Switch receptors and modified immune cells

Abstract

The present disclosure relates to modified immune cells comprising chimeric switch receptors, and methods of using and making immune cells comprising chimeric switch receptors. The present disclosure also relates to modified immune cells comprising membrane-tethered cytokines, and methods of altering the inflammatory phenotype of a population of cells. The present disclosure provides a system for establishing genetic control over immune cell (e.g., stem cell, macrophage, monocyte, and / or dendritic cell) phenotype using cytokine-based signaling.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application Nos. 63 / 356,336, filed June 28, 2022, 63 / 394,829, filed August 3, 2022, and 63 / 422,524, filed November 4, 2022, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Macrophages are potent modulators of the immune response and can generally adopt either a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. The precise balance of M1 / M2 macrophages is crucial for resolving the body's response to disease and injury, and various diseases involve a dysregulated M1 / M2 phenotype. For example, macrophages in the tumor microenvironment (TME) are often biased toward the tumor-protective M2 phenotype, whereas M1 macrophages in atherosclerotic tissues promote plaque progression.

[0003] Therefore, there is a need to establish engineered immune cells for cell therapy and methods to genetically control, modify, and / or maintain M1 / M2 polarization of immune cells in subjects. Summary of the Invention

[0004] The present disclosure encompasses, inter alia, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising a chimeric switch receptor, as well as methods of making the compositions. The present disclosure also encompasses, inter alia, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising a membrane-tethered cytokine, as well as methods of making the compositions.

[0005] The present disclosure provides a system for establishing genetic control over immune cell (e.g., stem cell, macrophage, monocyte, and / or dendritic cell) phenotype using cytokine-based signaling. The present disclosure provides, among other things, the expression in immune cells of chimeric switch receptors comprising an extracellular domain from one receptor and an intracellular domain from another (i.e., different) receptor, such that the receptor can, for example, convert an anti-inflammatory signal into a pro-inflammatory signal (or vice versa). The present disclosure also provides, among other things, the expression in immune cells of membrane-tethered cytokines comprising a cytokine fused to a membrane tether, such that the cytokine stimulates neighboring cells in trans.

[0006] In one aspect, the disclosure provides a modified immune cell comprising a chimeric switch receptor, wherein the modified immune cell is a stem cell, a macrophage, a monocyte, or a dendritic cell, and wherein the chimeric switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and wherein the extracellular domain is derived from a first receptor, the intracellular domain is derived from a second receptor, and the second receptor is a cytokine receptor (e.g., a second cytokine receptor).

[0007] In another aspect, the disclosure provides a modified immune cell comprising one or more nucleic acids encoding a chimeric switch receptor, wherein the modified immune cell is a stem cell, a macrophage, a monocyte, or a dendritic cell, and the chimeric switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and wherein the extracellular domain is derived from a first receptor, the intracellular domain is derived from a second receptor, and the second receptor is a cytokine receptor (e.g., a second cytokine receptor).

[0008] In some embodiments, the transmembrane domain is derived from a first receptor or a second receptor. In some embodiments, the first receptor is a cytokine receptor (e.g., a first cytokine receptor). In some embodiments, the first cytokine receptor is a receptor for a pro-inflammatory cytokine (e.g., a pro-inflammatory cytokine receptor). In some embodiments, the first cytokine receptor is a receptor for an anti-inflammatory cytokine (e.g., an anti-inflammatory cytokine receptor). In some embodiments, the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the second cytokine receptor is a pro-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is an anti-inflammatory cytokine receptor and the second cytokine receptor is a pro-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is a pro-inflammatory cytokine receptor and the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is selected from Table 1. In some embodiments, the second cytokine receptor is selected from Table 2.

[0009] In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFN-λR1. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFNAR2. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFN-γR1. In some embodiments, the first cytokine receptor is IFNGR1 and the second cytokine receptor is IL10Ra. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is GCSFR. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is TNFR2. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is TREM2. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is MerTK. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is IL10Ra. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor comprises MyD88 and / or CD40. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is IL28R. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is CD30. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor comprises MyD88 and / or CD40.

[0010] In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 3. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 4. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6.

[0011] In some embodiments, the modified immune cells of the disclosure further comprise one or more additional chimeric switch receptors, wherein the one or more additional chimeric switch receptors comprise a combination of extracellular and intracellular domains that differ from the extracellular and intracellular domains of the chimeric switch receptor. In some embodiments, the first cytokine receptor is IL10Ra, the second cytokine receptor comprises MyD88 and CD40, and the one or more additional chimeric switch receptors comprise a TGFbR2 extracellular domain and an IFNAR2 intracellular domain, and a TGFbR1 extracellular domain and an IFNAR1 intracellular domain. In some embodiments, the first cytokine receptor is IL10Ra, the second cytokine receptor is IFNLR1, and the one or more additional chimeric switch receptors comprise a TGFbR2 extracellular domain, a CD40 intracellular domain, and a MyD88 intracellular domain.

[0012] In some embodiments, the modified immune cells of the present disclosure further comprise a chimeric antigen receptor (CAR) and / or a nucleic acid encoding a CAR.

[0013] In another aspect, the present disclosure provides a chimeric switch receptor comprising: (a) an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain, wherein the extracellular domain is derived from a first receptor selected from Table 1, and the intracellular domain is derived from a second receptor selected from Table 2, and wherein the second receptor is a cytokine receptor (e.g., a second cytokine receptor).

[0014] In some embodiments, the transmembrane domain is derived from a first receptor or a second receptor. In some embodiments, the first receptor is a cytokine receptor (e.g., a first cytokine receptor). In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFN-λR1. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFNAR2. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFN-γR1. In some embodiments, the first cytokine receptor is IFNGR1 and the second cytokine receptor is IL10Ra. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is GCSFR. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is TNFR2. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is TREM2. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is MerTK. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is IL10Ra. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor comprises MyD88 and / or CD40. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is IL28R. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is CD30. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor comprises MyD88 and / or CD40.

[0015] In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 3. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 4. In some embodiments, the chimeric switch receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 7.

[0016] In another aspect, the disclosure provides polynucleotides encoding one or more chimeric switch receptors, wherein each chimeric switch receptor comprises (a) an extracellular domain, (b) a transmembrane domain, and (c) an intracellular domain, wherein the extracellular domain is derived from a first receptor selected from Table 1, the intracellular domain is derived from a second receptor selected from Table 2, and wherein the second receptor is a cytokine receptor (e.g., a second cytokine receptor).

[0017] In some embodiments, the transmembrane domain is derived from a first receptor or a second receptor. In some embodiments, the first receptor is a cytokine receptor (e.g., a first cytokine receptor). In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFN-λR1. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFNAR2. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFN-γR1. In some embodiments, the first cytokine receptor is IFNGR1 and the second cytokine receptor is IL10Ra.

[0018] In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is GCSFR. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is TNFR2. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is TREM2. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is MerTK. In some embodiments, the first cytokine receptor is IL17Ra and the second cytokine receptor is IL10Ra. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor comprises MyD88 and / or CD40. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is IL28R. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is CD30. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor comprises MyD88 and / or CD40. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor comprises MyD88 and / or CD40. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor is IFNAR2. In some embodiments, the first cytokine receptor is TGFbR1 and the second cytokine receptor is IFNAR1. In some embodiments, the first cytokine receptor is IL10Ra and the second cytokine receptor is IFNLR1. In some embodiments, the first cytokine receptor is TGFbR2 and the second cytokine receptor comprises MyD88 and / or CD40.

[0019] In some embodiments, the extracellular domain is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, the intracellular domain is encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6. In some embodiments, the polynucleotide comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8.

[0020] In some embodiments, the polynucleotides of the disclosure encode one or more chimeric switch receptors as a single polypeptide chain. In some embodiments, the one or more chimeric switch receptors are separated by one or more cleavage peptide sites. In some embodiments, the one or more cleavage peptide sites are selected from the group consisting of P2A, F2A, E2A, and T2A.

[0021] In another aspect, the present disclosure provides a pharmaceutical composition comprising a modified immune cell of the present disclosure, a chimeric switch receptor of the present disclosure, or a polynucleotide of the present disclosure. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0022] In another aspect, the present disclosure provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present disclosure to the subject, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration. In some embodiments, the administering step is or includes intraarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery. In some embodiments, the method of the present disclosure includes introducing a polynucleotide of the present disclosure into an immune cell. In some embodiments, the polynucleotide comprises DNA or messenger RNA (mRNA).

[0023] In some embodiments, the polynucleotide comprises a modification selected from 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.

[0024] In some embodiments, the polynucleotide is a purified polynucleotide. In some embodiments, the purified polynucleotide 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.

[0025] In some embodiments, the polynucleotide is codon optimized. In some embodiments, the polynucleotide is codon optimized for expression in stem cells, monocytes, macrophages, or dendritic cells.

[0026] In some embodiments, the introducing comprises electroporation or transfection with the polynucleotide.

[0027] In some embodiments, the polynucleotide is encapsulated in a delivery vehicle. In some embodiments, the delivery vehicle is or comprises a liposome, lipid nanoparticle, polymer, adeno-associated virus (AAV) vector, adenoviral vector, 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.

[0028] In some embodiments, the methods of the present disclosure further include introducing an additional payload into the immune cells. In some embodiments, the additional payload is or includes a pathogen recognition receptor agonist, polyinosinic-polycytidylic acid (polyI: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 cytosolic DNA sensing agonist, 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, a melanoma-deficient factor 2 (AIM2) agonist, an aryl hydrocarbon receptor (AhR) ligand, or a combination thereof. In some embodiments, the polynucleotide and additional payload are encapsulated within a delivery vehicle.

[0029] In another aspect, the present disclosure provides a modified immune cell comprising a membrane-tethered cytokine, wherein the modified immune cell is a stem cell, a macrophage, a monocyte, or a dendritic cell, and wherein the membrane-tethered cytokine comprises an extracellular domain and a membrane tether.

[0030] In some embodiments, the extracellular domain is or comprises a pro-inflammatory cytokine. In some embodiments, the extracellular domain is or comprises an anti-inflammatory cytokine. In some embodiments, the extracellular domain is or comprises IFN-β. In some embodiments, the membrane tether is or comprises a B7 transmembrane domain (TMD), a B7 TMD with a matrix metalloproteinase (MMP) linker, a glycosylphosphatidylinositol (GPI) anchor, or a GPI anchor with a CD28 spacer. In some embodiments, the membrane tether can be released from the modified immune cell when the extracellular domain binds to a receptor expressed on another cell. In some embodiments, the modified immune cell of the present disclosure further comprises a chimeric antigen receptor (CAR).

[0031] In another aspect, the present disclosure provides a method of altering the inflammatory phenotype of a population of cells, the method comprising contacting the population of cells with a modified immune cell of the present disclosure. In some embodiments, the population of cells comprises macrophages, monocytes, dendritic cells, T cells, NK cells, or a combination thereof. In some embodiments, the inflammatory phenotype of the population of cells is altered from an anti-inflammatory to a non-activated state. In some embodiments, the inflammatory phenotype of the population of cells is altered from a pro-inflammatory to a non-activated state. In some embodiments, the inflammatory phenotype of the population of cells is altered from an anti-inflammatory to a pro-inflammatory state. In some embodiments, the inflammatory phenotype of the population of cells is altered from a pro-inflammatory to an anti-inflammatory state.

[0032] The drawings are for purposes of illustration only and not limitation. [Brief explanation of the drawings]

[0033] [Figure 1] An exemplary wild-type (WT) receptor and a switch receptor for converting extracellular M2 signals to intracellular M1 signaling are shown.

[0034] [Figure 2] An exemplary experimental timeline of switch receptor transduction into primary macrophages, IL10 treatment, and cell marker and cytokine expression analysis is shown.

[0035] [Figure 3A] An exemplary flow cytometry gating strategy is shown when assessing whole cells, singlets, live cells, and untreated cells (UTD) for construct expression. [Figure 3B] An exemplary flow cytometry gating strategy is shown for assessing SR-expressing cells for whole cells, singlets, live cells, and construct expression. [Figure 3C] An exemplary flow cytometry gating strategy is shown when assessing CAR-expressing cells for whole cells, singlets, live cells, and construct expression.

[0036] [Figure 4A] Exemplary phenotypic markers, as measured by flow cytometry, are shown for UTD, SR-expressing, and CAR-expressing cells 24 hours after treatment with IL10 at concentrations of 0.1, 1, and 10 ng / mL. Expression of M2 markers (CD163) and M1 markers (CD86, CD40, HLA-DR) was assessed. [Figure 4B] Exemplary phenotypic markers, as measured by flow cytometry, are shown for UTD, SR-expressing, and CAR-expressing cells 48 hours after treatment with IL10 at concentrations of 0.1, 1, and 10 ng / mL. Expression of M2 markers (CD163) and M1 markers (CD86, CD40, HLA-DR) was assessed. [Figure 4C]Exemplary phenotypic markers, as measured by flow cytometry, are shown for UTD, SR-expressing, and CAR-expressing cells 72 hours after treatment with IL10 at concentrations of 0.1, 1, and 10 ng / mL. Expression of M2 markers (CD163) and M1 markers (CD86, CD40, HLA-DR) was assessed.

[0037] [Figure 5] Exemplary cytokine levels in culture supernatants, as measured by MSD, are shown for UTD, SR-expressing, and CAR-expressing cells 72 hours after IL10 treatment at concentrations of 0.1, 1, and 10 ng / mL. TNFα and IL6 expression were assessed.

[0038] [Figure 6] Exemplary cytokine levels in culture supernatants are shown, as measured by MSD in SR-expressing cells and normalized to UTD cells, at different MOIs and under different cytokine treatment conditions. IL6, TNFα, and IL4 expression were assessed.

[0039] [Figure 7] Exemplary cytokine levels in culture supernatants are shown, as measured by MSD in SR-expressing cells and normalized to UTD cells, at different MOIs and under different cytokine treatment conditions. IL13, IL12p70, and IL1β expression were assessed.

[0040] [Figure 8] An exemplary experimental timeline of switch receptor transduction into primary human monocytes, GM-CSF or M-CSF stimulation for differentiation, IL10 treatment, and cell marker analysis is shown.

[0041] [Figure 9A] An exemplary flow cytometry gating strategy is shown for evaluating GM-CSF stimulated differentiated UTD and SR-expressing cells for whole cells, singlets, live cells, and construct expression. [Figure 9B]An exemplary flow cytometry gating strategy is shown for evaluating M-CSF stimulated differentiated UTD and SR-expressing cells for total cells, singlets, live cells, and construct expression.

[0042] [Figure 10A] Representative phenotypic markers, as measured by flow cytometry, in UTD and SR-expressing cells from N=3 donors were shown. The cells were differentiated with GM-CSF stimulation and treated with or without IL10. Expression of M2 markers (CD163, CD206) and M1 markers (CD86, CD40, HLA-DR) was assessed. [Figure 10B] Representative phenotypic markers, as measured by flow cytometry, are shown for UTD and SR-expressing cells from N=3 donors. The cells were differentiated with M-CSF and treated with or without IL10. Expression of M2 markers (CD163, CD206) and M1 markers (CD86, CD40, HLA-DR) was assessed.

[0043] [Figure 11] An exemplary experimental timeline of switch receptor mRNA transduction into mouse bone marrow-derived macrophages (BMDMs), M-CSF stimulation for differentiation, IL10 treatment, and cell marker analysis is shown.

[0044] [Figure 12A] 1 shows an exemplary flow cytometry gating strategy when assessing whole cells, singlets, live cells, and mock electroporated cells for construct expression. [Figure 12B] An exemplary flow cytometry gating strategy is shown for assessing whole cells, singlets, live cells, and cells electroporated with 100 nM mRNA encoding SR for construct expression. [Figure 12C]An exemplary flow cytometry gating strategy is shown for assessing whole cells, singlets, live cells, and cells electroporated with 300 nM mRNA encoding SR for construct expression.

[0045] [Figure 13] Exemplary phenotypic markers are shown as measured by flow cytometry in mock and mRNA-treated cells treated with or without IL10. M2 marker (CD163) expression and M1 marker (CD86, CD40, IA / IE) expression were assessed.

[0046] [Figure 14] Representative Western blots of STAT phosphorylation in UTD macrophages treated with IL-10 or untreated, lentivirus (LV) control macrophages, and IL-10 switch receptor (SR) macrophages are shown.

[0047] [Figure 15] 1 shows exemplary heat maps of cytokine production in untreated untransduced (UTD) macrophages, LV control macrophages, and IL-10 SR macrophages compared to IL-10-treated UTD macrophages, LV control macrophages, and IL-10 macrophages.

[0048] [Figure 16] 1 shows exemplary graphs of quantified cytokine expression of TNFα, IL-6, IP-10, FLT3L, IL-15, and TRAIL in untreated untransduced (UTD) macrophages, LV control macrophages, and IL-10 SR macrophages compared to IL-10-treated UTD macrophages, LV control macrophages, and IL-10 macrophages.

[0049] [Figure 17]Exemplary phenotypic markers are shown as measured by flow cytometry in control M0, M2A, and M2C bystander cells. CD80, CD86, CD163, CD206, CD40, and HLA-DR expression was assessed.

[0050] [Figure 18] An exemplary experimental timeline for the preparation and co-culture of UTD effector cells, CAR-expressing effector cells, or SR-expressing effector cells, and M0, M2A, and M2C bystander cells is shown.

[0051] [Figure 19] FIG. 1 shows an exemplary flow cytometry gating strategy for separating effector and bystander cells in firefly co-cultures by fluorescence.

[0052] [Figure 20] Exemplary phenotypic markers as measured by flow cytometry are shown for UTD, CAR-, and SR-expressing effector cells grown alone (monoculture) or cocultured with M0, M2A, or M2C bystander cells, with or without IL10. CD86 and CD163 expression was assessed.

[0053] [Figure 21-1] Exemplary phenotypic markers as measured by flow cytometry are shown for M0, M2A, and M2C bystander cells cocultured with UTD effector cells, CAR-expressing effector cells, or SR-expressing effector cells with or without IL10. CD86, CD163, CD206, and PD1 expression were assessed. [Figure 21-2] Exemplary phenotypic markers as measured by flow cytometry are shown for M0, M2A, and M2C bystander cells cocultured with UTD effector cells, CAR-expressing effector cells, or SR-expressing effector cells with or without IL10. CD86, CD163, CD206, and PD1 expression were assessed.

