFN-β fusion protein).

[0219] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the present disclosure maintain an anti-inflammatory phenotype and / or resist destruction when induced by proinflammatory cytokines. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose response curve of an anti-inflammatory marker by treating modified immune cells comprising the anti-inflammatory fusion protein with increasing concentrations of a proinflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose response curve of an anti-inflammatory marker by treating modified immune cells comprising the anti-inflammatory fusion protein with increasing concentrations of an anti-inflammatory cytokine (e.g., by quantifying the effect of soluble IL-10 on modified immune cells comprising the IL-10 fusion protein).

[0220] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the present disclosure have minimal effects on surrounding cells. In some embodiments, the effects of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a fusion protein of the present disclosure on unmodified cells (e.g., immune cells not comprising a fusion protein of the present disclosure) can be tested by co-culturing the modified immune cells with the unmodified immune cells and analyzing the expression of pro-inflammatory and anti-inflammatory markers in the unmodified cells using flow cytometry. In some embodiments, modified and unmodified immune cells can be co-cultured in a culture dish where the modified and unmodified immune cells are in contact with each other. In some embodiments, modified and unmodified immune cells can be co-cultured in a culture dish where the modified and unmodified immune cells are separated by a transwell assay membrane. In some embodiments, a positive control for testing the effects of modified immune cells on unmodified immune cells includes modified immune cells that express soluble cytokines that may diffuse and stimulate surrounding cells instead of the fusion protein of the present disclosure.

[0221] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising the fusion proteins of the present disclosure have minimal cytotoxic effects on surrounding cells. In some embodiments, modifying immune cells to comprise the fusion proteins of the present disclosure is not cytotoxic to the modified immune cells. In some embodiments, RNAseq data from the modified immune cells is examined to determine whether there is upregulation of genes indicative of a cytotoxic effect.

[0222] In some embodiments, expression of a fusion protein of the present disclosure in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) that also comprises a CAR does not reduce the desired effector function (e.g., phagocytosis, target cytotoxicity, antigen presentation, or cytokine secretion) of the modified immune cell compared to a modified immune cell that comprises a CAR but does not comprise the fusion protein. In some embodiments, expression of a fusion protein of the present disclosure in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) that also comprises a CAR increases the desired effector function (e.g., phagocytosis, target cytotoxicity, antigen presentation, or cytokine secretion) of the modified immune cell compared to a modified immune cell that comprises a CAR but does not comprise the fusion protein.

[0223] Cytokines The present disclosure provides a fusion protein comprising a cytokine. In some embodiments, the cytokine is or comprises a proinflammatory (M1) cytokine. In some embodiments, the proinflammatory cytokine is or comprises a type I interferon (IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β, IFN-ω, IFN-ε, or IFN-κ), type II interferon (IFN-γ), type III interferon (IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4), TNF-α, IL-1β, IL-6, IL-12, IL-17, IL-23, or GM-CSF. In some embodiments, the cytokine is or comprises an anti-inflammatory (M2) cytokine, hi some embodiments, the anti-inflammatory cytokine is or comprises IL-4, IL-10, IL-13, IL-18, M-CSF, or TGF-β. In some embodiments, the cytokine is or comprises a type I interferon (IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, IFN-α21, IFN-β, IFN-ω, IFN-ε, or IFN-κ), a type II interferon (IFN-γ), a type III interferon (IFN-λ1, IFN-λ2, IFN-λ3, or IFN-λ4), TNF-α, IL-1β, IL-6, IL-12, IL-17, IL-23, GM-CSF, IL-4, IL-10, IL-13, IL-18, M-CSF, or TGF-β. In some embodiments, the cytokine is selected from Table 1.

[0224] In some embodiments, the fusion proteins of the invention comprise a cytokine that comprises the same linear amino acid sequence as an endogenous cytokine (e.g., a sequence in Table 2a and Table 2b). In some embodiments, the fusion proteins of the invention comprise a cytokine that comprises an engineered amino acid sequence (e.g., a sequence in Table 6). In some embodiments, the engineered cytokine amino acid sequence is derived from an endogenous cytokine sequence.

