Engineered immune cells expressing TLR receptors

Abstract

The present application provides modified immune cells that express a TLR receptor. The modified immune cells further comprise an engineered receptor, such as a chimeric antigen receptor (CAR). The present application also provides methods and pharmaceutical compositions for using the modified immune cells to treat cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / CN2021 / 113239 filed on August 18, 2021, International Application No. PCT / CN2021 / 122129 filed on September 30, 2021, International Application No. PCT / CN2021 / 133061 filed on November 25, 2021, and International Application No. PCT / CN2022 / 112578 filed on August 15, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following ASCII text file submitted are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: P11234-PCT.220817.Sequence listing.xml, Recorded: August 17, 2022, Size: 117KB).

[0003] Field This application relates to modified immune cells that express TLR receptors and methods of using same to treat diseases or conditions such as cancer. [Background technology]

[0004] Chimeric antigen receptor (CAR) T cells are cells that have already been modified to induce immune responses by producing engineered T cell receptors. For example, CAR-T cells can be engineered to more effectively recognize cancer cells to improve cancer therapy. Although CAR-T therapy has been successful, these methods often suffer from poor in vivo T cell expansion and exhaustion, resulting in poor durability of clinical remission in patients with hematological malignancies (e.g., acute myeloid leukemia). There remains a need for efficient cell-based cancer immunotherapy.

[0005] NKG2D is a transmembrane protein and belongs to the NKG2 family of C-type lectin-like receptors. In humans, it is expressed by NK cells, γδT cells, and CD8+ αβT cells. NKG2D ligands are derived self-proteins from the MIC and RAET1 / ULBP families, which are absent or present at low levels on the surface of normal cells but are overexpressed by infected, transformed, senescent, and stressed cells (Zingoni, A et al., 2018, Front Immunol. 9:476 (Non-Patent Document 1)).

[0006] Toll-like receptors (TLRs) are pattern recognition receptors that detect invading pathogens and activate innate and adaptive immune responses. TLRs function as effective costimulatory molecules in T cells and are expressed on the cell surface of activated T cells (e.g., memory CD4+ and CD8+ T cells). Therefore, activation of TLRs in T cells directly enhances T cell activation, function, and survival induced by T cell receptor (TCR) signals (Gelman, AE et al., 2004, 172(10):6065-6073). Given their important role in the immune system, TLR activation has been used to enhance immune responses. Conversely, inhibitors of TLR activation can reduce autoimmunity and other undesirable immune responses (Lu, H, 2014, Front. in Immunol. 5:83). Studies have shown that engineered immune cells, such as T cells, that express CARs can provide enhanced antitumor activity by being armed with toll / interleukin-1 (IL-1) receptor (TIR) ​​domains. See, e.g., Manavalan, B. et al., 2011, Front. Physiol. 2:41 (Non-Patent Document 4).

[0007] The disclosures of all publications, patents, patent applications, and patent applications disclosed herein are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Zingoni, A et al., 2018, Front Immunol.9:476 [Non-patent document 2] Gelman, AE et al., 2004, 172(10):6065-6073 [Non-patent document 3] Lu, H, 2014, Front. in Immunol. 5:83 [Non-patent document 4] Manavalan, B et al., 2011, Front. Physiol. 2:41 Summary of the Invention

[0009] The present application provides modified immune cells that express TLR receptors and methods of using same to treat diseases or conditions such as cancer.

[0010] In one aspect of the present application, there is provided an engineered immune cell comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling.

[0011] In some embodiments of the modified immune cells, the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule. In some embodiments, the subunits of the multimeric target molecule are the same. In some embodiments, the subunits of the multimeric target molecule are different. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule. In some embodiments, the first target binding domain and the second binding domain each bind to the same target site on the target molecule. In some embodiments, the first target binding domain and the second target binding domain are the same.

[0012] In another aspect of the present application, there is provided an engineered immune cell comprising an engineered immune cell, wherein a first target binding domain and a second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different non-overlapping target sites on the single target molecule.

[0013] In some embodiments of any one of the above modified immune cells, the first TLR transmembrane domain and the first TLR signaling domain are derived from the same TLR molecule. In some embodiments, the second TLR transmembrane domain and the second TLR signaling domain are derived from the same TLR molecule. In some embodiments, the first TLR transmembrane domain and the second TLR transmembrane domain are the same. In some embodiments, the first TLR signaling domain and the second TLR signaling domain are the same. In some embodiments, the first TLR transmembrane domain and / or the first TLR signaling domain is derived from TLR4. In some embodiments, the first TLR transmembrane domain and the second TLR transmembrane domain are different. In some embodiments, the first TLR signaling domain and the second TLR signaling domain are different. In some embodiments, the first TLR transmembrane domain and / or the first TLR signaling domain is derived from TLR2. In some embodiments, the second TLR transmembrane domain and / or the first TLR signaling domain is derived from TLR6. In some embodiments, the second TLR transmembrane domain and / or the second TLR signaling domain is derived from TLR1.

[0014] In some embodiments of any one of the above modified immune cells, the first target binding domain and / or the second target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain and / or the second target binding domain is an scFv or sdAb. In some embodiments, the scFv or sdAb specifically binds to CD33, CLL1, CD123, CD19, CD20, CD22, BCMA, GPRC5D, and GPC3.

[0015] In some embodiments of any one of the above modified immune cells, the target molecule is an immune checkpoint protein, hi some embodiments, the target molecule is selected from the group consisting of PD-1, CD70, CD27, SIRPα, and TIGIT.