[0054] [Figure 22] Exemplary heatmaps are shown for assessing bystander macrophage phenotype under various conditions. One plot was generated per effector cell construct (CAR or SR), per M2 bystander cell type (M2A or M2C), and per cytokine treatment condition (with or without IL10). Each row represents a marker, and each column represents a flow cytometry time point (day 1, day 3, or day 5). Red squares indicate significant changes in bystander marker expression compared to bystanders cocultured with UTD effector cells under the same conditions.

[0055] [Figure 23A] An exemplary flow cytometry gating strategy is shown when assessing construct expression in UTD macrophages and CAR-expressing macrophages. [Figure 23B] Exemplary flow cytometry gating strategies for assessing construct expression in UTD- and SR-expressing macrophages are shown. SR construct variants contain intracellular domains derived from IFN-λR1, IFNAR2, IFN-γR1, or STAT1min.

[0056] [Figure 24] Exemplary phenotypic markers as measured by flow cytometry on UTD macrophages, CAR-expressing macrophages, and SR-expressing macrophages are shown. M2 marker (CD163) expression and M1 marker (CD86) expression were assessed.

[0057] [Figure 25] An exemplary wild-type (WT) receptor and a switch receptor for converting extracellular M1 signals to intracellular M2 signaling are shown.

[0058] [Figure 26]Exemplary flow cytometry gating strategies for assessing construct expression in UTD and SR-expressing macrophages are shown. SR construct variants contain intracellular domains derived from IL10Rα or STAT3min.

[0059] [Figure 27] Exemplary phenotypic markers as measured by flow cytometry on UTD macrophages, CAR-expressing macrophages, and SR-expressing macrophages are shown. M2 marker (CD163) expression and M1 marker (CD86) expression were assessed.

[0060] [Figure 28] 1 shows an exemplary experimental timeline for preparing macrophages containing switched receptors from monocytes differentiated with GM-CSF or M-CSF.

[0061] [Figure 29A-1] 1 shows an exemplary flow cytometry gating strategy when assessing total cells, singlets, live cells, and GM-CSF-differentiated UTD and SR cells for IFNGR expression. [Figure 29A-2] 1 shows an exemplary flow cytometry gating strategy when assessing total cells, singlets, live cells, and GM-CSF-differentiated UTD and SR cells for IFNGR expression. [Figure 29B-1] 1 shows an exemplary flow cytometry gating strategy when assessing M-CSF-differentiated UTD and SR-expressing cells for total cells, singlets, live cells, and IFNGR expression. [Figure 29B-2] 1 shows an exemplary flow cytometry gating strategy when assessing M-CSF-differentiated UTD and SR-expressing cells for total cells, singlets, live cells, and IFNGR expression.

[0062] [Figure 30A]Figure 1 shows exemplary graphs of phenotypic markers as measured by flow cytometry in GM-CSF-differentiated UTD macrophages and SR-expressing monocyte-derived macrophages. Expression of M2 marker (CD163) and M1 marker (CD86) was assessed. [Figure 30B] Figure 1 shows exemplary graphs of phenotypic markers as measured by flow cytometry in UTD macrophages and SR-expressing monocyte-derived macrophages differentiated with M-CSF. Expression of M2 marker (CD163) and M1 marker (CD86) was assessed. [Figure 30C] Figure 1 shows exemplary graphs of phenotypic markers as measured by flow cytometry in UTD macrophages and SR-expressing monocyte-derived macrophages differentiated with GM-CSF or M-CSF. Expression of M2 marker (CD163) and M1 marker (CD86) was assessed.

[0063] [Figure 31A] 1 shows exemplary heat maps of cytokine production in naive, untransduced (UTD) macrophages and IFNγ SR monocyte-derived macrophages differentiated with GM-CSF compared to IFNγ-treated UTD macrophages and IFNγ SR monocyte-derived macrophages differentiated with GM-CSF. [Figure 31B] 1 shows exemplary heat maps of cytokine production in naive, untransduced (UTD) macrophages and IFNγ SR monocyte-derived macrophages differentiated with M-CSF compared to IFNγ-treated UTD macrophages and IFNγ SR monocyte-derived macrophages differentiated with M-CSF.

[0064] [Figure 32A] 1 shows exemplary graphs of cytokine production in naive, untransduced (UTD) macrophages and IFNγ SR monocyte-derived macrophages differentiated with GM-CSF compared to IFNγ-treated UTD macrophages and IFNγ SR monocyte-derived macrophages differentiated with GM-CSF. [Figure 32B] 1 shows exemplary graphs of cytokine production in naive, untransduced (UTD) macrophages and IFNγ SR monocyte-derived macrophages differentiated with M-CSF compared to IFNγ-treated UTD macrophages and IFNγ SR monocyte-derived macrophages differentiated with M-CSF.

[0065] [Figure 33] An exemplary wild-type (WT) receptor and a switch receptor for converting extracellular M1 signals (IL17) to intracellular M2 signaling are shown.

[0066] [Figure 34-1] Exemplary flow cytometry gating strategies for assessing construct expression in UTD, CAR, and SR-expressing macrophages are shown. SR construct variants contain different intracellular and transmembrane domains. [Figure 34-2] Exemplary flow cytometry gating strategies for assessing construct expression in UTD, CAR, and SR-expressing macrophages are shown. SR construct variants contain different intracellular and transmembrane domains.

[0067] [Figure 35] Illustrated are exemplary graphs of phenotypic markers as measured by flow cytometry in UTD macrophages, LV control macrophages, and SR-expressing macrophages. M2 marker (CD163) expression and M1 marker (CD86) expression were assessed.

[0068] [Figure 36] 1 shows an exemplary experimental timeline for preparing macrophages containing switched receptors from monocytes differentiated with GM-CSF or M-CSF.

[0069] [Figure 37A-1]An exemplary flow cytometry gating strategy is shown when assessing GM-CSF-differentiated UTD and SR cells for total cells, singlets, live cells, and IL17RA expression. [Figure 37A-2] An exemplary flow cytometry gating strategy is shown when assessing GM-CSF-differentiated UTD and SR cells for total cells, singlets, live cells, and IL17RA expression. [Figure 37B-1] An exemplary flow cytometry gating strategy is shown when assessing M-CSF-differentiated UTD and SR-expressing cells for total cells, singlets, live cells, and IL17RA expression. [Figure 37B-2] An exemplary flow cytometry gating strategy is shown when assessing M-CSF-differentiated UTD and SR-expressing cells for total cells, singlets, live cells, and IL17RA expression.

[0070] [Figure 38A] Figure 1 shows exemplary graphs of phenotypic markers as measured by flow cytometry in GM-CSF-differentiated UTD macrophages and SR-expressing monocyte-derived macrophages. Expression of M2 marker (CD163) and M1 marker (CD86) was assessed. [Figure 38B] Figure 1 shows exemplary graphs of phenotypic markers as measured by flow cytometry in UTD macrophages and SR-expressing monocyte-derived macrophages differentiated with M-CSF. Expression of M2 marker (CD163) and M1 marker (CD86) was assessed. [Figure 38C] Figure 1 shows exemplary graphs of phenotypic markers as measured by flow cytometry in UTD macrophages and SR-expressing monocyte-derived macrophages differentiated with GM-CSF or M-CSF. Expression of M2 marker (CD163) and M1 marker (CD86) was assessed.

[0071] [Figure 39] 1 shows an exemplary membrane-tethered IFNβ construct.

[0072] [Figure 40] Exemplary flow cytometry gating strategies for assessing construct expression in UTD effector cells and membrane-tethered IFNβ-expressing effector cells are shown. Membrane-tethered IFNβ construct variants were tethered to the membrane using either a protein-based anchor or a lipid-based anchor.

[0073] [Figure 41] An exemplary experimental timeline for the preparation and co-culture of UTD effector cells, CAR-expressing effector cells, or membrane-tethered IFNβ-expressing effector cells, and M2A or M2C bystander cells is shown.

[0074] [Figure 42] Exemplary phenotypic markers as measured by flow cytometry are shown for M2A and M2C bystander cells cocultured with UTD effector cells, CAR-expressing effector cells, or membrane-tethered IFNβ-expressing effector cells with or without IL10. CD80, CD86, CD163, and CD206 expression was assessed.

[0075] [Figure 43] Exemplary viability of M2A and M2C bystander cells co-cultured with UTD effector cells, CAR-expressing effector cells, or membrane-tethered IFNβ-expressing effector cells with or without IL10 is shown.

[0076] [Figure 44A] Exemplary endogenous receptors and engineered switch receptors to convert extracellular M2 signals to intracellular M1 signaling through STATs are shown. [Figure 44B] Diverse tumor-derived M2 signals are shown.

[0077] [Figure 45]1 shows an exemplary STAT3 / 1 chimeric molecule for converting a general M2 signal to an M1 DNA program.

[0078] [Figure 46] An exemplary experimental timeline of STAT3 / 1 chimera transduction into primary macrophages, anti-inflammatory cytokine treatment, and cell marker and cytokine expression analysis is shown.

[0079] [Figure 47A] An exemplary flow cytometry gating strategy is shown when assessing UTD cells for whole cells, singlets, live cells, and construct expression. [Figure 47B] 1 shows an exemplary flow cytometry gating strategy when assessing chimeric STAT3 / 1 expressing cells for whole cells, singlets, live cells, and construct expression.

[0080] [Figure 48] An exemplary experimental timeline for the preparation and co-culture of UTD effector cells, CAR-expressing effector cells, or chimeric STAT3 / 1-expressing effector cells, and M2A bystander cells is shown.

[0081] [Figure 49A] Exemplary phenotypic markers, as measured by flow cytometry, are shown for effector cells in monoculture and coculture experiments under various anti-inflammatory cytokine treatment conditions, respectively. CD80, CD86, and CD163 expression was assessed. [Figure 49B] Exemplary phenotypic markers, as measured by flow cytometry, are shown in bystander cells under various anti-inflammatory cytokine treatment conditions, in monoculture and coculture experiments, respectively. CD80, CD86, and CD163 expression was assessed.

[0082] [Figure 50]An exemplary experimental timeline of switch receptor transduction into primary macrophages, TGFβ treatment, and cell marker and cytokine expression analysis is shown.

[0083] [Figure 51A] An exemplary flow cytometry gating strategy is shown when assessing UTD cells for whole cells, singlets, live cells, and construct expression. [Figure 51B] An exemplary flow cytometry gating strategy is shown for assessing SR-expressing cells for whole cells, singlets, live cells, and construct expression.

[0084] [Figure 52A] Exemplary flow cytometry gating strategies are shown for assessing construct expression in UTD macrophages. SR construct variants include the intracellular domain of ΔICD (dominant negative, i.e., mutated catalytic domain), CD40-Myd88, Myd88-CD40, Myd88, IFNλR1, or CD30. [Figure 52B] Exemplary flow cytometry gating strategies for assessing construct expression in SR-expressing macrophages are shown. SR construct variants include the intracellular domain of ΔICD (dominant negative, i.e., mutated catalytic domain), CD40-Myd88, Myd88-CD40, Myd88, IFNλR1, or CD30. [Figure 52C] Exemplary flow cytometry gating strategies for assessing construct expression in SR-expressing macrophages are shown. SR construct variants include the intracellular domain of ΔICD (dominant negative, i.e., mutated catalytic domain), CD40-Myd88, Myd88-CD40, Myd88, IFNλR1, or CD30.

[0085] [Figure 53]Exemplary TGFβ cytokine levels in culture supernatants as measured by ELISA in UTD cells and cells expressing different SR construct variants 48 hours after treatment with TGFβ at various concentrations are shown.

[0086] [Figure 54A] Exemplary phenotypic markers, as measured by flow cytometry and cytokine / chemokine supernatant levels, in UTD cells and cells expressing different SR construct variants are shown. CD80 expression was assessed. Data are presented as absolute values. [Figure 54B] Exemplary phenotypic markers, as measured by flow cytometry and cytokine / chemokine supernatant levels, are shown in UTD cells and cells expressing different SR construct variants. CD80 expression was assessed. Data are presented normalized to the UTD control. [Figure 54C] Exemplary phenotypic markers, as measured by flow cytometry and supernatant levels of cytokines / chemokines, in UTD cells and cells expressing different SR construct variants are shown. TNFα, IFNγ, IL12p70, and eotaxin cytokines / chemokines were assessed. Data are expressed as absolute values. [Figure 54D] Exemplary phenotypic markers, as measured by flow cytometry and cytokine / chemokine supernatant levels, are shown in UTD cells and cells expressing different SR construct variants. TNFα, IFNγ, IL12p70, and eotaxin cytokines / chemokines were assessed. Data are normalized to UTD control.

[0087] [Figure 55] An exemplary experimental timeline of switch receptor transduction into primary macrophages, IL10 treatment, and cell marker and cytokine expression analysis is shown.

[0088] [Figure 56] Figure 1 shows exemplary phenotypic marker expression as measured by flow cytometry and cytokine supernatant levels in cells expressing SR constructs at different LV volumes with or without IL10 treatment. CD80 marker expression and MIP-1β and TNFα cytokine levels were assessed. NT means no treatment (i.e., cells were not treated with IL10).

[0089] [Figure 57] An exemplary means by which multiple SRs can be combined in a single cell to detect both IL10 and TGFβ is shown.

[0090] [Figure 58] An exemplary experimental timeline of switch receptor transduction into primary macrophages, IL10 and / or TGFβ treatment, and cell marker and cytokine expression analysis is shown.

[0091] [Figure 59] Exemplary % viability and SR expression as measured by flow cytometry in cells expressing SR constructs at different LV volumes are shown.

[0092] [Figure 60] Figure 1 shows exemplary phenotypic marker expression as measured by flow cytometry and cytokine supernatant levels in cells expressing SR constructs at different LV volumes, with or without IL10 and / or TGFβ treatment. CD80 marker expression and MIP-1β and IP-10 cytokine levels were assessed. NT means no treatment (i.e., cells were not treated with IL10). DETAILED DESCRIPTION OF THE INVENTION

[0093] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of these and other terms are also set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide further detail regarding its practice are incorporated herein by reference.

[0094] 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.

[0095] Approximately or About: As used herein, the term "approximately" or "about," when applied to one or more values of interest, refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from the context, the term "approximately" or "about" refers to a range of values that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (above or below) of the stated reference value (except where such number exceeds 100% of the possible values).

[0096] Activated: As used herein, the term "activated" refers to the state of a cell, e.g., a monocyte, macrophage, or dendritic cell, that has been sufficiently stimulated to induce detectable cell proliferation or to exert its effector function. Activation may also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, and / or antigen processing and presentation.

[0097] Activated Monocyte / Macrophage / Dendritic Cell: As used herein, the term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a monocyte / macrophage / dendritic cell that is undergoing cell division or exerting an effector function. The term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a cell that is performing an effector function or exhibiting some activity not found in the resting state, including phagocytosis, cytokine secretion, proliferation, changes in gene expression, changes in metabolism, and other functions.

[0098] Drug: As used herein, the term "drug" (or "biological agent" or "therapeutic agent") refers to a molecule that can be expressed, released, secreted, or delivered to a target by the modified cells described herein. Drugs include, but are not limited to, nucleic acids, antibiotics, anti-inflammatory agents, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, proteins (e.g., RNAse inhibitors), peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. Drugs can bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on the target or target cell. Drugs can diffuse or be transported into the cell and act within the cell.

[0099] Antibody: As used herein, the term "antibody" refers to a polypeptide containing sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As known in the art, intact antibodies, as produced in nature, are tetrameric agents of approximately 150 kD, composed of two identical heavy chain polypeptides (approximately 50 kD each) and two identical light chain polypeptides (approximately 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each approximately 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (located at the base tip of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain consists of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch." An intact antibody tetramer consists of two heavy-light chain dimers, in which the heavy and light chains are linked to each other by one disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to form a tetramer. Naturally produced antibodies are typically glycosylated in the CH2 domain. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold," formed by two beta sheets (e.g., three-, four-, or five-stranded sheets) packed together into an antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a natural antibody folds, the FR regions form beta sheets, providing a structural framework for the domain, and the CDR loop regions of both the heavy and light chains join in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, e.g., effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention (e.g., as components of chimeric switch receptors or CARs) comprise a glycosylated Fc domain, e.g., an Fc domain with modified or engineered glycosylation. In some embodiments, any polypeptide, or complex of polypeptides, that comprises 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., by an animal in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, as used herein, the term "antibody" can refer, in appropriate embodiments (unless otherwise specified or clear from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative embodiments.For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), single-chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced in nature. In some embodiments, the antibody can include a covalent modification (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0100] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may contain one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may contain one or more antibody sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative embodiments.For example, in some embodiments, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), small modular antibodies (e.g., IgG, IgE, IgM, IgA, IgB, IgC, IgD, IgE ... ImmunoPharmaceuticals ("SMIPs™"), single-chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody agent may lack covalent modifications (e.g., glycan attachment) that it would have if produced in nature. In some embodiments, an antibody agent can include a covalent modification (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs).In some embodiments, an antibody agent is a polypeptide comprising at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody, or comprises a polypeptide comprising at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are either identical in sequence or contain one to five amino acid substitutions compared to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs are deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR in that one to five amino acids within the included CDR are deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain, or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is not a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain, and / or does not comprise a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent can be or comprise a molecule or composition that does not comprise immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule comprising at least one antigen-binding domain).

[0101] 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.

[0102] 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.

[0103] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0104] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0105] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include either antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen," as the term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be synthetically produced or derived from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or body fluids.

[0106] Anti-tumor effect: As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention in preventing the development of an initial tumor.

[0107] Autologous: As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual.

[0108] Allogeneic: As used herein, the term "allogeneic" refers to any material (e.g., a population of cells) derived from a different animal of the same species.

[0109] Xenogeneic: As used herein, the term "xenogeneic" refers to any material (e.g., a population of cells) derived from an animal of a different species.

[0110] Cancer: As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream or lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. In certain embodiments, the cancer is medullary thyroid cancer.

[0111] Conservative sequence modification: As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies for various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for their ability to bind to antigen using the functional assays described herein.

[0112] Costimulatory Ligand: As used herein, the term "costimulatory ligand" refers to a molecule on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a monocyte / macrophage / dendritic cell, thereby providing a signal that mediates a monocyte / macrophage / dendritic cell response, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages / dendritic cells, including, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0113] Cytotoxic: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging cells. In one embodiment, metabolically enhanced cellular cytotoxicity is improved, e.g., macrophage cytolytic activity is increased.