[0225] In some embodiments, the engineered cytokine is or includes a circular permutation. A circular permutation is a version of a protein in which a section of amino acids is rearranged such that an amino acid region in the middle of the endogenous protein is instead located at the N-terminus or C-terminus, but the resulting protein retains a similar overall three-dimensional shape to the endogenous protein. In some embodiments, a fusion protein including a circular permutation of a cytokine will result in stronger binding of the cytokine to a cytokine receptor compared to a fusion protein including a cytokine that includes the same linear amino acid sequence as the endogenous cytokine. In some embodiments, a fusion protein including a circular permutation of a cytokine will result in increased signaling compared to a fusion protein including a cytokine that includes the same linear amino acid sequence as the endogenous cytokine. In some embodiments, a fusion protein including a circular permutation of a cytokine will include a shorter linker compared to a fusion protein including a cytokine that includes the same linear amino acid sequence as the endogenous cytokine. In some embodiments, the fusion protein of the invention includes a circular permutation of IL-1β, IL-4, IL-10, or IL-13.

[0226] In some embodiments, the engineered cytokine is or comprises a single chain cytokine. As used herein, a single chain cytokine comprises two or more copies of a cytokine fused together. In some embodiments, the two or more copies of a cytokine in a single chain cytokine are separated by a linker. In some embodiments, a single chain cytokine allows the fusion protein of the present disclosure to mimic endogenous cytokines that act as multimers when bound to a cytokine receptor. In some embodiments, the fusion protein of the present disclosure comprises a single chain cytokine comprising two or more copies of IFN-γ, TNF-α, IL-12, or IL-10.

[0227] In some embodiments, the engineered cytokine is or comprises a monomeric cytokine. As used herein, a monomeric cytokine is engineered such that it does not need to multimerize (e.g., dimerize or trimerize) to bind to its corresponding cytokine receptor. In some embodiments, the fusion protein of the present invention comprises a monomeric cytokine, including an engineered version of IFN-γ, TNF-α, IL-12, or IL-10.

[0228] In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 2a, Table 2b, or Table 6. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 2a, Table 2b, or Table 6.

[0229] In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 80% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 85% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 90% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 95% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 96% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 97% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is at least 98% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is at least 99% identical to a sequence selected from Table 4a, Table 4b, or Table 7. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is identical to a sequence selected from Table 4a, Table 4b, or Table 7.

[0230] Linker In some embodiments, the fusion protein of the present disclosure includes a linker. In some embodiments, the fusion protein of the present disclosure includes a linker between the cytokine and the cytokine receptor. In some embodiments, the linker is a flexible linker. In some embodiments, the flexible linker includes primarily small amino acids, either non-polar (e.g., serine or threonine) or polar (e.g., glycine). In some embodiments, the flexible linker includes amino acid substitutions (e.g., lysine, glutamic acid, glutamine, aspartic acid, and / or asparagine) compared to known linkers to improve the solubility of the linker. In some embodiments, the flexibility of the linker is determined using circular dichroism spectroscopy to test whether the linker is folded into a helical (i.e., rigid structure) or unstructured (i.e., flexible) coil. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker includes 5-50 amino acids. In some embodiments, the linker includes 19-26 amino acids. In some embodiments, the linker includes 26 amino acids. In some embodiments, the linker is at least 90 angstroms in length. In some embodiments, the linker comprises amino acids selected from glycine (G), serine (S), threonine (T), lysine (K), proline (P), glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), or alanine (A).

[0231] In some embodiments, the linker is (G4S) n linker (wherein n=1-5), Whitlow linker, and linker 26.

[0232] In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 80% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 85% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 90% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 95% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 96% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 97% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 98% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 99% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is identical to a linker sequence selected from Table 8.

[0233] In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 80% identical to a linker sequence selected from Table 8. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 85% identical to a linker sequence selected from Table 8. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 90% identical to a linker sequence selected from Table 8. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 90% identical to a linker sequence selected from Table 10. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 96% identical to a linker sequence selected from Table 8. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 97% identical to a linker sequence selected from Table 8. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids comprising a sequence at least 98% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is at least 99% identical to a linker sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is identical to a linker sequence selected from Table 8.