[0114] 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.

[0115] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity carried out by an immune cell in response to immune cell stimulation. For example, an effector function of a macrophage is to engulf and digest cellular debris, foreign bodies, microorganisms, cancer cells, and other unhealthy cells through phagocytosis.

[0116] Encode: As used herein, "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0117] Endogenous: As used herein, "endogenous" refers to any substance that originates or is produced within a particular organism, cell, tissue, or system.

[0118] 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.

[0119] Expansion: As used herein, the term "expansion" refers to an increase in number, as in increasing the number of cells, e.g., monocytes, macrophages, and / or dendritic cells. In one embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number compared to the number initially present in the culture. In another embodiment, ex vivo expanded monocytes, macrophages, or dendritic cells are increased in number compared to other cell types in the culture. In some embodiments, expansion can occur in vivo. As used herein, the term "ex vivo" refers to cells removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0120] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) generation of an RNA template from the DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0121] Expression Vector: As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0122] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that comprises a distinct portion of a whole, but lacks one or more portions found in the whole structure. In some embodiments, the fragment consists of such a distinct portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., nucleic acid) found throughout the nucleotide sequence. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the nucleotide whole. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the whole.

[0123] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, various algorithms are available that allow for comparison of sequences to determine the degree of homology, including, for example, allowing gaps of a specified length in one sequence relative to another sequence when considering which residues in different sequences "correspond" to each other. Calculating the percent homology between two nucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the reference sequence. Next, nucleotides at corresponding nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position; when a position in the first sequence is occupied by a nucleotide that is similar to the corresponding position in the second sequence, the molecules are similar at that position.The percent homology between two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0124] Identity: As used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, for example, if each position in two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid-long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0125] 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.

[0126] Immune cell: As used herein, the term "immune cell" refers to a cell that is involved in an immune response, for example, promoting an immune response. Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, or B-lymphocytes. The source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0127] Immune response: As used herein, the term "immune response" refers to a cellular and / or systemic response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen.

[0128] Immunoglobulin: As used herein, the terms "immunoglobulin" or "Ig" refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin 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.

[0129] 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.

[0130] Modified: As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0131] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level and / or change in the nature of a response in a subject compared to the level and / or nature of the response in the subject in the absence of the treatment or compound, and / or compared to the level and / or nature of the response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0132] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids include RNA. In some embodiments, nucleic acids are single-stranded. In some embodiments, nucleic acids are double-stranded. In some embodiments, nucleic acids include both single-stranded and double-stranded portions. In some embodiments, nucleic acids include a backbone that includes one or more phosphodiester linkages. In some embodiments, nucleic acids include a backbone that includes both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, nucleic acids can include a backbone that includes one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide linkages, e.g., as in "peptide nucleic acids." In some embodiments, nucleic acids include one or more or all naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, nucleic acids include one or more or all non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide.In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., replication in a recombinant cell or system, in vivo or in vitro, or chemical synthesis). In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length.

[0133] Operably linked: As used herein, the term "operably linked" refers to a functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous, in the same reading frame, and, where necessary, to join two protein-coding regions.

[0134] Overexpressed tumor antigen: As used herein, the term "overexpressed" tumor antigen or "overexpression" of a tumor antigen refers to an abnormal level of expression of the tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0135] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR™, etc., as well as by synthetic means.

[0136] 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 can comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide can include one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, and the like, e.g., combinations thereof. In some embodiments, a polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides.For each such class, the present specification provides, and / or one of skill in the art will be aware of, exemplary polypeptides within the class whose amino acid sequence and / or function are known. In some embodiments, such exemplary polypeptides are reference polypeptides of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (and in some embodiments with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with the reference polypeptide of the class (and in some embodiments, with all polypeptides in the class). For example, in some embodiments, a member polypeptide exhibits an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region that, in some embodiments, is or may contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and often up to 20 or more, amino acids, and in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide, hi some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0137] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a series of at least two amino acids linked together by a peptide bond). A protein can contain moieties other than amino acids (e.g., it can be a glycoprotein, proteoglycan, etc.) and / or can be otherwise processed or modified. Those of skill in the art will understand that a "protein" can be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may include, for example, two or more polypeptide chains linked by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known to those of skill in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0138] Signal Transduction Pathway: As used herein, the term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0139] 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 via 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.

[0140] Specific binding: As used herein, the term "specific binding," with respect to an antigen-binding domain such as an antibody agent, refers to an antigen-binding domain or antibody agent that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antigen-binding domain or antibody agent that specifically binds to an antigen from one species may also bind to antigens from more than one species. However, such cross-species reactivity does not, in itself, alter the classification of the antigen-binding domain or antibody agent as specific. In another example, an antigen-binding domain or antibody agent that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, alter the classification of the antigen-binding domain or antibody agent as specific. In some cases, the terms "specific binding" or "specifically bind" can be used in reference to the interaction of an antigen-binding domain or antibody agent, protein, or peptide with a second chemical species and may mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antigen-binding domain or antibody agent recognizes and binds to a specific protein structure rather than the entire protein. If an antigen-binding domain or antibody agent is specific for epitope "A," then in a reaction involving labeled "A" and an antigen-binding domain or antibody agent, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0141] Stimulation: As used herein, the term "stimulation" refers to a primary response induced by a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR / CD3 complex) binding to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signaling via the Fc receptor mechanism, via a chimeric switch receptor, or via a synthetic CAR. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structure. As used herein, the term "stimulatory molecule" refers to a molecule on a monocyte, macrophage, or dendritic cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. In some embodiments, the stimulatory molecule comprises an FcR extracellular domain comprising a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, FcαRI (CD89), or CD40 domain. In some embodiments, the stimulatory molecule comprises a TLR extracellular domain comprising a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, macrophage, dendritic cell, B cell, etc.) or tumor cell, specifically binds to a cognate binding partner (referred to herein as a "stimulatory molecule") on a monocyte, macrophage, or dendritic cell, thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, Toll-like receptor (TLR) ligands, anti-toll-like receptor antibodies, agonists, and antibodies against monocyte / macrophage receptors. In addition, cytokines such as interferon-gamma are potent stimulators of macrophages.

[0142] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog). In some embodiments, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is afflicted with a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is predisposed to a disease, disorder, or condition; in some embodiments, a predisposed subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or a reference population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit certain symptoms (e.g., clinical symptoms of the disease) or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or therapy is to be administered.

[0143] Substantially purified: As used herein, the term "substantially purified," e.g., when applied to cells, refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous cell population. In other instances, the term simply refers to cells that have been separated from cells with which they are naturally associated in their native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0144] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that is the subject of the provided methods, systems, and / or compositions, e.g., a cell, tissue, organ, or site in the body that is in need of treatment or that is preferentially bound by, for example, an antibody (or fragment thereof) or chimeric switch receptor, i.e., a CAR.

[0145] Target site: As used herein, the term "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0146] T cell receptor: As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, TCRs are composed of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains: a variable domain and a constant domain. In some embodiments, TCRs can be modified on any cell containing a TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0147] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, ameliorating, or eradicating a disease state.

[0148] 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.

[0149] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to the partial or complete alleviation, amelioration, delay in onset, inhibition, prevention, mitigation, and / or reduction in incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who does not exhibit signs or characteristics of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or characteristics of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, or condition. In some embodiments, treatment can include administering to an immune cell (e.g., a monocyte, macrophage, or dendritic cell) or contacting an immune cell with a modulator of a pathway activated by in vitro transcribed mRNA.

[0150] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor can include precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a manifestation of cancer. In some embodiments, a tumor can be a dispersed tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0151] 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. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0152] Throughout this disclosure, various aspects of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range. (Mode for Carrying Out the Invention)

[0153] Macrophages and monocytes are potent modulators of the immune response. Macrophages generally can adopt either a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. The precise balance of M1 / M2 macrophages is important in the body's response to disease and injury, and various diseases involve a dysregulated M1 / M2 phenotype. For example, macrophages in the tumor microenvironment (TME) are often biased toward the tumor-protective M2 phenotype, whereas M1 macrophages in atherosclerotic tissues typically promote plaque progression. Therefore, methods that allow external control over macrophage phenotype, either by repolarizing existing macrophages or by introducing macrophages of the desired phenotype (e.g., introducing M1 macrophages into the TME or M2 macrophages into atherosclerotic tissue), are promising therapeutic strategies.

[0154] Because natural classification systems use cytokines as potent regulators of the M1 / M2 phenotype, engineered cytokine signaling networks represent an attractive system for engineering macrophages. To generate immune cell (e.g., macrophage, monocyte, or dendritic cell) phenotypes distinct from those that would arise under endogenous conditions, an ideal technology would (i) identify the desired M1 / M2 phenotype and (ii) maintain this phenotype in the disease microenvironment, resulting from the use of chimeric switch receptors or membrane-tethered cytokines. The present disclosure provides, among other things, such a technology.

[0155] immune cells The present disclosure provides, among other things, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor described herein. In some embodiments, the modified immune cells of the disclosure comprise a chimeric switch receptor described herein and a chimeric antigen receptor (CAR) described herein. In some embodiments, the modified immune cells of the disclosure comprise a membrane-tethered cytokine described herein. In some embodiments, the modified immune cells of the disclosure comprise a membrane-tethered cytokine described herein and a CAR described herein. In some embodiments, the modified immune cells of the disclosure comprise a chimeric switch receptor described herein, a membrane-tethered cytokine described herein, and a CAR described herein.

[0156] In some embodiments, the immune cell populations described herein comprise stem cells, monocytes, macrophages, dendritic cells, and / or their precursors. In some embodiments, the immune cell populations comprise substantially purified populations or cell lines of stem cells, monocytes, macrophages, or dendritic cells.

[0157] In some embodiments, immune cells are activated, e.g., the immune cells exhibit increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, e.g., compared to inactive cells. In some embodiments, activated immune cells exhibit altered gene expression, e.g., induced pro-inflammatory gene expression, e.g., compared to inactive cells. In some embodiments, activated immune cells exhibit altered gene expression, e.g., induced anti-inflammatory gene expression, e.g., compared to inactive cells. In certain embodiments, activated immune cells undergo cell division. In some embodiments, the target effector activity of immune cells is enhanced by inhibition of CD47 and / or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating immune cells with anti-CD47 or anti-SIRPα antibodies, or by any method known to those of skill in the art.

[0158] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. The immune cells may be autologous or provided from an allogeneic or universal donor. Cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to those of skill in the art, such as Ficoll separation. In some embodiments, cells from a subject's circulating blood are obtained by apheresis or leukapheresis. Cells collected by apheresis can be washed to remove the plasma fraction and resuspended in various buffers (e.g., phosphate-buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g., monocytes) involves plastic adherence. In some embodiments, differentiation of enriched immune cells (e.g., monocytes) involves stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukopheresis composition (e.g., leukopheresis) is used for enrichment. In some embodiments, the leukopheresis composition (e.g., leukopheresis) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g., monocytes) is followed by mobilization with GM-CSF. In certain embodiments, selection of immune cells (e.g., monocytes) involves CD14-positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, immune cell precursors (e.g., precursors of macrophages, monocytes, or dendritic cells, including, but not limited to, induced pluripotent stem cells, or iPSCs) are used in the compositions and methods described herein. Immune cell precursors can be differentiated into immune cells in vivo or ex vivo. Non-limiting examples of immune progenitor cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof.For example, induced pluripotent stem cells can be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSCs) can be derived from normal human tissues such as peripheral blood, fibroblasts, skin, keratinocytes, and renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs can differentiate into myeloid lineages (e.g., monocytes, macrophages, dendritic cells, or their precursors).

[0159] Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein can be isolated from peripheral blood, e.g., by lysing red blood cells and depleting lymphocytes and red blood cells, e.g., by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. Specific subpopulations of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20. In some embodiments, enrichment of immune cell populations, e.g., by negative selection, can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. As non-limiting examples, cell selection can also include negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

[0160] During the isolation of a desired population of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) as described herein by positive or negative selection, the concentration and surface (e.g., particles such as beads) of the immune cells can be varied. To ensure maximum contact area between the cells and beads, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together.

[0161] In some embodiments, prior to administration, the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein) are treated with an inducing agent. In some embodiments, treatment with an inducing agent activates an anti-inflammatory (i.e., M2) phenotype in the immune cells described herein. In some embodiments, the inducing agent comprises or is a pro-inflammatory cytokine. In some embodiments, the inducing agent comprises or is IFN-α, IFN-β, IFN-λ, IFN-γ, TNF-α, IL-6, IL-1b, IL-2, IL-8, IL-12, IL-15, IL-18, IL-17, IL-1a, IL-3, IL-21, IL-33, IL-23, IL-37, or IL-36. In some embodiments, the pro-inflammatory agent comprises or is an IFN-γ agonist (eg, IFN-γ).

[0162] In some embodiments, prior to administration, the immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells (e.g., comprising one or more of a chimeric switch receptor, a membrane-tethered cytokine, and / or a CAR) are treated with an anti-inflammatory agent. In some embodiments, treatment with the anti-inflammatory agent activates a pro-inflammatory (i.e., M1) phenotype in the immune cells described herein. In some embodiments, the anti-inflammatory agent comprises or is an anti-inflammatory cytokine. In some embodiments, the anti-inflammatory agent comprises or is an IL-10, TGF-β, IL-4, IL-13, IL-11, or IL-35. In some embodiments, the anti-inflammatory agent comprises or is an IL-10 agonist (e.g., IL-10).

[0163] In some embodiments, the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising one or more of a chimeric switch receptor, membrane-tethered cytokine, and / or CAR described herein) are administered to a subject in combination with an inducing agent. In some embodiments, the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising one or more of a chimeric switch receptor, membrane-tethered cytokine, and / or CAR described herein) are administered to a subject substantially simultaneously with, before, or after the inducing agent. In some embodiments, administration with the inducing agent activates an anti-inflammatory (i.e., M2) phenotype in the immune cells described herein. In some embodiments, the inducing agent includes or is an IFN-γ agonist (e.g., IFN-γ).

[0164] In some embodiments, the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising one or more of a chimeric switch receptor, membrane-tethered cytokine, and / or CAR described herein) are administered to a subject in combination with an anti-inflammatory agent. In some embodiments, the immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising one or more of a chimeric switch receptor, membrane-tethered cytokine, and / or CAR described herein) are administered to a subject substantially simultaneously with, before, or after the anti-inflammatory agent. In some embodiments, administration with the anti-inflammatory agent increases the anti-tumor activity of the immune cells described herein. In some embodiments, administration with the anti-inflammatory agent activates a pro-inflammatory (i.e., M1) phenotype in the immune cells described herein. In some embodiments, the anti-inflammatory agent includes or is an IL-10 agonist (e.g., IL-10).

[0165] 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, cellular debris, or other foreign bodies, 2) cytotoxicity against tumor cells, and 3) presentation of tumor antigens to orchestrate adaptive antitumor immune responses.

[0166] 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 a greater number of M2-like TAMs, which further contributes to the general suppression of antitumor immune responses. However, recent studies have confirmed that TAMs can be "reprogrammed" via proinflammatory signals and that a switch from an M2 phenotype to a more M1 phenotype is associated with a productive antitumor immune response. Engineering macrophages that induce endogenous TAMs to switch to M1 cells and are unable to subvert to M2 would significantly improve antitumor immunotherapy, representing a significant advance in the field.

[0167] In some embodiments, the macrophages comprise or are undifferentiated or M0 macrophages. In certain embodiments, the macrophages comprise or express one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Upon exposure to various stimuli, M0 macrophages are induced to polarize into several distinct populations that can be identified by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0168] In some embodiments, the macrophages include or are polarized macrophages. Under classical conditions of activation, M0 macrophages can be exposed to proinflammatory signals, such as LPS, IFNγ, or GM-CSF, and polarize into 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 various "alternatively activated" or anti-inflammatory (i.e., M2) macrophage populations.

[0169] In some embodiments, the macrophages comprise or are proinflammatory (M1) macrophages. In some embodiments, the macrophages express one or more markers of proinflammatory (M1) macrophages (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, ten, eleven, twelve, thirteen, thirteen, fourteen, fifteen, six, seven ...

[0170] In some embodiments, macrophages that comprise or express one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein, e.g., produce relatively higher levels of one or more inflammatory cytokines (e.g., one, two, three, four, five, six, seven, or eight of IL-1, TNF, IL-12, IL-18, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-6, IL-8, or IL33) compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. , 8, 9, 10, 11, or 12) or chemokines (e.g., one or both of the CC chemokines or the CXC chemokines) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the CXC chemokines; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the CC chemokines; e.g., one or both of the CXC chemokines). In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein stimulate an immune response and / or inflammation, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0171] In some embodiments, the macrophages include or are anti-inflammatory (M2) macrophages (e.g., M2a, M2b, M2c, and M2d macrophages). M2a macrophages can be induced by IL-4, IL-13, and / or fungal infection. M2b macrophages can be induced by IL-1R ligands, immune complexes, and / or LPS. M2c macrophages can be induced by IL-10 and / or TGFβ. M2d macrophages can be induced by IL-6 and / or adenosine. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein reduce the immune response of a subject, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, the macrophages express one or more markers of anti-inflammatory (M2) macrophages (e.g., one, two, or three of CD206, CD163, or CD209). In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL-10 or TGFβ), e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0172] In some embodiments, the macrophages comprise at least one upregulated pro-inflammatory (M1) marker and / or at least one downregulated anti-inflammatory (M2) marker compared to control macrophages that do not contain one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs provided herein, and / or the same macrophages prior to introduction of one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs 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 macrophages. In some embodiments, at least one anti-inflammatory (M2) marker (e.g., CD206, CD163, and / or CD209) is downregulated in the macrophages.

[0173] In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased phagocytosis, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased tumor antigen presentation (e.g., post-phagocytic presentation) and / or increased antigen processing, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased tumor killing (e.g., increased phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0174] In some embodiments, macrophages that comprise or express one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs 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), or decreased expression of one or more genes (e.g., CD163, CD206, TGFβ, IL-10, and / or IL4) typically associated with decreased effector function (e.g., phagocytosis, target cell cytotoxicity, antigen presentation, or cytokine secretion), e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased production of ROS, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit metabolic reprogramming (e.g., interferon signaling pathway, TH1 pathway, PTEN signaling, PI3K signaling, MTOR signaling, TLR signaling, CD40 signaling, 41BB signaling, 41BBL signaling, macrophage maturation signaling, dendritic cell maturation signaling, CD3 zeta signaling, FcRγ signaling, CD64 signaling, CD32a signaling, CD32c signaling, CD16a signaling, TLR1 signaling, TLR2 signaling, TLR3 signaling, TLR4 signaling, TLR5 signaling, TLR6 signaling, TLR7 signaling, TLR8 signaling, TLR9 signaling, TLR10 signaling, TLR11 signaling, TLR12 signaling, TLR13 signaling, TLR14 signaling, TLR15 signaling, TLR16 signaling, TLR17 signaling, TLR18 signaling, TLR19 signaling, TLR20 signaling, TLR21 signaling, TLR22 signaling, TLR23 signaling, TLR24 signaling, TLR25 signaling, TLR26 signaling, TLR27 signaling, TLR28 signaling, TLR29 signaling, TLR29 signaling, TLR210 signaling, TLR211 signaling, TLR212 signaling, TLR213 signaling, TLR214 ...TLR6 signaling, TLR7 signaling, TLR8 signaling, TLR9 signaling, ALK signaling, AXL signaling, DDR2 signaling, EGFR signaling, EphA1 signaling, INSR signaling, cMET signaling, MUSK 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, In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit induction of cell survival mechanisms, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit induction of cell death mechanisms, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. ...For example, the macrophages may exhibit one, two, three, four, or five of the following, compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein: increased resistance to phagocytic checkpoints; increased expression of chemokine receptors to aid trafficking; increased expression of chemokines to recruit other immune cells; increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity); and / or increased proliferation. In some embodiments, macrophages that comprise or express one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit one, two, three, or four of the following, compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein: improved duration of chimeric switch receptor expression; improved stability of the chimeric switch receptor at the cell surface; increased levels of chimeric switch receptor expression; and / or reduced background activity of the chimeric switch receptor.

[0175] In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein reduce one or more signs and / or symptoms of an infection (e.g., infectious pathogen) in a subject, e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, the infectious pathogen comprises or is a virus, a protozoan (e.g., Trypanosoma, Malaria, or Toxoplasma), a bacterium (e.g., Mycobacterium, Salmonella, or Listeria), a fungus (e.g., Candida), or a combination thereof. In some embodiments, the virus includes a hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C, or hepatitis E), a retrovirus, a human immunodeficiency virus (e.g., HIV1 or HIV2), a T-cell leukemia virus, a lymphotropic virus (e.g., HTLV1 or HTLV2), a herpes simplex virus (e.g., herpes simplex virus type 1 or 2), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, rubella virus, Japanese encephalitis virus, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, a coronavirus (e.g., severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), Ebola virus, West Nile virus, or a variant or combination thereof.

[0176] In some embodiments, macrophages comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein reduce the formation and / or degradation of existing aggregates via phagocytosis of at least one protein aggregate in a subject (e.g., a subject suffering from a neurodegenerative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, an amyloidosis, or a combination thereof), e.g., compared to macrophages that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, the neurodegenerative disease is selected from the group consisting of a tauopathy, a synucleopathy, presenile dementia, senile dementia, Alzheimer's disease, progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, a polyglutamine disease, a trinucleotide repeat disease, and a prion disease. In some embodiments, the inflammatory disease is selected from the group consisting of systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn's disease, chronic infections, hereditary periodic fevers, malignancies, systemic vasculitis, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, immunodeficiency, Muckle-Wells (MWS) disease, and familial Mediterranean fever (FMF). In some embodiments, the amyloidosis is selected from the group consisting of primary amyloidosis (AL), secondary amyloidosis (AA), familial amyloidosis (ATTR), beta2 microglobulin amyloidosis, localized amyloidosis, heavy chain amyloidosis (AH), light chain amyloidosis (AL), primary systemic amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, apolipoprotein C2 amyloidosis, apolipoprotein C3 amyloidosis, corneal lactoferrin amyloidosis, transthyretin-related amyloidosis, dialysis amyloidosis, fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and lysozyme amyloidosis.In some embodiments, the cardiovascular disease is selected from the group consisting of atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, and heart failure. In some embodiments, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation-induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, bone marrow fibrosis, and skin fibrosis.

[0177] Monocytes Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen and generally do not proliferate in a steady state. Monocytes vary greatly in size, ranging from approximately 10 to 30 μm in diameter. The nucleus-to-cytoplasm ratio of monocytes can range from approximately 2:1 to approximately 1:1. Monocytes typically contain chemokine receptors and pathogen recognition receptors that mediate migration from the blood to tissues, such as during infection. Monocytes produce proinflammatory cytokines, internalize cells and / or toxic molecules, and can differentiate into dendritic cells or macrophages.

[0178] In some embodiments, monocytes comprise or express one or more phenotypic markers. Examples of phenotypic markers for 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, CDw121b, CDw123, CD127, CDw128, CDw131, CD147, CD155, CD156a, CD157, and CD162. Examples of phenotypic markers for mouse monocytic cells include, but are not limited to, CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD2132, CDw210, CD226, CD281, CD282, CD284, and CD286. Examples of phenotypic markers for mouse monocytic cells include, but are not limited to, CD11a, CD11b, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdw131, CD281, CD282, CD284, CD286, F4 / 80, and CD49b. In certain embodiments, monocytes comprise one, two, or three of CD11b, CD14, or CD16. In certain embodiments, the monocytes comprise CD14+ CD16- monocytes, CD14+ CD16+ monocytes, or CD14- CD16+ monocytes.

[0179] In some embodiments, monocytes are differentiated into macrophages. In some embodiments, monocytes are differentiated into dendritic cells (DCs). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages is induced by macrophage colony-stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with IL-4.

[0180] In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased phagocytosis, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit enhanced survival, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit enhanced differentiation into macrophages (e.g., M1 or M2 macrophages), e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit enhanced differentiation into DCs (e.g., resident DCs or migratory DCs, and / or in lymphoid or non-lymphoid tissues), e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased cytotoxicity against tumor cells, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased tumor antigen presentation (e.g., post-phagocytic presentation) and / or increased antigen processing, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased tumor killing (e.g., phagocytosis, lysis, apoptosis, or production of tumor-killing cytokines (e.g., TNFα)), e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0181] In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs 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 that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing at least one chimeric switch receptor described herein exhibit increased production of ROS, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit metabolic reprogramming, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit induction of cell survival mechanisms, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, monocytes comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit induction of cell death mechanisms, e.g., compared to monocytes that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.In some embodiments, monocytes comprising or expressing at least one chimeric switch receptor described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity), or increased proliferation, compared to, e.g., monocytes that comprise or express at least one chimeric switch receptor described herein and at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of chimeric switch receptor expression, improved stability of the chimeric switch receptor at the cell surface, increased level of chimeric switch receptor expression, and / or reduced background activity of the chimeric switch receptor, compared to, e.g., monocytes that comprise or express at least one chimeric switch receptor described herein and at least one CAR described herein, compared to, e.g., monocytes that comprise or express at least one chimeric switch receptor described herein and at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of chimeric switch receptor expression, improved stability of the chimeric switch receptor at the cell surface, increased level of chimeric switch receptor expression, and / or reduced background activity of the chimeric switch receptor, compared to, e.g., monocytes that do not comprise a chimeric switch receptor described herein.

[0182] dendritic cells Dendritic cells (DCs) are specialized bone marrow-derived antigen-presenting cells that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells can be found in both lymphoid and non-lymphoid organs and are generally thought to originate from either the lymphoid or myeloid lineages.

[0183] 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.

[0184] Immature DCs may be characterized by a high capacity for antigen capture but a relatively low capacity for T cell stimulation. Inflammatory mediators promote the maturation of DCs. When DCs reach a mature stage, their properties change dramatically compared to immature DCs, such as a decreased capacity for antigen capture and / or an increased capacity for T cell stimulation. In some embodiments, the DCs include or are immature DCs. In other embodiments, the DCs include or are mature DCs.

[0185] Without wishing to be bound by theory, it is believed that by modifying DC cells to include or express one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein, mature DCs can simultaneously exhibit increased antigen capture capacity and T cell stimulation, e.g., compared to DCs that do not include one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs that include or express one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein mediate tumor antigen presentation, e.g., increase tumor antigen presentation, compared to DCs that do not include one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs that include or express one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein mediate tumor T cell stimulation, e.g., increase T cell stimulation, compared to DCs that do not include one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0186] In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased phagocytosis, e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased tumor antigen presentation (e.g., post-phagocytic presentation), increased antigen processing, increased antigen cross-presentation, increased T cell priming, and / or T cell stimulation, e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0187] In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit either or both increased expression of preferred genes or decreased expression of undesired genes compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit increased production of ROS, e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit metabolic reprogramming, e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit induction of cell survival mechanisms, e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein.

[0188] In some embodiments, DCs comprising or expressing one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein exhibit induction of cell death mechanisms, e.g., compared to DCs that do not comprise one or more of the chimeric switch receptors, membrane-tethered cytokines, and / or CARs described herein. In some embodiments, DCs comprising or expressing at least one chimeric switch receptor described herein exhibit one, two, three, four, or five of the following, e.g., increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM-degrading enzymes (e.g., MMPs that degrade tumor ECM and / or exhibit anti-fibrotic activity), or increased proliferation, compared to DCs that do not comprise the chimeric switch receptors described herein. In some embodiments, DCs comprising or expressing at least one chimeric switch receptor described herein and at least one CAR described herein exhibit one, two, three, or four of the following, e.g., improved duration of chimeric switch receptor expression, improved stability of the CAR at the cell surface, increased levels of chimeric switch receptor expression, and / or reduced background activity of the chimeric switch receptor, compared to DCs that do not comprise a chimeric switch receptor described herein.

[0189] Methods for modifying immune cells The present disclosure provides, inter alia, methods of modifying immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells), the methods comprising introducing into the immune cells a nucleic acid construct comprising one or more nucleic acids encoding one or more of a chimeric switch receptor, a membrane-tethered cytokine, and / or a CAR, or fragments thereof. The method can comprise introducing into the immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) a nucleic acid construct comprising one or more nucleic acids encoding a chimeric switch receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain is derived from a first receptor and the intracellular domain is derived from a second receptor, and wherein the second receptor is a cytokine receptor. In some embodiments, the nucleic acid construct of the present disclosure comprises one or more nucleic acids encoding a chimeric switch receptor and a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid construct of the present disclosure comprises one or more nucleic acids encoding a chimeric switch receptor and a membrane-tethered cytokine. In some embodiments, a nucleic acid construct of the present disclosure comprises one or more nucleic acids encoding a membrane-tethered cytokine and a chimeric antigen receptor (CAR). In some embodiments, a nucleic acid construct of the present disclosure comprises one or more nucleic acids encoding a chimeric switch receptor, a membrane-tethered cytokine, and a chimeric antigen receptor (CAR).

[0190] How to install Nucleic acid constructs comprising one or more nucleic acid sequences encoding at least one chimeric switch receptor, at least one membrane-tethered cytokine, at least one CAR, or combinations thereof described herein can be introduced into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) by physical, chemical, or biological methods. In some embodiments, the present disclosure provides methods for modifying immune cells, comprising producing the modified immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) ex vivo. In some embodiments, the present disclosure provides methods for modifying immune cells, comprising producing the modified immune cells (e.g., stem cells, monocytes, macrophages, or dendritic cells) within a subject (i.e., in vivo).

[0191] 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, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof. Nucleic acid constructs can be introduced into immune cells using commercially available methods, such as electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.), Gene Pulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg, Germany)). Nucleic acid constructs can also be introduced into immune cells using mRNA transfection, e.g., cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001), which is incorporated herein by reference in its entirety).

[0192] Biological methods for introducing the nucleic acid constructs described herein into immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) include the use of DNA and RNA vectors. In one embodiment, the vector comprises a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., PiggyBac, Sleeping Beauty), a site-specific insertion vector (e.g., CRISPR, zinc finger nuclease, TALEN), a suicide expression vector, or another vector known in the art. 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 by reference in their entireties). Retroviral vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer, allowing for long-term and stable integration of the transgene and its propagation in daughter cells. In some embodiments, the lentiviral vector is packaged with a Vpx protein (e.g., as described in International Publication No. WO2017 / 044487, 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, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein are transfected with a lentiviral vector packaged with a Vpx protein. In some embodiments, Vpx inhibits at least one antiviral factor in immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein.In some embodiments, lentiviral vectors packaged with Vpx protein exhibit increased transfection efficiency of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein, e.g., compared to lentiviral vectors not packaged with Vpx protein. In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein are electroporated and / or transfected with at least one VPX mRNA prior to transfection with a viral vector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)).

[0193] 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, liposomes, and lipofectamine-nucleic acid complexes).

[0194] An exemplary system for introducing the nucleic acid constructs described herein is a lipid-based system. The nucleic acid constructs described herein may be encapsulated in the aqueous interior of liposomes, dispersed within lipid bilayers, attached to liposomes via linking molecules, attached to lipid nanoparticles (LNPs) via linking molecules, entrapped in liposomes, entrapped in LNPs, complexed with liposomes, complexed with LNPs, dispersed in a solution or suspension containing lipids, mixed with lipids, complexed with micelles, or otherwise associated with lipids. The lipids used in the methods described herein may be naturally occurring or synthetic lipids. Lipids may also be obtained from commercial sources. For example, dimyristylphosphatidylcholine 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 dimyristylphosphatidylglycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. In some embodiments, the lipid-based system can 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 preferentially 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 the delivery vehicle.

[0195] targeting part In some embodiments, the delivery vehicle may include one or more targeting moieties. In some embodiments, the targeting moiety may facilitate passive targeting of the composition to a desired target. In some embodiments, the targeting moiety may facilitate active targeting of the composition to a desired target.

[0196] In some embodiments, the targeting moiety is or may include one or more antibodies (e.g., monoclonal, polyclonal, synthetic, human, humanized, non-human antibodies) or any fragment thereof, such as an scFv, an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule, such as a molecule that binds to one or more of CD14, CD11b, CD163, CD206, CD33, CD209. In some embodiments, the targeting moiety is or may include a small molecule.

[0197] In some embodiments, the targeting moiety may be or include a combination of specific lipids or hydrophobic entities, for example, present on or forming the outer surface of a liposome or lipid nanoparticle (e.g., for targeting to a specific cell type(s)).

[0198] nucleic acid molecule In some embodiments of the present disclosure, the one or more nucleic acid molecules are or comprise DNA. In some embodiments of the present disclosure, the one or more nucleic acid molecules are or comprise messenger RNA (mRNA). In some embodiments, mRNA according to the present disclosure can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to increase stability. Modification of mRNA can include, for example, modifications of nucleotides of the RNA. Thus, modified mRNA according to the present disclosure can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, modifying the mRNA includes including modified nucleotides, modifications to the 5' or 3' untranslated region (UTR), a cap structure, and / or a poly(A) tail in the mRNA.

[0199] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding chimeric switch receptors, mRNAs encoding membrane-tethered cytokines, and / or mRNAs encoding CARs) may contain RNA backbone modifications. Typically, backbone modifications are modifications in which the backbone phosphates of nucleotides contained in the RNA are chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium groups, etc., which include replacing phosphodiester bonds with other anionic, cationic, or neutral groups.

[0200] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding chimeric switch receptors, mRNAs encoding membrane-tethered cytokines, and / or mRNAs encoding CARs) can contain sugar modifications. Exemplary sugar modifications are chemical modifications of the sugars 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-alkyloligoribonucleotides, 2'-deoxy Sugar modifications include, but are not limited to, sugar modifications selected from the group consisting of 2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-aracytidine 5'-triphosphate, 2'-arauidine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0201] In some embodiments, an mRNA of the disclosure (e.g., an mRNA encoding a chimeric switch receptor, an mRNA encoding a membrane-tethered cytokine, and / or an mRNA encoding a CAR) comprises modified nucleotides including pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or a combination thereof.

[0202] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a chimeric switch receptor, an mRNA encoding a membrane-tethered cytokine, and / or an mRNA encoding a CAR) 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.

[0203] 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.

[0204] Thus, in some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a chimeric switch receptor, an mRNA encoding a membrane-tethered cytokine, and / or an mRNA encoding a CAR) comprises a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to generate a 5' triphosphate bond; and the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A) and G(5')ppp(5')G). In some embodiments, the cap comprises a Cap0 structure. The Cap0 structure lacks 2'-O-methyl residues on the ribose attached to bases 1 and 2. In some embodiments, the cap comprises an AGCap1 structure. The AGCap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap comprises a Cap2 structure. The Cap2 structure has 2'-O-methyl residues attached to both bases 2 and 3. In some embodiments, the cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, modified mRNAs of the disclosure comprise m6AGCap1 and modified nucleotides including pseudouridine (PsU).

[0205] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a chimeric switch receptor, an mRNA encoding a membrane-tethered cytokine, and / or an mRNA encoding a CAR) comprises a 3' poly(A) tail structure. The poly(A) tail at the 3' end of the mRNA typically comprises about 10-400 adenosine nucleotides (SEQ ID NO: 200) (e.g., about 100-400 adenosine nucleotides, about 10-200 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-100 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA comprises a 3' poly(C) tail structure. A suitable poly(C) tail at the 3' end of an mRNA typically contains about 10 to 200 cytosine nucleotides (SEQ ID NO: 201) (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly(C) tail may be in addition to or in place of a poly(A) tail.

[0206] In some embodiments, an mRNA of the present disclosure (e.g., an mRNA encoding a chimeric switch receptor, an mRNA encoding a membrane-tethered cytokine, and / or an mRNA encoding a CAR) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron-responsive element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length.

[0207] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 or more nucleotides in length.

[0208] Administration of additional payloads In some embodiments, the methods of the disclosure include one or more steps of treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) during the process of modifying the immune cells. In some embodiments, the methods of the disclosure include one or more steps of administering to the subject an additional payload to modulate the 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 for the one or more nucleic acid molecules.

[0209] In some embodiments, the methods of the present disclosure include treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a modulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, the additional payload is or can include a modulator of a pathway activated by in vitro transcribed mRNA. 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 via ribonuclease L (RNase L), and overexpression and inhibition of self-amplifying mRNA replication are relevant to the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0210] In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase L, RNase T2, or RNase 1 inhibitor. In some embodiments, the modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase L inhibitor. In some embodiments, the RNase L inhibitor comprises sunitinib. In some embodiments, the RNase L inhibitor comprises ABCE1.