[0234] Cytokine Receptors The present disclosure provides a fusion protein comprising a cytokine receptor. In some embodiments, the cytokine receptor is or comprises a proinflammatory (M1) cytokine receptor. In some embodiments, the proinflammatory cytokine receptor is or comprises IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, TNFR1, TNFR2, IL-1R1, IL-1R3, IL-6Rα, gp130, IL-12Rβ1, IL-12Rβ2, IL-17RA, IL-17RB, IL-17RC, IL-23R, CSF2-Rα, or CSF2-Rβ. In some embodiments, the cytokine receptor is or comprises an anti-inflammatory (M2) cytokine receptor. In some embodiments, the anti-inflammatory cytokine receptor is or includes IL-4Rα, IL-4Rα1, IL-2Rγc, IL-10R1, IL-10R2, IL-13Rα1, IL-18Rα, IL-18Rβ, CSF1-R, TGF-βR1, or TGF-βR2. In some embodiments, the cytokine receptor is or comprises IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, TNFR1, TNFR2, IL-1R1, IL-1R3, IL-6Rα, gp130, IL-12Rβ1, IL-12Rβ2, IL-17RA, IL-17RB, IL-17RC, IL-23R, CSF2-Rα, CSF2-Rβ, IL-4Rα, IL-4Rα1, IL-2Rγc, IL-10R1, IL-10R2, IL-13Rα1, IL-18Rα, IL-18Rβ, CSF1-R, TGF-βR1, or TGF-βR2.

[0235] In some embodiments, the fusion proteins of the invention comprise a cytokine receptor that comprises the same linear amino acid sequence as an endogenous cytokine (e.g., a sequence in Tables 3a and 3b). In some embodiments, the fusion proteins of the invention comprise a cytokine receptor that comprises an engineered amino acid sequence. In some embodiments, the engineered cytokine amino acid sequence is derived from an endogenous cytokine sequence.

[0236] Many cytokines, including IFN-β and IL-10, signal through heterodimeric receptor complexes. In some embodiments, the fusion proteins of the present disclosure include a cytokine and either cytokine receptor subunit (see Table 1). In some embodiments, the cytokine of the present disclosure binds to its tethered receptor subunit (the receptor subunit included with the cytokine in the fusion protein) and then attracts a second endogenously expressed receptor subunit. For example, a fusion protein including IFN-β and IFNAR-1 will attract endogenous IFNAR-2. Alternatively, a fusion protein including IFN-β and IFNAR-2 will attract endogenous IFNAR-1. In some embodiments, the fusion proteins of the present disclosure include a cytokine and both receptor subunits (e.g., a cytokine selected from Table 1 and both receptor 1 and receptor 2 in the same row of Table 1). In some embodiments, the fusion proteins of the present disclosure including a cytokine and both receptor subunits further include a cleavable linker between the receptor subunits.

[0237] In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the present disclosure comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 3a or Table 3b. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is identical to a sequence selected from Table 3a or Table 3b.

[0238] In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 80% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 85% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 90% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 95% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 96% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the present disclosure are encoded by one or more nucleic acids comprising a sequence at least 97% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids that include a sequence that is at least 98% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids that include a sequence that is at least 99% identical to a sequence selected from Table 5a or Table 5b. In some embodiments, the fusion proteins of the disclosure are encoded by one or more nucleic acids that include a sequence that is identical to a sequence selected from Table 5a or Table 5b.

[0239] Signal peptide In some embodiments, the fusion protein of the present disclosure comprises a signal peptide. In some embodiments, the fusion protein of the present disclosure comprises a signal peptide at the N-terminus. In some embodiments, the nucleic acid encoding the signal peptide comprises a nucleic acid encoding a signal peptide. In some embodiments, the signal peptide is or comprises a human signal peptide. In some embodiments, the signal peptide of the present disclosure is derived from a membrane-expressed or membrane-secreted protein. In some embodiments, the signal peptide of the present disclosure is derived from a membrane-expressed or membrane-secreted cytokine. In some embodiments, the signal peptide of the present disclosure is or comprises any signal peptide that results in cell membrane localization of the variant protein. In some embodiments, the signal peptide comprises a CD8 signal peptide.

[0240] In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 80% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 85% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 90% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 95% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 96% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 97% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 98% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is at least 99% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure comprises an amino acid sequence that is identical to a signal peptide sequence selected from Table 8.

[0241] In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 80% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 85% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 90% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 95% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 96% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 97% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 98% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is at least 99% identical to a signal peptide sequence selected from Table 8. In some embodiments, a fusion protein of the disclosure is encoded by one or more nucleic acids that include a sequence that is at least 99% identical to a signal peptide sequence selected from Table 8.