[0211] In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases mRNA stability in the modified immune cells compared to mRNA stability in modified immune cells of the same type that were not treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases chimeric switch receptor expression in the modified immune cells compared to chimeric switch receptor expression in modified immune cells of the same type that were not treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases membrane-tethered cytokine expression in the modified immune cells compared to membrane-tethered cytokine expression in modified immune cells of the same type that were not treated with the RNaseL inhibitor. In some embodiments, treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases CAR expression in the modified immune cells compared to CAR expression in modified immune cells of the same type that were not treated with the RNaseL inhibitor. In some embodiments, treatment of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with an RNase L inhibitor enhances effector activity in the modified immune cells compared to effector activity in the same type of modified immune cells that have not been treated with the RNase L inhibitor.

[0212] In some embodiments, administration of an RNaseL inhibitor to a subject increases mRNA stability in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) compared to mRNA stability in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered. In some embodiments, administration of an RNaseL inhibitor to a subject increases expression of a chimeric switch receptor in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) compared to chimeric switch receptor expression in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered. In some embodiments, administration of an RNaseL inhibitor to a subject increases membrane-tethered cytokine expression in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) compared to membrane-tethered cytokine expression in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered. In some embodiments, administration of an RNaseL inhibitor to a subject increases CAR expression in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) compared to CAR expression in modified immune cells of the same type in a subject to which the RNaseL inhibitor was not administered. In some embodiments, administration of an RNaseL inhibitor to a subject enhances effector activity in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) compared to effector activity in the same type of modified immune cell in a subject to which the RNaseL inhibitor was not administered.

[0213] In some embodiments of the present disclosure, the step of treating immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) occurs before the step of introducing mRNA into the immune cells. 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 mRNA to the subject.

[0214] In some embodiments, the methods of the disclosure include culturing immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or an immunostimulatory recombinant protein. In some embodiments, the methods of the disclosure include administering to a subject a cytokine or an immunostimulatory recombinant protein. In some embodiments, the cytokine is selected from the group consisting of IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonist, 4-1BB ligand, recombinant 4-1BB, CD19 agonist, TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, or TLR-9), TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3), glucocorticoids, and the like. Id, 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, CD1 54, lymphotoxin beta (LT-β), A proliferation-inducing ligand (APRIL), CD70, CD153, glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L (CD252), TALL-1 (tumor necrosis factor ligand superfamily member 13B-TNFSF13B), TNF-related apoptosis-inducing ligand (TRAIL), and TNF-related weak apoptosis-inducing factor (TNF-IL). WEAK), TNF-related activation-inducing cytokine (TRANCE), erythropoietin (Epo), thyroid peroxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), nucleotide-binding oligomerization domain-containing protein (NOD) ligand (e.g., NOD1, NOD2, or NOD1 / 2 agonist),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, ionomycin, PHA-P, or PMA), or combinations thereof. In some embodiments, the cytokine comprises IFN-β.

[0215] In some embodiments of the present disclosure, the step of culturing immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) occurs after the step of introducing mRNA into the immune cells. In some embodiments of the present disclosure, the step of administering a cytokine or immunostimulatory recombinant protein to the subject occurs after the step of administering a composition comprising mRNA to the subject.

[0216] In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the viability of the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases expression of a protein (e.g., a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof) in the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases the longevity of protein (e.g., a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof) expression compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or immunostimulatory recombinant protein enhances the effector activity of the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein. In some embodiments, culturing modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) with a cytokine or immunostimulatory recombinant protein increases pro-inflammatory (M1) polarization of the modified immune cells compared to modified immune cells of the same type that have not been cultured with the cytokine or immunostimulatory recombinant protein.

[0217] In some embodiments, administration of a cytokine or immunostimulatory recombinant protein to a subject increases the survival rate of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject, compared to modified immune cells of the same type 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 expression of a protein (e.g., a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof) in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject, compared to modified immune cells of the same type 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 longevity of expression of a protein (e.g., a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof) in modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject, compared to modified immune cells of the same type 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 enhances the effector activity of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject compared to the same type of modified immune 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 pro-inflammatory (M1) polarization of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) in the subject compared to the same type of modified immune cells in a subject that has not been administered the cytokine or immunostimulatory recombinant protein.

[0218] Methods for altering the inflammatory phenotype of a population of cells In some embodiments, the methods of the present disclosure include altering the inflammatory phenotype of a population of cells. In some embodiments, the method of altering the inflammatory phenotype of a population of cells includes contacting the population of cells with a modified immune cell (e.g., a stem cell, a macrophage, a monocyte, or a dendritic cell) described herein. In some embodiments, the population of cells includes a macrophage, a monocyte, a dendritic cell, a T cell, a NK cell, or a combination thereof.

[0219] In some embodiments, the inflammatory phenotype of a population of cells is changed from an anti-inflammatory to a non-active state. In some embodiments, the inflammatory phenotype of a population of cells is changed from a pro-inflammatory to a non-active state. In some embodiments, the inflammatory phenotype of a population of cells is changed from anti-inflammatory to pro-inflammatory. In some embodiments, the inflammatory phenotype of a population of cells is changed from pro-inflammatory to anti-inflammatory.

[0220] modified immune cells In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) are generated by the methods of the present disclosure. In some embodiments, the modified immune cells comprise a chimeric switch receptor. In some embodiments, the modified immune cells comprise one or more nucleic acids encoding the chimeric switch receptor. In some embodiments, the chimeric switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain is derived from a first receptor and the intracellular domain is derived from a second receptor. In some embodiments, the first receptor is a cytokine receptor (i.e., a first cytokine receptor). In some embodiments, the first cytokine receptor binds a cytokine described herein. In some embodiments, the first receptor binds an antigen described herein. In some embodiments, the second receptor is a cytokine receptor (i.e., a second cytokine receptor). In some embodiments, the second cytokine receptor binds a cytokine described herein. In some embodiments, the transmembrane domain is derived from the first receptor or the second receptor.

[0221] In some embodiments, the first cytokine receptor is a receptor for a pro-inflammatory cytokine (i.e., a pro-inflammatory cytokine receptor). In some embodiments, the first cytokine receptor is a receptor for an anti-inflammatory cytokine (i.e., an anti-inflammatory cytokine receptor). In some embodiments, the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the second cytokine receptor is a pro-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is an anti-inflammatory cytokine receptor and the second cytokine receptor is a pro-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is a pro-inflammatory cytokine receptor and the second cytokine receptor is an anti-inflammatory cytokine receptor.

[0222] In some embodiments, the first cytokine receptor is selected from Table 1. [Table 1-1] [Table 1-2]

[0223] In some embodiments, the second cytokine receptor is selected from Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0224] In some embodiments, the first cytokine receptor is or comprises IL10Ra, and the second cytokine receptor is or comprises IFN-λR1. In some embodiments, the first cytokine receptor is or comprises IL10Ra, and the second cytokine receptor is or comprises IFNAR2. In some embodiments, the first cytokine receptor is or comprises IL10Ra, and the second cytokine receptor is or comprises IFN-γR1. In some embodiments, the first cytokine receptor is or comprises IFNGR1, and the second cytokine receptor is or comprises IL10Ra.

[0225] In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 3. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 4.

[0226] In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6.

[0227] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the disclosure comprises a chimeric switch receptor described herein, wherein the chimeric switch receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 7.

[0228] In some embodiments, the modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) of the present disclosure comprise a chimeric switch receptor that comprises a first receptor that 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 CD19, CD123, CD22, CD30, CD171, CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B-cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), protease-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21 (testisin or or PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor),Carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, 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), intratumor Skin marker 7-related (TEM7R), 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 receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase,Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), rat sarcoma tumor (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Proteins) Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mut) hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), or immunoglobulin lambda-like polypeptide 1 (IGLL1).In certain embodiments, the tumor antigen comprises ERBB2 (Her2 / neu). In certain embodiments, the tumor antigen comprises PSMA. In certain embodiments, the tumor antigen comprises mesothelin.

[0229] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises a membrane-tethered cytokine. In some embodiments, a modified immune cell comprising a membrane-tethered cytokine can stimulate a receptor on a neighboring cell. In some embodiments, the membrane-tethered cytokine comprises an extracellular domain and a membrane tether. In some embodiments, the extracellular domain is or comprises a pro-inflammatory cytokine. In some embodiments, the extracellular domain is or comprises an anti-inflammatory cytokine. In some embodiments, the extracellular domain is or comprises IFN-β. In some embodiments, the membrane tether is or comprises a B7 transmembrane domain (TMD), a B7 TMD with a matrix metalloproteinase (MMP) linker, a glycosylphosphatidylinositol (GPI) anchor, or a GPI anchor with a CD28 spacer. In some embodiments, the membrane tether can be released from the modified immune cell when the extracellular domain binds to a receptor expressed on another cell.

[0230] In some embodiments, the modified immune cells of the present disclosure further comprise a chimeric antigen receptor (CAR) and / or a nucleic acid encoding a CAR.

[0231] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, exhibit increased survival compared to modified immune cells of the same type comprising unmodified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, exhibit increased expression of mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, compared to modified immune cells of the same type comprising unmodified mRNA encoding a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, exhibit increased expression of the chimeric switch receptor, membrane-tethered cytokine, CAR, or a combination thereof compared to modified immune cells of the same type comprising unmodified mRNA encoding the chimeric switch receptor, membrane-tethered cytokine, CAR, or a combination thereof.In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, exhibit increased longevity of the mRNA encoding the chimeric switch receptor, membrane-tethered cytokine, CAR, or combination thereof compared to modified immune cells of the same type comprising unmodified mRNA encoding the chimeric switch receptor, membrane-tethered cytokine, CAR, or combination thereof. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or combination thereof, exhibit increased longevity of the chimeric switch receptor, membrane-tethered cytokine, CAR, or combination thereof compared to modified immune cells of the same type comprising unmodified mRNA encoding the chimeric switch receptor, membrane-tethered cytokine, CAR, or combination thereof. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, exhibit enhanced effector activity compared to modified immune cells of the same type comprising unmodified mRNA encoding a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising modified mRNA encoding a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, exhibit increased pro-inflammatory (M1) polarization compared to modified immune cells of the same type comprising unmodified mRNA encoding a chimeric switch receptor, a membrane-tethered cytokine, a CAR, or a combination thereof.

[0232] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof maintain a pro-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof maintain a pro-inflammatory phenotype 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 chimeric switch receptor, membrane-tethered cytokine, CAR, or a combination thereof.

[0233] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof maintain an anti-inflammatory phenotype over time. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof maintain an anti-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and / or 28 days after the immune cells have been modified with a nucleic acid encoding the chimeric switch receptor, membrane-tethered cytokine, CAR, or a combination thereof.

[0234] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof maintain a pro-inflammatory phenotype and / or resist phenotypic reversal when affected by 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 pro-inflammatory marker by treating modified immune cells comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof 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 pro-inflammatory marker by treating modified immune cells comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof with increasing concentrations of a pro-inflammatory cytokine (e.g., quantifying the effect of soluble IFN-γ on modified immune cells comprising an IFN-γ chimeric switch receptor).

[0235] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof maintain an anti-inflammatory phenotype and / or resist phenotypic reversal when affected by pro-inflammatory cytokines. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve for an anti-inflammatory marker by treating modified immune cells comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof with increasing concentrations of a pro-inflammatory cytokine. In some embodiments, the sensitivity of modified immune cells to environmental cytokines is measured by generating a dose-response curve for an anti-inflammatory marker by treating modified immune cells comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof with increasing concentrations of an anti-inflammatory cytokine (e.g., quantifying the effect of soluble IL-10 on modified immune cells comprising an IL-10 chimeric switch receptor).

[0236] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof have a minimal effect on neighboring cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof have a significant effect on neighboring cells. In some embodiments, the effect of modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof on unmodified cells (e.g., immune cells that do not comprise a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof) 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, the 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, hi some embodiments, the 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.

[0237] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof have minimal cytotoxic effects on neighboring cells. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof have significant cytotoxic effects on neighboring cells (e.g., cancer cells). In some embodiments, modifying immune cells to comprise a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof results in no cytotoxicity 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.

[0238] In some embodiments, expression of a chimeric switch receptor or membrane-tethered cytokine 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 a desired effector function of the modified immune cell (e.g., phagocytosis, target cytotoxicity, antigen presentation, or cytokine secretion) compared to a modified immune cell that comprises a CAR but does not comprise a chimeric switch receptor or membrane-tethered cytokine. In some embodiments, expression of a chimeric switch receptor or membrane-tethered cytokine 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 a desired effector function of the modified immune cell (e.g., phagocytosis, target cytotoxicity, antigen presentation, or cytokine secretion) compared to a modified immune cell that comprises a CAR but does not comprise a chimeric switch receptor or membrane-tethered cytokine.

[0239] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a chimeric switch receptor provided herein, a membrane-tethered cytokine provided herein, a CAR provided herein, or a combination thereof, can comprise one or more regulatory systems, such as a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional regulation (e.g., a cell-specific promoter, a cell state-specific promoter, a downstream promoter of chimeric switch receptor, membrane-tethered cytokine, or CAR activation, a downstream promoter of an endogenous signaling pathway, or drug-inducible transcription), a chimeric switch receptor, membrane-tethered cytokine, or CAR These include, but are not limited to, post-transcriptional control of mRNA (e.g., RNA-based inhibition by endogenous or recombinant miRNA), or post-translational control of the structure or stability of a chimeric switch receptor, membrane-tethered cytokine, or CAR (e.g., a chimeric switch receptor, membrane-tethered cytokine, or CAR whose intracellular domain conditionally associates with the complete structure by drug / light-induced association (allowing signaling) or dissociation (inhibiting signaling), or whose stability is regulated by a drug for inducible stabilization (allowing signaling) or degradation (inhibiting signaling)).These control systems can be combined to create logic gates, e.g., AND gates (e.g., a chimeric switch receptor, membrane-tethered cytokine, or CAR with an inducible promoter and cytosolic domain that associates in a drug-dependent manner and thus requires the presence of the chimeric switch receptor, membrane-tethered cytokine, CAR activation, and a small molecule), OR gates (e.g., a chimeric switch receptor, membrane-tethered cytokine, or CAR under the control of a promoter that is transcriptionally activated after either chimeric switch receptor, membrane-tethered cytokine, or CAR activation or the addition of a small molecule), and / or NOT gates (e.g., a chimeric switch receptor, membrane-tethered cytokine, or CAR whose mRNA is degraded by an endogenous miRNA (a miRNA that is upregulated through a specific cytokine signaling pathway and therefore expresses the chimeric switch receptor, membrane-tethered cytokine, or CAR only in the absence of this cytokine) that is expressed in a native immune cell signaling state).

[0240] Assay Various assays can be performed to confirm the presence of a nucleic acid construct described herein and / or the presence of a protein (e.g., a chimeric switch receptor or CAR) in immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells). For example, such assays include molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, as well as biochemical assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting). Other assays of the present disclosure include, for example, fluorescence-activated cell sorting (FACS), immunofluorescence microscopy, MSD cytokine analysis, mass spectrometry (MS), RNA-Seq, and functional assays.

[0241] Various 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 lifespan, protein (e.g., chimeric switch receptor, membrane-tethered cytokine, CAR, or combinations thereof) expression, protein (e.g., chimeric switch receptor, membrane-tethered cytokine, CAR, or combinations thereof) lifespan, effector activity, and pro-inflammatory (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.

[0242] Chimeric Switch Receptor The term "chimeric switch receptor," as used herein, refers to an artificial chimeric protein that includes an extracellular domain from a first receptor and an intracellular domain from a second (i.e., different) receptor, such that the receptor can transduce a signal into another signal. In some embodiments, a switch receptor of the present disclosure converts an anti-inflammatory signal into a pro-inflammatory signal. In some embodiments, a switch receptor of the present disclosure converts a pro-inflammatory signal into an anti-inflammatory signal.

[0243] In some embodiments, the chimeric switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain is derived from a first receptor and the intracellular domain is derived from a second receptor. In some embodiments, the first receptor is a cytokine receptor (i.e., a first cytokine receptor). In some embodiments, a chimeric switch receptor comprising an extracellular domain from a first cytokine receptor binds to a cytokine described herein. In some embodiments, the first receptor binds to an antigen described herein. In some embodiments, the second receptor is a cytokine receptor (i.e., a second cytokine receptor). In some embodiments, binding of a ligand to the extracellular portion of the chimeric switch receptor results in an intracellular domain from the second cytokine receptor that generates a signal substantially similar to the signal that results from binding of the ligand to a naturally occurring (i.e., intact) second cytokine receptor, as described herein. As a non-limiting example, in some embodiments, in a chimeric switch receptor comprising the extracellular domain of IL10 and the intracellular domain of IFN-γ, binding of IL10 to the extracellular portion of the chimeric switch receptor produces a signal substantially similar to the signal produced when IFN-γ binds to the intact IFN-γ receptor, hi some embodiments, the transmembrane domain is derived from the first receptor or the second receptor.

[0244] In some embodiments, the first cytokine receptor is a receptor for a pro-inflammatory cytokine (i.e., a pro-inflammatory cytokine receptor). In some embodiments, the first cytokine receptor is a receptor for an anti-inflammatory cytokine (i.e., an anti-inflammatory cytokine receptor). In some embodiments, the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the second cytokine receptor is a pro-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is an anti-inflammatory cytokine receptor and the second cytokine receptor is a pro-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is a pro-inflammatory cytokine receptor and the second cytokine receptor is an anti-inflammatory cytokine receptor.

[0245] In some embodiments, the chimeric switch receptor of the present disclosure is membrane bound. In some embodiments, the chimeric switch receptor of the present disclosure is not membrane bound.

[0246] In some embodiments, the first cytokine receptor is selected from Table 1. In some embodiments, the second cytokine receptor is selected from Table 2. In some embodiments, the first cytokine receptor is or comprises IL10Ra and the second cytokine receptor is or comprises IFN-λR1. In some embodiments, the first cytokine receptor is or comprises IL10Ra and the second cytokine receptor is or comprises IFNAR2. In some embodiments, the first cytokine receptor is or comprises IL10Ra and the second cytokine receptor is or comprises IFN-γR1. In some embodiments, the first cytokine receptor is or comprises IFNGR1 and the second cytokine receptor is or comprises IL10Ra.

[0247] As non-limiting examples, in some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a STAT1 / 2 intracellular domain converts what would be STAT1 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a STAT3 intracellular domain converts what would be STAT1 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a STAT5 intracellular domain converts what would be STAT1 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a STAT6 intracellular domain converts what would be STAT1 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a TRAF1 / 2 intracellular domain converts what would be STAT1 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a TRAF2 intracellular domain converts what would be STAT1 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a TRAF2 / 3 intracellular domain converts what would be STAT1 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a TRAF2 / 5 intracellular domain converts what would be STAT1 signaling to TRAF2 / 5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would be STAT1 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be STAT1 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a DAP10 / 12 intracellular domain converts what would be STAT1 signaling to DAP10 / 12 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 extracellular domain and a DAP12 intracellular domain converts what would be STAT1 signaling to DAP12 signaling (based on an extracellular signal).

[0248] In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a STAT1 intracellular domain converts what would otherwise be STAT1 / 2 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a STAT3 intracellular domain converts what would otherwise be STAT1 / 2 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a STAT5 intracellular domain converts what would otherwise be STAT1 / 2 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a STAT6 intracellular domain converts what would otherwise be STAT1 / 2 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a TRAF1 / 2 intracellular domain converts what would otherwise be STAT1 / 2 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a TRAF2 intracellular domain converts what would otherwise be STAT1 / 2 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a TRAF2 / 3 intracellular domain converts what would otherwise be STAT1 / 2 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a TRAF2 / 5 intracellular domain converts what would otherwise be STAT1 / 2 signaling to TRAF2 / 5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would otherwise be STAT1 / 2 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be STAT1 / 2 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a DAP10 / 12 intracellular domain converts what would be STAT1 / 2 signaling to DAP10 / 12 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT1 / 2 extracellular domain and a DAP12 intracellular domain converts what would be STAT1 / 2 signaling to DAP12 signaling (based on an extracellular signal).

[0249] In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a STAT1 intracellular domain converts what would be STAT3 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a STAT1 / 2 intracellular domain converts what would be STAT3 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a STAT5 intracellular domain converts what would be STAT3 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a STAT6 intracellular domain converts what would be STAT3 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a TRAF1 / 2 intracellular domain converts what would be STAT3 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a TRAF2 intracellular domain converts what would be STAT3 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a TRAF2 / 3 intracellular domain converts what would be STAT3 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a TRAF2 / 5 intracellular domain converts what would be STAT3 signaling to TRAF2 / 5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would be STAT3 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be STAT3 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a DAP10 / 12 intracellular domain converts what would be STAT3 signaling to DAP10 / 12 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT3 extracellular domain and a DAP12 intracellular domain converts what would be STAT3 signaling to DAP12 signaling (based on an extracellular signal).

[0250] In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a STAT1 intracellular domain converts resulting STAT5 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a STAT1 / 2 intracellular domain converts resulting STAT5 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a STAT3 intracellular domain converts resulting STAT5 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a STAT6 intracellular domain converts resulting STAT5 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a TRAF1 / 2 intracellular domain converts resulting STAT5 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a TRAF2 intracellular domain converts resulting STAT5 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a TRAF2 / 3 intracellular domain converts what would be STAT5 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a TRAF2 / 5 intracellular domain converts what would be STAT5 signaling to TRAF2 / 5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would be STAT5 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be STAT5 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a DAP10 / 12 intracellular domain converts what would be STAT5 signaling to DAP10 / 12 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT5 extracellular domain and a DAP12 intracellular domain converts what would be STAT5 signaling to DAP12 signaling (based on an extracellular signal).

[0251] In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a STAT1 intracellular domain converts resulting STAT6 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a STAT1 / 2 intracellular domain converts resulting STAT6 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a STAT3 intracellular domain converts resulting STAT6 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a STAT5 intracellular domain converts resulting STAT6 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a TRAF1 / 2 intracellular domain converts resulting STAT6 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a TRAF2 intracellular domain converts resulting STAT6 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a TRAF2 / 3 intracellular domain converts what would be STAT6 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a TRAF2 / 5 intracellular domain converts what would be STAT6 signaling to TRAF2 / 5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would be STAT6 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be STAT6 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a DAP10 / 12 intracellular domain converts what would be STAT6 signaling to DAP10 / 12 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a STAT6 extracellular domain and a DAP12 intracellular domain converts what would be STAT6 signaling to DAP12 signaling (based on an extracellular signal).

[0252] In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a STAT1 intracellular domain converts what would be SMAD2 / 3 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a STAT1 / 2 intracellular domain converts what would be SMAD2 / 3 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a STAT3 intracellular domain converts what would be SMAD2 / 3 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a STAT5 intracellular domain converts what would be SMAD2 / 3 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a STAT6 intracellular domain converts what would be SMAD2 / 3 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a TRAF1 / 2 intracellular domain converts what would be SMAD2 / 3 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a TRAF2 intracellular domain converts what would be SMAD2 / 3 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a TRAF2 / 3 intracellular domain converts what would be SMAD2 / 3 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a TRAF2 / 5 intracellular domain converts what would be SMAD2 / 3 signaling to TRAF2 / 5 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would be SMAD2 / 3 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be SMAD2 / 3 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a DAP10 / 12 intracellular domain converts what would be SMAD2 / 3 signaling to DAP10 / 12 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a SMAD2 / 3 extracellular domain and a DAP12 intracellular domain converts what would be SMAD2 / 3 signaling to DAP12 signaling (based on an extracellular signal).

[0253] In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a STAT1 intracellular domain converts what would otherwise be DAP10 / 12 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a STAT1 / 2 intracellular domain converts what would otherwise be DAP10 / 12 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a STAT3 intracellular domain converts what would otherwise be DAP10 / 12 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a STAT5 intracellular domain converts what would otherwise be DAP10 / 12 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a STAT6 intracellular domain converts what would otherwise be DAP10 / 12 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a TRAF1 / 2 intracellular domain converts what would otherwise be DAP10 / 12 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a TRAF2 intracellular domain converts what would otherwise be DAP10 / 12 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a TRAF2 / 3 intracellular domain converts what would otherwise be DAP10 / 12 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a TRAF2 / 5 intracellular domain converts what would otherwise be DAP10 / 12 signaling to TRAF2 / 5 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would otherwise be DAP10 / 12 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and an IRAK1 / 4 intracellular domain converts what would otherwise be DAP10 / 12 signaling to IRAK1 / 4 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP10 / 12 extracellular domain and a DAP12 intracellular domain converts what would otherwise be DAP10 / 12 signaling to DAP12 signaling (based on an extracellular signal).

[0254] In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a STAT1 intracellular domain converts what would otherwise be DAP12 signaling to STAT1 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a STAT1 / 2 intracellular domain converts what would otherwise be DAP12 signaling to STAT1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a STAT3 intracellular domain converts what would otherwise be DAP12 signaling to STAT3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a STAT5 intracellular domain converts what would otherwise be DAP12 signaling to STAT5 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a STAT6 intracellular domain converts what would otherwise be DAP12 signaling to STAT6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a TRAF1 / 2 intracellular domain converts what would otherwise be DAP12 signaling to TRAF1 / 2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a TRAF2 intracellular domain converts what would otherwise be DAP12 signaling to TRAF2 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a TRAF2 / 3 intracellular domain converts what would otherwise be DAP12 signaling to TRAF2 / 3 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a TRAF2 / 5 intracellular domain converts what would otherwise be DAP12 signaling to TRAF2 / 5 signaling (based on an extracellular signal).In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and a TRAF2 / 3 / 6 intracellular domain converts what would be DAP12 signaling to TRAF2 / 3 / 6 signaling (based on an extracellular signal). In some embodiments, a chimeric switch receptor comprising a DAP12 extracellular domain and an IRAK1 / 4 intracellular domain converts what would be DAP12 signaling to IRAK1 / 4 signaling (based on an extracellular signal).

[0255] In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 3. In some embodiments, the first cytokine receptor comprises an amino acid sequence that is identical to a sequence selected from Table 3.

[0256] In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 4. In some embodiments, the second cytokine receptor comprises an amino acid sequence that is identical to a sequence selected from Table 4.

[0257] In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 5. In some embodiments, the first cytokine receptor comprises a nucleic acid sequence that is identical to a sequence selected from Table 5.

[0258] In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 6. In some embodiments, the second cytokine receptor comprises a nucleic acid sequence that is identical to a sequence selected from Table 6.

[0259] In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 80% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 85% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 90% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 95% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 96% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 97% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 98% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence that is at least 99% identical to a sequence selected from Table 7. In some embodiments, a chimeric switch receptor of the present disclosure comprises an amino acid sequence identical to a sequence selected from Table 7.

[0260] In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 85% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 90% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 95% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 96% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 97% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 98% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence that is at least 99% identical to a sequence selected from Table 8. In some embodiments, a chimeric switch receptor of the present disclosure comprises a nucleic acid sequence identical to a sequence selected from Table 8.

[0261] In some embodiments, the chimeric switch receptor comprises a chimeric switch receptor extracellular domain, a chimeric switch receptor transmembrane domain, and a chimeric switch receptor intracellular domain.

[0262] In some embodiments, the chimeric switch receptor comprises a spacer domain or hinge between the chimeric switch receptor extracellular domain and the chimeric switch receptor transmembrane domain. In some embodiments, the chimeric switch receptor comprises a spacer domain or hinge between the chimeric switch receptor intracellular domain and the chimeric switch receptor transmembrane domain. As used herein, the term "spacer domain" or "hinge" refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular or intracellular domain of a polypeptide chain. In some embodiments, the spacer domain or hinge can 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, can form the link between the transmembrane domain and the intracellular domain of the chimeric switch receptor. An example of a linker includes a glycine-serine doublet.

[0263] In some embodiments, the chimeric switch receptor comprises a chimeric switch receptor extracellular domain operably linked to another domain of the chimeric switch receptor, such as a chimeric switch receptor transmembrane domain or a chimeric switch receptor intracellular domain, for expression in an immune cell. In some embodiments, the nucleic acid encoding the chimeric switch receptor extracellular domain is operably linked to a nucleic acid encoding the chimeric switch receptor transmembrane domain, and the nucleic acid encoding the chimeric switch receptor transmembrane domain is operably linked to a nucleic acid encoding the chimeric switch receptor intracellular domain.

[0264] In some embodiments, the effector activity of an immune cell comprising the chimeric switch receptor is directed against a target cell that comprises an antigen that specifically binds to the antigen-binding domain of the chimeric switch receptor, hi some embodiments, the target effector activity directed against the target cell is or includes phagocytosis, target cytotoxicity, antigen presentation, or cytokine secretion.

[0265] In some embodiments, an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a chimeric switch receptor may comprise one or more regulatory systems, including, but not limited to, a safety switch (e.g., an on switch and an off switch, a suicide switch), a logic gate, e.g., an AND gate (e.g., two or more chimeric switch receptors, each of which lacks one or more signaling domains such that activation of both / all chimeric switch receptors is required for activation or function of the intact immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell)), an OR gate (e.g., two or more chimeric switch receptors, each of which has an intracellular domain such as CD3ζ and a costimulatory domain), and / or a NOT gate (e.g., two or more chimeric switch receptors, one of which contains an inhibitory domain that antagonizes the function of the other chimeric switch receptor(s)).

[0266] Chimeric switch receptor extracellular domain The present disclosure provides chimeric switch receptors comprising an extracellular domain, in some embodiments, the chimeric switch receptor extracellular domain binds to a cytokine of the present disclosure. In some embodiments, the chimeric switch receptor extracellular domain binds to a cytokine selected from IL-10, IL-4 / IL-13, IL-7 / TSLP, IL-9, IL-21, IL-2 / IL-15, IL-2 / 15 / 4 / 7 / 9 / 21, TSLP, IL-6, IL-11, IL-6 / 11 / 27 / 31 / LIF / CNTF, GCSF, IL-3, IL-5, GM-CSF, IL-3 / 5 / GM-CSF, LIF, IL-31, CNTF / CT-1, IL-27, EPO, GH, PRL, IFN-α / β / ω / ε / κ, IFN-γ, IFN-λ1 / λ2 / λ3, IL-26 / 19 / 20 / 24, IL-22 / 20 / 24, TGF-β, TREM1, and TREM2. In some embodiments, the chimeric switch receptor extracellular domain binds to an antigen of the disclosure. In some embodiments, the chimeric switch receptor extracellular domain comprises a CAR extracellular domain of the present disclosure. In some embodiments, the CAR extracellular domain comprises an Fc receptor (FcR) extracellular domain of the present disclosure. In some embodiments, the CAR extracellular domain comprises a toll-like receptor (TLR) extracellular domain of the present disclosure. In some embodiments, the CAR extracellular domain comprises a leader domain of the present disclosure. In some embodiments, the CAR extracellular domain comprises an antigen-binding domain of the present disclosure. In some embodiments, the CAR extracellular domain comprises a hinge domain of the present disclosure. In some embodiments, the CAR 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 CAR 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 CAR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0267] In some embodiments, the chimeric switch receptor antigen-binding domain comprises any domain that binds to an antigen. In some embodiments, the chimeric switch receptor 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, e.g., an scFv. In some embodiments, the chimeric switch receptor 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 chimeric switch receptor antigen-binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, the chimeric switch receptor antigen-binding domain is derived, in whole or in part, from the same species in which the chimeric switch receptor will ultimately be used. For example, when used in humans, the antigen-binding domain of the chimeric switch receptor comprises a human antibody, a humanized antibody, or a fragment thereof (e.g., an scFv). In some embodiments, the chimeric switch receptor antigen-binding domain can be a domain endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the chimeric switch receptor 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).

[0268] Chimeric switch receptor transmembrane domains In some embodiments, the chimeric switch receptor comprises a transmembrane domain, for example, connecting the extracellular domain to the intracellular domain. In some embodiments, the chimeric switch receptor transmembrane domain is naturally associated with one or more other domain(s) of the chimeric switch receptor. In some embodiments, to minimize interactions with other members of the receptor complex, the chimeric switch receptor transmembrane domain can be modified to avoid binding to the transmembrane domain of other surface membrane proteins. In some embodiments, the chimeric switch receptor transmembrane domain can be derived from either naturally occurring or synthetic sources. In some embodiments, the chimeric switch receptor transmembrane domain is derived from a naturally occurring membrane-associated or transmembrane protein. In some embodiments, the chimeric switch receptor transmembrane domain is or comprises a human transmembrane domain. In some embodiments, the chimeric switch receptor 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 chimeric switch receptor transmembrane domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0269] In some embodiments, the chimeric switch receptor transmembrane domain comprises a CAR transmembrane domain of the present disclosure. In some embodiments, the CAR transmembrane domain comprises an Fc receptor (FcR) transmembrane domain of the present disclosure. In some embodiments, the CAR transmembrane domain comprises a toll-like receptor (TLR) transmembrane domain of the present disclosure.

[0270] In some embodiments, the chimeric switch receptor 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, 41 BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3 zeta, FcRγ, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPβ, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain.

[0271] Chimeric switch receptor intracellular domain In some embodiments, the chimeric switch receptor comprises one or more intracellular domains. In some embodiments, the chimeric switch receptor intracellular domain is or comprises a human intracellular domain or a portion thereof. In some embodiments, the chimeric switch receptor 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 chimeric switch receptor intracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0272] In some embodiments, the chimeric switch receptor intracellular domain comprises a CAR intracellular domain of the present disclosure. In some embodiments, the CAR intracellular domain comprises an Fc receptor (FcR) intracellular domain of the present disclosure. In some embodiments, the CAR intracellular domain comprises a toll-like receptor (TLR) intracellular domain of the present disclosure.

[0273] In some embodiments, the intracellular domain and / or other cytoplasmic domain of the chimeric switch receptor participates in activation of a cell (e.g., an immune cell) in which the chimeric switch receptor is expressed. In some embodiments, the intracellular domain of the chimeric switch receptor participates in signal activation and / or transduction in an immune cell that contains the chimeric switch receptor.

[0274] In some embodiments, the intracellular domain of the chimeric switch receptor comprises at least one domain involved in signal activation and / or transduction. In some embodiments, the chimeric switch receptor intracellular domain is or comprises at least one of a costimulatory molecule and a signaling domain. In some embodiments, the intracellular domain of the chimeric switch receptor comprises dual signaling domains. In some embodiments, the intracellular domain of the chimeric switch receptor comprises three or more signaling domains.

[0275] In some embodiments, the one or more chimeric switch receptor intracellular signaling domains are selected from the group consisting of CD3-zeta, FcR γ, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40 , CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, S IRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b , SIRPβ, CD22, PIR-B, LILRB1, Syk, 41BB ligand (41BBL; TNFSF9), CD27, OX40L, CD32b, CD11b, ITGAM, SLAMF7, CD206, CD163, CD209, CD89, FLT3, Dectin-2, or one or more cytokine receptor signaling domains (e.g., IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, Dectin-1, or ICOSL intracellular signaling domain).

[0276] In some embodiments, the intracellular domain of the chimeric switch receptor comprises dual signaling domains, e.g., 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, Dectin-1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, in any combination with any of the signaling domains listed in the paragraph above.

[0277] In some embodiments, the chimeric switch receptor intracellular domain comprises a cytoplasmic portion of a surface receptor. In some embodiments, the chimeric switch receptor intracellular domain comprises a costimulatory molecule. In some embodiments, the chimeric switch receptor intracellular domain comprises a molecule that acts to initiate signaling in an immune cell.

[0278] As used herein, a "costimulatory molecule" or "costimulatory domain" refers to a molecule in an immune cell that is used to enhance or attenuate initial stimulation. For example, pathogen-associated pattern recognition receptors such as TLRs or the CD47 / SIRPα axis are molecules on immune cells that enhance or attenuate initial stimulation, respectively. In some embodiments, the costimulatory domain is selected from the group consisting of TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, and LIG. HT, NKG2C, B7-H3, CD83-specific binding ligand, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, I TGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), Costimulatory molecules include CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0279] In some embodiments, the chimeric switch receptor costimulatory domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the chimeric switch receptor costimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0280] As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal, such as activation of a chimeric switch receptor on an immune cell, results in activation of the immune cell.