[0242] target immune cells The present disclosure provides, inter alia, immune cells in a subject that can be modified by the methods described herein.

[0243] The disclosure provides, among other things, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject, where the subject is administered a composition described herein comprising (a) one or more nucleic acid molecules encoding, at least in part, a CAR, a fusion protein, and / or a peptide agent, and (b) a delivery vehicle. Thus, in some embodiments, after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) to generate a modified immune cell comprising a CAR, a fusion protein, and / or a peptide agent. In some embodiments, the modified immune cell comprising a CAR, a fusion protein, and / or a peptide agent has target effector activity.

[0244] In some embodiments, the immune cell comprising the fusion protein comprises a cytokine (e.g., a cytokine described herein), a linker (e.g., a linker described herein), and a cytokine receptor (e.g., a cytokine receptor described herein), where the cytokine binds to the cytokine receptor (see Figures 3A and 4A). In some embodiments, the immune cell further comprises at least one chimeric antigen receptor (CAR) (see Figures 3B and 4B). Thus, in some embodiments, 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).

[0245] In some embodiments, the immune cell populations described herein comprise stem cells, monocytes, macrophages, and / or dendritic cells.

[0246] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) that comprise one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit increased survival compared to modified immune cells of the same type that comprise, at least in part, one or more unmodified nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent.

[0247] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit increased expression of one or more nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent provided herein compared to a modified immune cell of the same type comprising, at least in part, one or more unmodified nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent.

[0248] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) that comprise one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit increased CAR, fusion protein, and / or peptide agent expression compared to modified immune cells of the same type that comprise, at least in part, one or more unmodified nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent.

[0249] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit extended lifespan of the one or more nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent provided herein compared to a modified immune cell of the same type comprising, at least in part, one or more unmodified nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent provided herein.

[0250] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit extended longevity of the CAR, fusion protein, and / or peptide agent compared to modified immune cells of the same type comprising, at least in part, one or more unmodified nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent.

[0251] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) that comprise one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit increased effector activity compared to a modified immune cell of the same type that comprises one or more unmodified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent.

[0252] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) that comprise one or more modified nucleic acid molecules, at least in part, encoding a CAR, fusion protein, and / or peptide agent provided herein, exhibit increased pro-inflammatory (M1) polarization compared to modified immune cells of the same type that comprise, at least in part, one or more unmodified nucleic acid molecules encoding a CAR, fusion protein, and / or peptide agent.

[0253] In some embodiments, the 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, the activated immune cells exhibit altered gene expression, e.g., induction of pro-inflammatory gene expression (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 inactive cells. In some embodiments, the activated immune cells exhibit altered gene expression, e.g., induction of anti-inflammatory gene expression, e.g., compared to inactive cells. In certain embodiments, the activated immune cells undergo cell division. In some embodiments, the target effector activity of the immune cells is enhanced by inhibition of CD47 and / or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating the immune cells with anti-CD47 or anti-SIRPα antibodies, or by any method known to one of skill in the art.

[0254] Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject described herein are treated with a pro-inflammatory agent. In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject described herein are administered a composition described herein in combination with treatment with a pro-inflammatory agent. In some embodiments, a composition of the present disclosure is administered to a subject substantially simultaneously with, before, or after treatment with a pro-inflammatory agent. In some embodiments, treatment with a pro-inflammatory agent increases the anti-tumor activity of an immune cell described herein. In some embodiments, treatment with a pro-inflammatory agent promotes a pro-inflammatory (i.e., M1) phenotype in an immune cell described herein (e.g., a switch from an anti-inflammatory (M2) phenotype to a pro-inflammatory (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).

[0255] Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject described herein are treated with an anti-inflammatory agent. In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in a subject described herein are administered a composition described herein in combination with treatment with a pro-inflammatory agent. In some embodiments, a composition of the present disclosure is administered to a subject substantially simultaneously with, before, or after treatment with an anti-inflammatory agent. In some embodiments, treatment with an anti-inflammatory agent promotes an anti-inflammatory (i.e., M2) phenotype in immune cells described herein (e.g., a switch from a pro-inflammatory (M1) phenotype to an anti-inflammatory (M2) phenotype). In some embodiments, the anti-inflammatory agent includes or is an IL-10 agonist. In some embodiments, the anti-inflammatory agent includes or is a TGFβ agonist.