[0281] In some embodiments, the intracellular domain of the chimeric switch receptor comprises any portion of one or more costimulatory molecules, including at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0282] Chimeric Switch Receptor Peptide Agents As used herein, a chimeric switch receptor peptide agent refers to a peptide that is co-expressed with a chimeric switch receptor in an immune cell. In some embodiments, a chimeric switch receptor peptide agent is co-expressed with a chimeric switch receptor to ensure stoichiometric balance and optimal signaling of the chimeric switch receptor. In some embodiments, a chimeric switch receptor peptide agent forms a homodimer with an identical chimeric switch receptor peptide agent. In some embodiments, a chimeric switch receptor peptide agent forms a heterodimer with a different chimeric switch receptor peptide agent. In some embodiments, a nucleic acid described herein comprises one or more nucleic acid sequences encoding one or more chimeric switch receptor peptide agents. In some embodiments, a chimeric switch receptor peptide agent is or comprises an FcR gamma chain.

[0283] In some embodiments, a chimeric switch receptor peptide agent comprises any peptide, protein, receptor, secreted antibody, or fragment thereof (e.g., scFv, Fab, Fab', F(ab')2, Fc, or nanobody). In some embodiments, a chimeric switch receptor peptide agent comprises one or more cytokines (e.g., one or more of IL-1, IL-2, IL-6, IL-8, TNF-α, IFNα, IFNβ, IFN-γ, GMCSF, or MCSF), CD40-L, dominant negative SIRPα, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC10, or dominant negative LILRB.

[0284] Membrane-tethered cytokines The term "membrane-tethered cytokine," as used herein, refers to an artificial chimeric protein comprising a cytokine fused to a membrane tether such that the cytokine stimulates neighboring cells in trans. Membrane-tethered cytokines can be used as therapeutic agents, for example, by adoptive cell transfer. For example, in some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) are removed from a patient (e.g., from the blood, tumor, or ascites) and modified to express the membrane-tethered cytokine. In some embodiments, such modified immune cells are then reintroduced into the same or a different patient as a therapeutic agent. In some embodiments, the use of modified immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) comprising the membrane-tethered cytokine avoids the toxicity associated with systemic delivery of cytokines.

[0285] In some embodiments, the membrane-tethered cytokine comprises an extracellular domain and a membrane tether (see Figure 30). In some embodiments, the extracellular domain is or comprises a pro-inflammatory (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), 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, or GM-CSF. In some embodiments, the extracellular domain is or comprises an anti-inflammatory (M2) cytokine. In 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 membrane-tethered cytokine is or comprises an extracellular domain comprising 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 extracellular domain is or comprises IFN-β.

[0286] In some embodiments, a "membrane tether" comprises a membrane anchor region and a spacer region. In some embodiments, the membrane anchor region comprises a protein-based transmembrane domain (TMD) or a lipid-based post-translational modification (e.g., a glycolipid). In some embodiments, the protein-based TMD comprises up to 30 amino acids, preferably 18-25 amino acids. In some embodiments, the protein-based TMD adopts an alpha-helical secondary structure. In some embodiments, the protein-based TMD comprises more than 50% hydrophobic residues (e.g., isoleucine, valine, leucine, tryptophan, alanine, or methionine). In some embodiments, the protein-based TMD adopts an alpha-helical secondary structure and comprises more than 50% hydrophobic residues (e.g., isoleucine, valine, leucine, tryptophan, alanine, or methionine). In some embodiments, the protein-based TMD is derived from B7, CD86, EGFR, PDGFRA, PDGFRB, PDL1, Notch, CD4, CD8, ICAM-1, VCAM-1, EPCAM, PECAM1, or NCAM-1. In some embodiments, the lipid-based membrane anchor region comprises a glycosylphosphatidylinositol (GPI) anchor. In some embodiments, the lipid-based membrane anchor region comprises a C-terminal amino acid signal sequence (i.e., a GPI signal sequence) that directs post-translational addition of the GPI anchor. In some embodiments, the GPI signal sequence is derived from Thy1, CD52, CD55, CD59, GP2, CD87, or folate receptor 1. In some embodiments, the membrane anchor region is fused to a spacer region. In some embodiments, the spacer region comprises up to 120 amino acids, preferably 10 to 48 amino acids. In some embodiments, the spacer sequence comprises a glycine-serine linker; an MMP-cleavable peptide; an immunoglobulin (Ig)-like domain from CD28, CD33, IL1R1, IL1R2, SIGLEC1, SIGLEC7, or Thy1; or at least 20 amino acids from the above Ig-like domains.In some embodiments, the membrane tether is or comprises a B7 transmembrane domain (TMD) membrane anchor region, a B7 TMD membrane anchor region with a matrix metalloproteinase (MMP) linker, a glycosylphosphatidylinositol (GPI) membrane anchor region, or a GPI membrane anchor region with a CD28 spacer region. In some embodiments, the membrane tether can be released from the modified immune cell upon binding of the extracellular domain to a receptor expressed on another cell.

[0287] In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 85% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 96% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 98% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the present disclosure comprises an amino acid sequence that is at least 99% identical to an amino acid sequence selected from Table 9. In some embodiments, a membrane-tethered cytokine of the present disclosure comprises an amino acid sequence identical to an amino acid sequence selected from Table 9.

[0288] In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 80% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 85% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 90% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 95% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 96% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 97% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence at least 98% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane-tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence that is at least 99% identical to a nucleotide sequence selected from Table 9. In some embodiments, a membrane-tethered cytokine of the disclosure is encoded by one or more nucleic acids comprising a sequence that is identical to a nucleotide sequence selected from Table 9.

[0289] Chimeric antigen receptor (CAR) As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically target the cells and / or bind to an antigen. CARs can be used as a therapy, for example, by adoptive cell transfer. For example, in some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, and / or dendritic cells) are removed from a patient (e.g., from the blood, tumor, or ascites) and modified to express a receptor specific for a particular form of antigen. In some embodiments, such modified immune cells are then reintroduced into the same or a different subject as a therapeutic agent. In some embodiments, the CAR is specifically expressed against an antigen, for example, a tumor-associated antigen. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0290] In some embodiments, modified immune cells, e.g., modified stem cells, macrophages, monocytes, or dendritic cells, are generated by expressing a CAR therein. In some embodiments, the immune cells comprise a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and the immune cells comprise stem cells, macrophages, monocytes, or dendritic cells.

[0291] In some embodiments, the CAR may further comprise one or more extracellular leader domains, one or more extracellular hinge domains, and one or more intracellular costimulatory domains.

[0292] In some embodiments, the CAR comprises a spacer domain or hinge between the extracellular domain and the transmembrane domain. In some embodiments, the CAR comprises a spacer domain or hinge between the intracellular domain and the transmembrane domain. As used herein, the term "spacer domain" or "hinge" refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular or intracellular domain of a polypeptide chain. In some embodiments, the spacer domain or hinge may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, a short oligopeptide or polypeptide linker, preferably between 2-10 amino acids in length, may form the link between the transmembrane and intracellular domains of the CAR. An example of a linker includes a glycine-serine doublet.

[0293] In some embodiments, an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR may comprise one or more regulatory systems, including, but not limited to, a safety switch (e.g., an on switch and an off switch, a suicide switch), a logic gate, e.g., an AND gate (e.g., two or more CARs, each lacking one or more signaling domains such that activation of both / all CARs is required for activation or function of the complete immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell)), an OR gate (e.g., two or more CARs, each having 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)).

[0294] The present 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.

[0295] In some embodiments, the CAR comprises an extracellular domain operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, for expression in an immune cell. In some embodiments, the nucleic acid encoding the extracellular domain is operably linked to the nucleic acid encoding the transmembrane domain, which is operably linked to the nucleic acid encoding the intracellular domain.

[0296] In some embodiments, the effector activity of an immune cell comprising a CAR is directed against a target cell that contains an antigen that specifically binds to the antigen-binding domain of the CAR. In some embodiments, the targeted effector activity directed against the target cell is or includes phagocytosis, target cytotoxicity, antigen presentation, or cytokine secretion.

[0297] 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 the 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 to CD47 and acts as a dominant-negative receptor, inhibiting SIRPα activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds to SIRPα comprises, e.g., an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction between CD47 and SIRPα. In some embodiments, a CAR is or comprises a phagocytosis logic gate.

[0298] In some embodiments, an immune cell described herein (e.g., comprising or expressing a chimeric switch receptor, membrane-tethered cytokine, or CAR described herein) comprises or expresses at least one variant or fragment of SIRPα (e.g., dominant-negative SIRPα or a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), PD1 (e.g., dominant-negative PD1 or HAC-I), anti-PD1 scFv (e.g., E27 or durvalumab), Siglec-10, Siglec-9, Siglec-11, and / or SHP-1. In some embodiments, the variant or fragment comprises a mutant intracellular domain. In some embodiments, the variant or fragment does not comprise or express at least one intracellular domain (e.g., the immune cell comprises or expresses an anti-CD47 scFv, a CD8 hinge domain, and a CD8 transmembrane domain). In some embodiments, an immune cell described herein (e.g., comprising or expressing a chimeric switch receptor, membrane-tethered cytokine, and / or CAR described herein) comprises a dominant-negative receptor, e.g., that blocks an inhibitory checkpoint.

[0299] In some embodiments, the CARs described herein further comprise a truncated peptide (e.g., a P2A, F2A, E2A, and / or T2A peptide) and at least one second CAR comprising at least one inhibitory domain of anti-phagocytic signaling. In some embodiments, the at least one second CAR comprises SIRPα (e.g., a high-affinity engineered variant of SIRPα (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., humanized B6H12 scFv), or a CD47-binding extracellular domain or a fragment thereof. In some embodiments, the at least one second CAR comprises a SIRPα transmembrane domain or a fragment thereof. In certain embodiments, the second CAR further comprises a hinge domain (e.g., a CD8 hinge domain). In certain embodiments, at least one second CAR comprises (i) a leader sequence (e.g., a CD8 leader), ii) an extracellular domain (e.g., a SIRPα, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain), and ii) a transmembrane domain (e.g., a SIRPα transmembrane domain). In some embodiments, the CARs described herein further comprise a truncated peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD20 or a fragment thereof, CD19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and / or a fluorescent protein).

[0300] In some embodiments, an immune cell described herein (e.g., comprising or expressing a chimeric switch receptor, membrane-tethered cytokine, and / or CAR described herein) comprises or expresses one or more phosphatase-dead domains (e.g., phosphatase-dead Shp1, phosphatase-dead 72-5ptase (INPP5E), phosphatase-dead Shp2, and / or phosphatase-dead SHIP-1 domains) and / or constitutively active kinase domains (e.g., constitutively active LYN domains). 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., phosphatase-dead Shp1, phosphatase-dead 72-5ptase (INPP5E), phosphatase-dead Shp2, and / or phosphatase-dead SHIP-1 domains) and / or a constitutively active kinase domain (e.g., a constitutively active LYN domain).

[0301] CAR extracellular domain The present disclosure provides chimeric antigen receptors (CARs) comprising an extracellular domain. In some embodiments, the CAR extracellular domain comprises an Fc receptor (FcR) extracellular domain. In some embodiments, the CAR extracellular domain comprises a toll-like receptor (TLR) extracellular domain. In some embodiments, the CAR extracellular domain comprises a leader domain. In some embodiments, the CAR extracellular domain comprises an antigen-binding domain. In some embodiments, the CAR extracellular domain comprises a hinge domain. In some embodiments, the CAR 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 CAR 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 CAR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0302] FcR extracellular domain In some embodiments, the FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, the FcR extracellular domain (or portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, the FcR extracellular domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the FcR extracellular domain comprises a CD64 (FcγRI), CD32a (FcγRIIa), CD32b (FcγRIIb), CD32c, CD16a (FcγRIIIa), CD16b (FcγRIIIb), FcεRI, FcεRII, or FcαRI (CD89) domain.

[0303] TLR extracellular domains In some embodiments, the TLR ectodomain comprises a full-length TLR ectodomain. In some embodiments, the TLR ectodomain comprises a portion of a full-length TLR ectodomain. In some embodiments, the TLR ectodomain (or portion thereof) is or comprises a human TLR ectodomain. In some embodiments, the TLR ectodomain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the TLR ectodomain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

[0304] Leader Domain In some embodiments, the CAR comprises one or more extracellular leader domains. In some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence encoding an extracellular leader domain, but the extracellular leader domain is cleaved from the CAR before the CAR is expressed in an immune cell. In some embodiments, the extracellular leader domain is or comprises a human extracellular leader domain. In some embodiments, the extracellular leader domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the extracellular leader domain comprises a CD8 extracellular leader domain. In some embodiments, the extracellular leader domain comprises a leader domain derived from a stimulatory or costimulatory domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88 domain).

[0305] antigen-binding domain In some embodiments, the CAR comprises an antigen binding domain that binds to, for example, an antigen on a target cell. In some embodiments, the CAR comprises an antigen binding domain that binds to an antigen associated with a viral infection, a bacterial infection, a parasitic infection, an autoimmune disease, and / or a cancer cell. In some embodiments, the CAR antigen binding domain recognizes an antigen that acts as a cell surface marker on target cells associated with a particular disease state.

[0306] In some embodiments, the CAR antigen binding domain binds to a tumor antigen, such as an antigen specific to a tumor or cancer of interest. In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B-cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), protease-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21 (testisin or PRSS21), vascular endothelial growth factor receptor 2 (V EGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface-associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, beta9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), and claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8),Melanoma antigen recognized by T cell 1 (MelanA or MART1), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Proteins) Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mut) hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), or immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, the tumor antigen isIn certain embodiments, the tumor antigen comprises ERBB2 (Her2 / neu). In certain embodiments, the tumor antigen comprises PSMA. In certain embodiments, the tumor antigen comprises mesothelin.

[0307] In some embodiments, the CAR antigen binding domain binds to a misfolded protein antigen or a protein in a protein aggregate, e.g., a protein specific to a disease / disorder of interest. In some embodiments, the disease / disorder is a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease / disorder, or amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or transthyretin). In some embodiments, the neurodegenerative disease / disorder is a tauopathy, amyloid nucleopathy, presenile dementia, senile dementia, Alzheimer's disease (mediated by beta-amyloid protein aggregates), Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine diseases, trinucleotide repeat diseases, familial British dementia, fatal familial insomnia, Gerstmann-Sträussler-Scheinker syndrome, hereditary cerebral hemorrhage with amyloidosis (Icelandic type) (HCHW). AI), sporadic fatal insomnia (sFI), variably protease-sensitive prion disease (VPSPr), familial Danish dementia, and prion diseases (such as Creutzfeldt-Jakob disease, CJD, and variant Creutzfeldt-Jakob disease (vCJD)).

[0308] In some embodiments, the CAR antigen-binding domain comprises any domain that binds to an antigen. In some embodiments, the CAR 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 CAR 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 CAR antigen-binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, the CAR 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 CAR antigen-binding domain can be a domain endogenous to a particular immune cell type (e.g., the modified immune cells provided herein). In some embodiments, the CAR 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).

[0309] In some embodiments, the CAR comprises one or more antigen-binding domains. In some embodiments, the CAR comprises two or more antigen-binding domains. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the immune cells comprise two or more different CARs comprising one or more antigen-binding domains. In some embodiments, immune cells comprising bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of two antigens. In some embodiments, the immune cells comprise bispecific CARs and / or two or more different CARs comprising one or more antigen-binding domains, where the CARs alone are insufficient to mediate activation of the modified cell, but together are synergistic and provide distinct signals that stimulate activation of the modified cell. In some embodiments, such a configuration may be referred to as an "AND" logic gate.

[0310] In some embodiments, immune cells containing bispecific CARs and / or two or more different CARs containing one or more antigen-binding domains can reduce off-target and / or on-target extratissue effects by requiring the presence of one antigen and the absence of a second, normal protein antigen before cellular activity is stimulated. In some embodiments, such a configuration may be referred to as a "NOT" logic gate. In contrast to AND gates, NOT-gate CAR-modified cells are activated by binding to a single antigen. However, binding of a second receptor to the second antigen functions to neutralize the activation signal perpetuated through the CAR. Typically, such inhibitory receptors target antigens that are abundantly expressed in normal tissues but absent in tumor tissues.

[0311] Hinge domain In some embodiments, the CAR comprises one or more extracellular hinge domains. In some embodiments, the CAR extracellular hinge domain is or comprises a human extracellular hinge domain. In some embodiments, the CAR extracellular hinge domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR extracellular hinge domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the one or more CAR extracellular hinge domains comprise a CD8a extracellular hinge domain or an IgG4 or CD28 extracellular hinge domain. In some embodiments, the CAR extracellular hinge domain optimizes the physicochemical parameters of the CAR, such as, for example, optimal size (e.g., allowing for exclusion of inhibitory molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and / or lack of homodimerization to tumor antigens.

[0312] CAR transmembrane domain In some embodiments, the CAR comprises a transmembrane domain, for example, connecting the extracellular domain to the intracellular domain. In some embodiments, the CAR transmembrane domain is naturally associated with one or more other domain(s) of the CAR. In some embodiments, to minimize interaction with other members of the receptor complex, the CAR transmembrane domain can be modified to avoid binding of other surface membrane proteins to the transmembrane domain. In some embodiments, the CAR transmembrane domain can be derived from either a naturally occurring source or a synthetic source. In some embodiments, the CAR transmembrane domain is derived from a naturally occurring membrane-associated or transmembrane protein. In some embodiments, the CAR transmembrane domain is or comprises a human transmembrane domain. In some embodiments, the CAR 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 CAR 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 CAR 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, including CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3 zeta, FcRγ, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPβ, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain.

[0313] 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.

[0314] 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.

[0315] CAR intracellular domain In some embodiments, a CAR comprises one or more intracellular domains. In some embodiments, the CAR intracellular domain is or comprises a human intracellular domain or portion thereof. In some embodiments, the CAR 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 CAR 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 CAR intracellular domain and / or other cytoplasmic domains of a CAR are involved in activation of the cell (e.g., immune cell) in which the CAR is expressed. In some embodiments, the CAR intracellular domain of a CAR is involved in signal activation and / or transduction in the immune cell comprising the CAR.

[0316] In some embodiments, the CAR intracellular domain of the CAR comprises at least one domain involved in signal activation and / or transduction. In some embodiments, the CAR intracellular domain is or comprises at least one of a costimulatory molecule and a signaling domain. In some embodiments, the CAR intracellular domain of the CAR comprises dual signaling domains. In some embodiments, the CAR intracellular domain of the CAR comprises three or more signaling domains.