[0256] 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 can perform at least three distinct antitumor functions: 1) phagocytosis of dead or dying cells, microorganisms, cancer cells, cell debris, or other foreign bodies, 2) cytotoxicity against tumor cells, and 3) presentation of tumor antigens to orchestrate adaptive antitumor immune responses.

[0257] Accumulating evidence suggests that macrophages are abundant in the tumor microenvironment of numerous cancers and can assume 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 contribute to a 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 are unable to induce endogenous TAMs to switch to M1-type cells and subvert to M2 would greatly improve antitumor immunotherapy, which represents a significant advance in the field.

[0258] 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 different populations that can be identified by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0259] 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, GM-CSF, and polarized into proinflammatory (i.e., M1) macrophages. Generally, proinflammatory (M1) macrophages are associated with proinflammatory immune responses such as Th1 and Th17 T cell responses. Exposure to other stimuli can polarize macrophages into a variety of "alternatively activated" macrophage populations or anti-inflammatory (i.e., M2) macrophage populations.

[0260] In some embodiments, the macrophage comprises or is a proinflammatory (M1) macrophage. In some embodiments, the macrophage expresses one or more markers of a proinflammatory (M1) macrophage (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, seven, eight, nine, ten, eleven ...

[0261] In some embodiments, modified macrophages in a subject administered a composition described herein exhibit 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., In some embodiments, modified macrophages in a subject administered a composition described herein stimulate an immune response and / or inflammation, e.g., as compared to macrophages in a subject that has not been administered a composition described herein.

[0262] In some embodiments, the macrophages include 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, the modified macrophages in a subject administered a composition described herein reduce an immune response in the subject. 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, modified macrophages in a subject administered a composition 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 in a subject that has not been administered a composition described herein.

[0263] In some embodiments, modified macrophages in a subject administered a composition described herein comprise at least one upregulated pro-inflammatory (M1) marker and / or at least one downregulated anti-inflammatory (M2) marker compared to macrophages in a subject not administered a composition described herein and / or the same macrophages in a subject prior to administration of a composition described herein. In some embodiments, at least one pro-inflammatory (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 the modified macrophages. In some embodiments, at least one anti-inflammatory (M2) marker (e.g., CD206, CD163, and / or CD209) is downregulated in the modified macrophages.

[0264] In some embodiments, modified macrophages in a subject administered a composition described herein exhibit increased phagocytosis, e.g., compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to macrophages in a subject not administered a composition described herein.

[0265] In some embodiments, modified macrophages in a subject administered a composition described herein exhibit one or both of increased expression of one or more 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) typically associated with increased effector function (e.g., phagocytosis, target cell cytotoxicity, antigen presentation, or cytokine secretion), as compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit increased production of ROS, as compared to, for example, in a subject not administered a composition described herein.In some embodiments, modified macrophages in a subject administered a composition described herein exhibit metabolic reprogramming (e.g., the interferon signaling pathway, the 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, etc.) compared to macrophages in a subject to which a composition described herein was not administered. 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, modified macrophages in a subject administered a composition described herein exhibit an induction of cell survival mechanisms, e.g., compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit an induction of cell death mechanisms, e.g., compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytosis 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), and / or increased proliferation, e.g., compared to macrophages in a subject not administered a composition described herein. In some embodiments, modified macrophages in a subject administered a composition described herein exhibit one, two, three, or four of the following, e.g., improved duration of the CAR, fusion protein, and / or peptide agent, improved stability of the CAR, fusion protein, and / or peptide agent on the cell surface, increased levels of the CAR, fusion protein, and / or peptide agent, and / or decreased background activity of the CAR, fusion protein, and / or peptide agent, compared to macrophages in a subject that has not been administered a composition described herein.

[0266] In some embodiments, modified macrophages in a subject administered a composition described herein reduce one or more signs and / or symptoms of an infection (e.g., of an infectious agent) in the subject, e.g., compared to macrophages in a subject to which a composition described herein has not been administered. In some embodiments, the infectious agent includes 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), an Epstein-Barr virus, a cytomegalovirus, a varicella-zoster virus, a poliovirus, a measles virus, a rubella virus, a Japanese encephalitis virus, a mumps virus, an influenza virus, an adenovirus, an enterovirus, a rhinovirus, a coronavirus (e.g., a severe acute respiratory syndrome (SARS) virus, a Middle East respiratory syndrome (MERS) virus, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), an Ebola virus, a West Nile virus, or a variant or combination thereof.