[0317] In some embodiments, the CAR intracellular domain comprises the cytoplasmic portion of a surface receptor. In some embodiments, the CAR intracellular domain comprises a costimulatory molecule. In some embodiments, the CAR intracellular domain comprises a molecule that acts to initiate signal transduction in immune cells.

[0318] In some embodiments, the intracellular domain of the CAR comprises any portion of one or more costimulatory molecules, for example, at least one signaling domain from CD3, Fc epsilon RI gamma chain, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0319] 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.

[0320] 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.

[0321] Signaling domains In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, the CAR intracellular signaling domain is or comprises a human intracellular signaling domain or a portion thereof. In some embodiments, the CAR signaling 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 CAR signaling domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0322] In some embodiments, one or more CAR intracellular signaling domains are selected from the group consisting of CD3 zeta, FcR γ, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL , DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD 40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V / I / LxYxxL / V, SIRPα, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, and one or more cytokine receptor signaling domains (e.g., IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, Dap10, CD36, Dectin-1, or ICOSL intracellular signaling domains).

[0323] In some embodiments, the intracellular domain of the CAR comprises dual signaling domains, e.g., 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain, CSF1-R, LRP1 / CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, Dectin-1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, in any combination with any of the signaling domains listed in the paragraph above.

[0324] 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, a CAR 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, L IGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, 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, ITGAE, 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.

[0325] In some embodiments, the CAR costimulatory domain can be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell provided herein). In some embodiments, the CAR costimulatory domain can be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell provided herein).

[0326] As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal, such as activation of a CAR on an immune cell, results in activation of the immune cell.

[0327] Cleaved peptide As used herein, a cleavage peptide refers to a peptide that can induce cleavage of a recombinant protein within a cell. In some embodiments, the cleavage peptide is a 2A peptide. In some embodiments, the cleavage peptide is or comprises a P2A, F2A, E2A, or T2A peptide. In some embodiments, the nucleic acids described herein comprise one or more nucleic acid sequences encoding one or more cleavage peptides. In some embodiments, the nucleic acid comprising the nucleic acid sequence encoding the cleavage peptide also comprises one or more nucleic acid sequences encoding one or more intracellular domains and one or more nucleic acid sequences comprising one or more peptide agents, wherein translation of the nucleic acid results in a protein comprising one or more intracellular domains separated from the one or more peptide agents by the cleavage peptides. In some embodiments, a first promoter is operably linked to one or more nucleic acids encoding a CAR, and a second promoter is operably linked to one or more nucleic acids encoding a peptide agent. In some embodiments, the nucleic acid sequence comprising a CAR and optionally one or more peptide agents further comprises an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA, facilitating initiation of translation.

[0328] CAR peptide agents As used herein, a CAR peptide agent refers to a peptide that is co-expressed with a CAR in an immune cell. In some embodiments, the CAR peptide agent is co-expressed with a CAR to ensure stoichiometric balance and optimal signaling of the CAR. In some embodiments, a CAR peptide agent forms a homodimer with the same peptide agent. In some embodiments, a CAR peptide agent forms a heterodimer with a different peptide agent. In some embodiments, a nucleic acid described herein comprises one or more nucleic acid sequences encoding one or more CAR peptide agents. In some em...

Claims

1. A modified immune cell comprising a chimeric switch receptor or one or more nucleic acids encoding a chimeric switch receptor, The modified immune cells are stem cells, macrophages, monocytes, or dendritic cells. The chimeric switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is derived from the first receptor, and the intracellular domain is derived from the second receptor. The second receptor is a cytokine receptor, as described above, on the modified immune cell.

2. The modified immune cell according to claim 1, wherein the transmembrane domain is derived from the first receptor or the second receptor.

3. (a) The first receptor is a pro-inflammatory cytokine receptor or an anti-inflammatory cytokine receptor, (b) The second receptor is an anti-inflammatory cytokine receptor or a pro-inflammatory cytokine receptor. (c) The first receptor is (i) an anti-inflammatory cytokine receptor, wherein the second receptor is either an anti-inflammatory cytokine receptor or (ii) an anti-inflammatory cytokine receptor, the second receptor being an anti-inflammatory cytokine receptor. (d) The first receptor is Table 11-1 Table 11-2 A cytokine receptor selected from and / or (e) The second receptor is Table 12-1 Table 12-2 Table 12-3 Table 12-4 Modified immune cells according to claim 1, wherein the cytokine receptor is selected from the above.

4. (a) The first receptor is IFNGR1, and the second receptor is IL10Ra, (b) The first receptor is IL17Ra, and the second receptor is GCSFR, (c) The first receptor is IL17Ra, and the second receptor is TNFR2, (d) The first receptor is IL17Ra, and the second receptor is TREM2, (e) The first receptor is IL17Ra, and the second receptor is MerTK, or (f) The modified immune cell according to claim 1, wherein the first receptor is IL17Ra and the second receptor is IL10Ra.

5. The first receptor is, Table 13-1 Table 13-2 Table 13-3 Table 13-4 Table 13-5 Table 13-6 Table 13-7 Table 13-8 Table 13-9 Table 13-10 Table 13-11 Table 13-12 Table 13-13 Table 13-14 Table 13-15 Table 13-16 Table 13-17 Table 13-18 Table 13-19 Table 13-20 Table 13-21 Table 13-22 Table 13-23 Table 13-24 Table 13-25 Table 13-26 Table 13-27 Table 13-28 Table 13-29 Table 13-30 Table 13-31 Table 13-32 Table 13-33 Table 13-34 Table 13-35 Modified immune cells comprising the chimeric switch receptor according to claim 1, which is a cytokine receptor comprising an amino acid sequence that is at least 80% identical to a sequence selected from.

6. The second receptor is, Table 14-1 Table 14-2 Table 14-3 Table 14-4 Table 14-5 Table 14-6 Table 14-7 Table 14-8 Table 14-9 Table 14-10 Table 14-11 Table 14-12 Table 14-13 Table 14-14 Table 14-15 Table 14-16 Table 14-17 Table 14-18 Table 14-19 Table 14-20 Table 14-21 Table 14-22 Table 14-23 Table 14-24 Table 14-25 Table 14-26 Table 14-27 Table 14-28 Table 14-29 Table 14-30 Table 14-31 Table 14-32 Table 14-33 Table 14-34 Table 14-35 Table 14-36 Table 14-37 Table 14-38 Table 14-39 Table 14-40 Table 14-41 Table 14-42 Modified immune cells comprising the chimeric switch receptor according to claim 1, which is a cytokine receptor comprising an amino acid sequence that is at least 80% identical to a sequence selected from.

7. The first receptor is, Table 15-1 Table 15-2 Table 15-3 Table 15-4 Table 15-5 Table 15-6 Table 15-7 Table 15-8 Table 15-9 Table 15-10 Table 15-11 Table 15-12 Table 15-13 Table 15-14 Table 15-15 Table 15-16 Table 15-17 Table 15-18 Table 15-19 Table 15-20 Table 15-21 Table 15-22 Table 15-23 Table 15-24 Table 15-25 Table 15-26 Table 15-27 Table 15-28 Table 15-29 Table 15-30 Table 15-31 Table 15-32 Table 15-33 Table 15-34 Table 15-35 Table 15-36 Table 15-37 Table 15-38 Table 15-39 Table 15-40 Table 15-41 Table 15-42 Table 15-43 Table 15-44 Table 15-45 Table 15-46 Table 15-47 Table 15-48 Table 15-49 Table 15-50 Table 15-51 Table 15-52 Table 15-53 Table 15-54 Table 15-55 Table 15-56 Table 15-57 Table 15-58 Table 15-59 Table 15-60 Table 15-61 Table 15-62 Table 15-63 Table 15-64 Table 15-65 Table 15-66 Table 15-67 Table 15-68 Table 15-69 Table 15-70 Table 15-71 Table 15-72 Table 15-73 Table 15-74 Table 15-75 Table 15-76 Table 15-77 Table 15-78 Table 15-79 Table 15-80 Table 15-81 Table 15-82 Table 15-83 Table 15-84 Table 15-85 Table 15-86 A modified immune cell comprising one or more nucleic acids encoding the chimeric switch receptor according to claim 1, which is a cytokine receptor encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from.

8. The second receptor is, Table 16-1 Table 16-2 Table 16-3 Table 16-4 Table 16-5 Table 16-6 Table 16-7 Table 16-8 Table 16-9 Table 16-10 Table 16-11 Table 16-12 Table 16-13 Table 16-14 Table 16-15 Table 16-16 Table 16-17 Table 16-18 Table 16-19 Table 16-20 Table 16-21 Table 16-22 Table 16-23 Table 16-24 Table 16-25 Table 16-26 Table 16-27 Table 16-28 Table 16-29 Table 16-30 Table 16-31 Table 16-32 Table 16-33 Table 16-34 Table 16-35 Table 16-36 Table 16-37 Table 16-38 Table 16-39 Table 16-40 Table 16-41 Table 16-42 Table 16-43 Table 16-44 Table 16-45 Table 16-46 Table 16-47 Table 16-48 Table 16-49 Table 16-50 Table 16-51 Table 16-52 Table 16-53 Table 16-54 Table 16-55 Table 16-56 Table 16-57 Table 16-58 Table 16-59 Table 16-60 Table 16-61 Table 16-62 Table 16-63 Table 16-64 Table 16-65 Table 16-66 Table 16-67 Table 16-68 Table 16-69 Table 16-70 Table 16-71 Table 16-72 Table 16-73 Table 16-74 Table 16-75 Table 16-76 Table 16-77 Table 16-78 Table 16-79 Table 16-80 Table 16-81 Table 16-82 Table 16-83 Table 16-84 Table 16-85 Table 16-86 Table 16-87 Table 16-88 Table 16-89 Table 16-90 Table 16-91 Table 16-92 Table 16-93 Table 16-94 A modified immune cell comprising one or more nucleic acids encoding the chimeric switch receptor according to claim 1, which is a cytokine receptor encoded by a nucleic acid sequence that is at least 80% identical to a sequence selected from.

9. Modified immune cells comprising the chimeric switch receptor according to claim 1, further comprising one or more additional chimeric switch receptors, the one or more additional chimeric switch receptors comprising a combination of extracellular and intracellular domains different from the extracellular and intracellular domains of the chimeric switch receptor, and / or further comprising a chimeric antigen receptor (CAR).

10. A polynucleotide encoding one or more chimeric switch receptors, where each chimeric switch receptor is (a) Extracellular domain, (b) Transmembrane domain, and (c) including intracellular domains, The extracellular domain is, Table 17-1 Table 17-2 Derived from a first receptor selected from, the intracellular domain is Table 18-1 Table 18-2 Table 18-3 Table 18-4 Derived from a second receptor selected from, and The second receptor is the polynucleotide, which is a cytokine receptor.

11. The polynucleotide according to claim 10, wherein the transmembrane domain is derived from the first receptor or the second receptor, and / or the first receptor is a cytokine receptor.

12. (a) The first cytokine receptor is IFNGR1, and the second cytokine receptor is IL10Ra, (b) The first cytokine receptor is IL17Ra, and the second cytokine receptor is GCSFR, (c) The first cytokine receptor is IL17Ra, and the second cytokine receptor is TNFR2, (d) The first cytokine receptor is IL17Ra, and the second cytokine receptor is TREM2, (e) The first cytokine receptor is IL17Ra, and the second cytokine receptor is MerTK, or (f) The polynucleotide according to claim 11, wherein the first cytokine receptor is IL17Ra and the second cytokine receptor is IL10Ra.

13. (a) The extracellular domain is Table 19-1 Table 19-2 Table 19-3 Table 19-4 Table 19-5 Table 19-6 Table 19-7 Table 19-8 Table 19-9 Table 19-10 Table 19-11 Table 19-12 Table 19-13 Table 19-14 Table 19-15 Table 19-16 Table 19-17 Table 19-18 Table 19-19 Table 19-20 Table 19-21 Table 19-22 Table 19-23 Table 19-24 Table 19-25 Table 19-26 Table 19-27 Table 19-28 Table 19-29 Table 19-30 Table 19-31 Table 19-32 Table 19-33 Table 19-34 Table 19-35 Table 19-36 Table 19-37 Table 19-38 Table 19-39 Table 19-40 Table 19-41 Table 19-42 Table 19-43 Table 19-44 Table 19-45 Table 19-46 Table 19-47 Table 19-48 Table 19-49 Table 19-50 Table 19-51 Table 19-52 Table 19-53 Table 19-54 Table 19-55 Table 19-56 Table 19-57 Table 19-58 Table 19-59 Table 19-60 Table 19-61 Table 19-62 Table 19-63 Table 19-64 Table 19-65 Table 19-66 Table 19-67 Table 19-68 Table 19-69 Table 19-70 Table 19-71 Table 19-72 Table 19-73 Table 19-74 Table 19-75 Table 19-76 Table 19-77 Table 19-78 Table 19-79 Table 19-80 Table 19-81 Table 19-82 Table 19-83 Table 19-84 Table 19-85 Table 19-86 Encoded by a nucleic acid sequence that is at least 80% identical to the sequence selected from, (b) The intracellular domain is Table 20-1 Table 20-2 Table 20-3 Table 20-4 Table 20-5 Table 20-6 Table 20-7 Table 20-8 Table 20-9 Table 20-10 Table 20-11 Table 20-12 Table 20-13 Table 20-14 Table 20-15 Table 20-16 Table 20-17 Table 20-18 Table 20-19 Table 20-20 Table 20-21 Table 20-22 Table 20-23 Table 20-24 Table 20-25 Table 20-26 Table 20-27 Table 20-28 Table 20-29 【Table 20-30】 【Table 20-31】 Table 20-32 Table 20-33 Table 20-34 Table 20-35 Table 20-36 Table 20-37 Table 20-38 Table 20-39 Table 20-40 Table 20-41 Table 20-42 Table 20-43 Table 20-44 Table 20-45 Table 20-46 Table 20-47 Table 20-48 Table 20-49 Table 20-50 Table 20-51 Table 20-52 Table 20-53 Table 20-54 Table 20-55 Table 20-56 Table 20-57 Table 20-58 Table 20-59 Table 20-60 Table 20-61 Table 20-62 Table 20-63 Table 20-64 Table 20-65 Table 20-66 Table 20-67 Table 20-68 Table 20-69 Table 20-70 Table 20-71 Table 20-72 Table 20-73 Table 20-74 Table 20-75 Table 20-76 Table 20-77 Table 20-78 Table 20-79 Table 20-80 Table 20-81 Table 20-82 Table 20-83 Table 20-84 Table 20-85 Table 20-86 Table 20-87 Table 20-88 Table 20-89 Table 20-90 Table 20-91 Table 20-92 Table 20-93 Table 20-94 Encoded by a nucleic acid sequence that is at least 80% identical to the sequence selected from, (c) Table 21-1 Table 21-2 Table 21-3 Table 21-4 Table 21-5 Table 21-6 Table 21-7 Table 21-8 Table 21-9 Table 21-10 Table 21-11 Table 21-12 Table 21-13 Table 21-14 Table 21-15 Table 21-16 Table 21-17 Table 21-18 Table 21-19 Table 21-20 Table 21-21 Table 21-22 Table 21-23 Table 21-24 Table 21-25 Table 21-26 Table 21-27 Table 21-28 Table 21-29 Table 21-30 Table 21-31 Table 21-32 Table 21-33 Table 21-34 Table 21-35 Table 21-36 Table 21-37 Table 21-38 Table 21-39 Table 21-40 Table 21-41 Table 21-42 Table 21-43 Table 21-44 Table 21-45 Table 21-46 Table 21-47 Table 21-48 Table 21-49 Table 21-50 Table 21-51 Table 21-52 Table 21-53 Table 21-54 Table 21-55 Table 21-56 Table 21-57 Table 21-58 Table 21-59 Table 21-60 Table 21-61 Table 21-62 Table 21-63 Table 21-64 Table 21-65 Table 21-66 A nucleic acid sequence that is at least 80% identical to a sequence selected from, (d) The polynucleotide encodes one or more chimeric switch receptors as a single polypeptide chain, and / or (e) The polynucleotide according to claim 12, wherein the one or more chimeric switch receptors are separated by one or more cleavage peptide sites.

14. The polynucleotide according to claim 13, wherein one or more cleavage peptide sites are selected from the group consisting of P2A, F2A, E2A, and T2A.

15. A composition for treating or preventing a target disease or disorder, comprising modified immune cells according to any one of claims 1 to 9 or polynucleotides according to any one of claims 10 to 14, characterized in that at least one sign or symptom of the disease or disorder is improved in the target after administration of the composition.

16. The composition according to claim 15, wherein the administration of the composition is by transarterial, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal delivery, or includes these methods.

17. A composition for use in a method for modifying immune cells, comprising a polynucleotide according to any one of claims 10 to 14, wherein the method comprises introducing the composition into the immune cells.

18. (a) The polynucleotide comprises DNA or messenger RNA (mRNA), (b) The polynucleotide includes modifications selected from modified nucleotides, modifications to the 5' untranslated region (UTR), modifications to the 3'UTR, cap structures, poly(A) tails, or combinations thereof. (c) The polynucleotide is a purified polynucleotide, and / or (d) The composition according to claim 17, wherein the polynucleotide is codon-optimized.

19. (a) The cap structure comprises AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA), and / or the modified nucleotide comprises pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudridine (5meC PsU), N1-methyl-pseudridine (N1mPsU), or a combination thereof. (b) The purified polynucleotides are prepared 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, and / or (c) The composition according to claim 18, wherein the polynucleotide is codon-optimized for expression in stem cells, monocytes, macrophages, or dendritic cells.

20. (a) The introduction includes electroporation or transfection of the polynucleotide, or (b) The composition according to claim 17, wherein the polynucleotide is encapsulated within the introduction vehicle.

21. The method further comprises introducing an additional payload to the immune cells, the additional payload being a pathogen recognition receptor agonist, polyinosine polycytidic acid (polyI: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, and a circular G of the interferon gene. MP-AMP synthase-stimulating factor (cGAS-STING) agonists, interferon-inducing protein 16 (IFI16) agonists, DEAD box helicase 41 (DDX41) agonists, LRR-binding FLII-interacting protein 1 (LRRFIP1) agonists, melanoma-deficient factor 2 (AIM2) agonists, aryl hydrocarbon receptor (AhR) ligands, or combinations thereof, or containing thereof, and / or The composition according to claim 17, wherein the polynucleotide and the additional payload are encapsulated within the introduction vehicle.

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