[0267] In some embodiments, modified macrophages in a subject administered a composition described herein reduce formation of at least one protein aggregate and / or degrade existing aggregates by phagocytosis in a subject (e.g., a subject having a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, an amyloidosis, or a combination thereof), e.g., compared to macrophages in a subject to which a composition described herein is not administered. 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's 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, malignant tumors, 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.

[0268] 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 about 10 to 30 μm in diameter. The nucleus to cytoplasm ratio of monocytes can range from about 2:1 to about 1:1. Monocytes typically contain chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, such as during infection. Monocytes can produce inflammatory cytokines, internalize cells and / or toxic molecules, and differentiate into dendritic cells or macrophages.

[0269] In some embodiments, the monocytes comprise or express one or more phenotypic markers. Examples of phenotypic markers of human monocytic cells include CD9, CD11b, CD11c, CDw12, CD13, 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, CD162 Examples of phenotypic markers of mouse monocytic cells include, but are not limited to, CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD213 2, CDw210, CD226, CD281, CD282, CD284, and CD286. Examples of phenotypic markers of 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, the 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.

[0270] 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, monocytes are differentiated into macrophages by macrophage colony-stimulating factor (M-CSF). Monocytes are differentiated into DCs by granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with IL-4.

[0271] In some embodiments, modified monocytes in a subject administered a composition 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 in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit increased phagocytosis, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit improved survival, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit enhanced differentiation into macrophages (e.g., M1 or M2 macrophages), e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition 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 in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis) and / or increased antigen processing, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to monocytes in a subject not administered a composition described herein.

[0272] In some embodiments, modified monocytes in a subject administered a composition described herein exhibit one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, target cell cytotoxicity, antigen presentation, or cytokine secretion) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, target cell cytotoxicity, antigen presentation, or cytokine secretion), e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit increased production of ROS, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit metabolic reprogramming, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit induction of cell survival mechanisms, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit an induction of cell death mechanisms, e.g., compared to monocytes in a subject not administered a composition described herein. In some embodiments, modified monocytes in a subject administered a composition described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytosis checkpoints, increased expression of chemokine receptors to aid in 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, compared to monocytes in a subject not administered a composition described herein.In some embodiments, modified monocytes in a subject administered a composition described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR, fusion protein, and / or peptide agent expression, improved stability of the CAR, fusion protein, and / or peptide agent on the cell surface, increased levels of CAR, fusion protein, and / or peptide agent expression, or decreased background activity of the CAR, fusion protein, and / or peptide agent, compared to monocytes in a subject that has not been administered a composition described herein.

[0273] Dendritic cells Dendritic cells (DCs) are specialized bone marrow-derived antigen-presenting cells involved in initiating immune responses and maintaining the 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.

[0274] 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.

[0275] Immature DCs may be characterized by a high antigen capture capacity but a relatively low T cell stimulating capacity. Inflammatory mediators promote DC maturation. When DCs reach a mature stage, their properties change dramatically compared to immature DCs, such as a decreased antigen capture capacity and / or an increased ability to stimulate T cells. In some embodiments, the DCs include or are immature DCs. In other embodiments, the DCs include or are mature DCs.

[0276] Without wishing to be bound by theory, it is believed that modification of DC cells in a subject administered a composition described herein can allow mature DCs to simultaneously exhibit increased antigen capture capacity and T cell stimulation, for example, compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein mediate tumor antigen presentation, for example, increase tumor antigen presentation, compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein mediate tumor T cell stimulation, for example, increase T cell stimulation, compared to DCs in a subject not administered a composition described herein.

[0277] In some embodiments, modified DCs in a subject administered a composition 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 in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit increased phagocytosis, e.g., compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit increased tumor antigen presentation (e.g., presentation after phagocytosis), increased antigen processing, increased antigen cross-presentation, increased T cell priming, and / or T cell stimulation, e.g., compared to DCs in a subject not administered a composition described herein.

[0278] In some embodiments, modified DCs in a subject administered a composition described herein exhibit one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit increased production of ROS, e.g., compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit metabolic reprogramming, e.g., compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit induction of cell survival mechanisms, e.g., compared to DCs in a subject not administered a composition described herein.

[0279] In some embodiments, modified DCs in a subject administered a composition described herein exhibit induction of cell death mechanisms, e.g., compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytosis checkpoints, increased expression of chemokine receptors to aid in 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, e.g., compared to DCs in a subject not administered a composition described herein. In some embodiments, modified DCs in a subject administered a composition described herein exhibit one, two, three, or four of the following, e.g., improved duration of CAR, fusion protein, and / or peptide agent expression, improved stability of the CAR, fusion protein, and / or peptide agent on the cell surface, increased levels of CAR, fusion protein, and / or peptide agent expression, or decreased background activity of the CAR, fusion protein, and / or peptide agent, compared to DCs in a subject that has not been administered a composition described herein. Example arrangement

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[0280] Equivalent It should be understood that various changes, modifications, and improvements to the present disclosure will be readily apparent 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 present invention. Accordingly, the foregoing description and drawings are by way of example only, and any inventions described in this disclosure are further described in detail by the following claims.

[0281] One of ordinary skill in the art will understand the typical standards of 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 additional details regarding its practice are hereby incorporated by reference in their entirety.

Claims

1. A composition for generating modified immune cells comprising a chimeric antigen receptor (CAR), the composition being administered to a subject, the composition comprising: (a) one or more nucleic acid molecules encoding, at least in part, said CAR; (b) a delivery vehicle; Including, after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell to generate a modified immune cell comprising the CAR; the immune cells are stem cells, monocytes, macrophages, or dendritic cells in the subject; The composition, wherein the modified immune cells comprising the CAR have targeted effector activity.

2. 1. A composition for treating a disease or disorder in a subject in need thereof, wherein the composition is administered to the subject, the composition comprising: (a) one or more nucleic acid molecules encoding, at least in part, a chimeric antigen receptor (CAR); (b) a delivery vehicle; Including, after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell to generate a modified immune cell comprising the CAR; the immune cells are stem cells, monocytes, macrophages, or dendritic cells in the subject; The composition, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration.

3. The composition of claim 1 or 2, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

4. 3. The composition of claim 1 or 2, wherein the one or more nucleic acid molecules comprise DNA or messenger RNA (mRNA).

5. 3. The composition of claim 1 or 2, wherein the one or more nucleic acid molecules comprise modifications including modified nucleotides, modifications to the 5' untranslated region (UTR), modifications to the 3' UTR, a cap structure, a poly(A) tail, or a combination thereof.

6. The composition of claim 5 , wherein the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA).

7. 6. The composition of claim 5, wherein the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof.

8. The composition of claim 1 or 2, wherein the one or more nucleic acid molecules are purified nucleic acid molecules.

9. 9. The composition of claim 8, wherein the purified nucleic acid molecule is produced by a method comprising silica membrane purification, high performance liquid chromatography (HPLC), Dynabeads, LiCl precipitation, phenol-chloroform extraction, resin-based purification, polyA isolation, RNeasy, or a combination thereof.

10. The composition of claim 1 or 2, wherein one or more nucleic acid molecules are codon-optimized.

11. 11. The composition of claim 10, wherein the one or more nucleic acid molecules are codon-optimized for expression in stem cells, monocytes, macrophages, or dendritic cells.

12. The composition of claim 1 or 2, wherein the composition further comprises an additional payload.

13. 13. The composition of claim 12, wherein the additional payload is or comprises a pathogen recognition receptor agonist, polyinosinic-polycytidylic acid (poly I:C), TLR7 / 8 agonist, CpG oligodeoxynucleotide, NOD-like receptor (NLR) agonist, RIG-I-like receptor (RLR) agonist, C-type lectin receptor (CLR) agonist, cytoplasmic DNA sensing, cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) agonist, interferon-inducible protein 16 (IFI16) agonist, DEAD-box helicase 41 (DDX41) agonist, LRR-binding FLII-interacting protein 1 (LRRFIP1) agonist, absentee in melanoma 2 (AIM2) agonist, aryl hydrocarbon receptor (AhR) ligand, or a combination thereof.

14. The composition of claim 12 , wherein the one or more nucleic acid molecules and the additional payload are encapsulated within the delivery vehicle.

15. 3. The composition of claim 1 or 2, wherein the delivery vehicle is or comprises a liposome, a lipid nanoparticle, a polymer, an adeno-associated virus (AAV) vector, an adenoviral vector, a retroviral vector, or a combination thereof.

16. 16. The composition of claim 15, wherein the liposome or lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, one or more PEG-modified lipids, or a combination thereof.

17. 16. The composition of claim 15, wherein the retroviral vector comprises a lentiviral vector or a gammaretroviral vector.

18. 18. The composition of claim 17, wherein the lentiviral vector is packaged with a Vpx protein.

19. 16. The composition of claim 15, wherein the adenoviral vector comprises an Ad2 vector or an Ad5 vector.

20. 20. The composition of claim 19, wherein the Ad5 vector comprises an Ad5f35 adenoviral vector.

21. 3. The composition of claim 1 or 2, wherein the administration is or includes intraarterial, subcutaneous, intravenous, intradermal, intratumoral, intralymph node, intramedullary, intramuscular, or intraperitoneal delivery.

22. 3. The composition of claim 1 or 2, wherein the targeted effector activity directed against the target cell is or includes phagocytosis, target cell cytotoxicity, antigen presentation, or cytokine secretion.

23. The composition of claim 1 or 2, wherein at least a portion of the one or more nucleic acid molecules encodes a peptide agent.

24. 3. The composition of claim 1 or 2, wherein at least a portion of the one or more nucleic acid molecules encodes a peptide agent that stimulates and / or polarizes the modified immune cells toward a pro-inflammatory phenotype.

25. 24. The composition of claim 23, wherein the peptide agent is or comprises a cytokine, cytokine receptor, chemokine, chemokine receptor, immune ligand, dominant negative receptor, switch receptor, secreted antibody or fragment thereof, transcription factor, angiopoietin receptor, or combinations thereof.

26. 26. The composition of claim 25, wherein after administration of the composition, the one or more nucleic acid molecules are translated in the immune cell to produce the modified immune cell comprising the cytokine, the cytokine receptor, the chemokine, the chemokine receptor, the immune ligand, the dominant negative receptor, the switch receptor, the secreted antibody or fragment thereof, the transcription factor, the angiopoietin receptor, or a combination thereof.

27. 24. The composition of claim 23, wherein the portion of the one or more nucleic acid molecules encoding a CAR and the portion of the one or more nucleic acid molecules encoding a peptide agent are present in the composition in a ratio of approximately 1000:1, 500:1, 250:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 1:250, 1:500, or 1:1000.

28. 28. The composition of claim 27, wherein the portion of the one or more nucleic acid molecules encoding a CAR is present in a first delivery vehicle and the portion of the one or more nucleic acid molecules encoding a peptide agent is present in a second delivery vehicle.

29. 30. The composition of claim 28, wherein the first delivery vehicle is or comprises a first population of lipid nanoparticles or liposomes and the second delivery vehicle is or comprises a second population of lipid nanoparticles or liposomes.

30. 24. The composition of claim 23, wherein the portion of the one or more nucleic acid molecules encoding a CAR and the portion of the one or more nucleic acid molecules encoding a peptide agent are separated by a cleavage peptide.

31. 3. The composition of claim 1 or 2, wherein the composition is administered alone or in combination with one or more therapeutic agents, procedures, or modalities.

32. 3. The composition of claim 1 or 2, wherein the immune cells are mobilized prior to administering the composition to the subject.

33. 33. The composition of claim 32, wherein the immune cells are mobilized by administering G-CSF, GM-CSF, FLT3-ligand, or plerixafor to the subject.

34. (a) one or more nucleic acid molecules encoding, at least in part, a CAR; and (b) a delivery vehicle; A composition comprising:

35. 35. The composition of claim 34, wherein the delivery vehicle comprises at least one targeting moiety.

36. 36. The composition of claim 35, wherein the targeting moiety facilitates passive targeting of the composition to a desired target.

37. 36. The composition of claim 35, wherein the targeting moiety facilitates active targeting of the composition to a desired target.

38. 38. The composition of claim 37, wherein the targeting moiety is or comprises an antibody or any fragment thereof.

39. 39. The composition of claim 38, wherein the antibody or any fragment thereof binds to CD14, CD11b, CD163, CD206, CD33, CD209, or a combination thereof.

40. 36. The composition of claim 35, wherein the targeting moiety is or comprises a small molecule.

41. 36. The composition of claim 35, wherein the targeting moiety is or comprises a specific lipid or hydrophobic element combination.

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