[0016] In some embodiments of any one of the above modified immune cells, the target molecule is a naturally occurring protein expressed on the immune cell. In some embodiments, the target molecule is NKG2D. In some embodiments, the target molecule is a mutant NKG2D. In some embodiments, the mutant NKG2D comprises a truncated sequence and / or amino acid substitutions, mutations, additions, and / or deletions.

[0017] In some embodiments, the target molecule is the extracellular antigen-binding domain of NKG2D. In some embodiments, the target molecule is the full-length sequence of NKG2D.

[0018] In some embodiments of any one of the above engineered immune cells, the engineered immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells. In some embodiments, the engineered immune cell is an NK cell. In some embodiments, the engineered immune cell is a cytotoxic T cell. In some embodiments, the engineered immune cell comprises an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered receptor is an engineered T cell receptor (TCR). In some embodiments, the engineered receptor is a T-cell antigen coupler (TAC) receptor.

[0019] In another aspect of the present application, there is provided an engineered immune cell according to any one of the above engineered immune cells, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes a non-overlapping target site on the same target molecule as the first polypeptide and / or the second polypeptide. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes any of CD19, CLL1, BCMA, and GPC3.

[0020] In some embodiments of any one of the above modified immune cells, the modified immune cell comprises a first nucleic acid encoding a first polypeptide and a second nucleic acid encoding a second polypeptide.

[0021] In some embodiments of any one of the above modified immune cells, the first polypeptide and the second polypeptide are the same, and the modified immune cell comprises a first nucleic acid encoding the first polypeptide and the second polypeptide.

[0022] In some embodiments of any one of the above modified immune cells, the modified immune cell comprises a third nucleic acid encoding an engineered receptor. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to separate promoters. In some embodiments, the first nucleic acid and the third nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid and the third nucleic acid are operably linked to separate promoters. In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are operably linked to the same promoter.

[0023] In some embodiments of any one of the above modified immune cells, the first target binding domain and the second target binding domain specifically recognize a subunit of CD20. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD33. In some embodiments, the first target binding domain specifically recognizes the V subunit of CD33, and the second target binding domain specifically recognizes the C2 subunit of CD33. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of BCMA. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of GPRC5D.

[0024] In some embodiments of any one of the above modified immune cells, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor. In some embodiments, the second polypeptide further comprises a second intracellular domain of a second cytokine receptor. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and the second polypeptide further comprises a second intracellular domain of a second cytokine receptor. In some embodiments, the first intracellular domain and the second intracellular domain are the same. In some embodiments, the first intracellular domain and the second intracellular domain are different. In some embodiments, the first cytokine receptor is selected from the group consisting of GM-CSF receptor, IL-18 receptor, IL-21 receptor, IL-15 receptor, and IL-23 receptor. In some embodiments, the second cytokine receptor is selected from the group consisting of GM-CSF receptor, IL-18 receptor, IL-21 receptor, IL-15 receptor, and IL-23 receptor. In some embodiments, the first intracellular domain of the first cytokine receptor comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the second intracellular domain of the second cytokine receptor comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the first intracellular domain of the first cytokine receptor and the second intracellular domain of the second cytokine receptor comprise an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the C-terminus of the first intracellular domain of the first cytokine receptor is fused to the N-terminus of the first TLR signaling domain. In some embodiments, the C-terminus of the second intracellular domain of the second cytokine receptor is fused to the N-terminus of the second TLR signaling domain. In some embodiments, the C-terminus of the first intracellular domain of the first cytokine receptor is fused to the N-terminus of the first TLR signaling domain and the C-terminus of the second intracellular domain of the second cytokine receptor is fused to the N-terminus of the second TLR signaling domain.In some embodiments, the N-terminus of the first intracellular domain of a first cytokine receptor is fused to the C-terminus of the TLR signaling domain. In some embodiments, the N-terminus of the second intracellular domain of a second cytokine receptor is fused to the C-terminus of the TLR signaling domain. In some embodiments, the N-terminus of the first intracellular domain of a first cytokine receptor is fused to the C-terminus of the TLR signaling domain and the N-terminus of the second intracellular domain of a second cytokine receptor is fused to the C-terminus of the TLR signaling domain.

[0025] In one aspect of the application, a method of producing a modified immune cell is provided, the method comprising introducing a first nucleic acid encoding a first polypeptide and, optionally, a second nucleic acid encoding a second polypeptide into a precursor immune cell.

[0026] In some embodiments according to any one of the above production methods, the precursor immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, NK cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells. In some embodiments, the precursor immune cells comprise an engineered receptor. In some embodiments, the production method further comprises introducing into the precursor immune cells a third nucleic acid encoding the engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a modified T cell receptor (TCR), or a T cell antigen coupler (TAC) receptor.

[0027] In some embodiments according to any one of the above methods of production, the first nucleic acid, the second nucleic acid, and / or the third nucleic acid are on the same vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, and derivatives thereof.

[0028] In some embodiments of any one of the above methods of production, the method further comprises isolating or enriching immune cells comprising the first nucleic acid, the second nucleic acid, and / or the third nucleic acid.

[0029] Further provided is an altered immune cell produced by any one of the above production methods.

[0030] Further provided is a pharmaceutical composition comprising a modified immune cell according to any one of the above modified immune cells and a pharmaceutically acceptable carrier.

[0031] In another aspect of the present application, there is provided a method for treating a disease in an individual, the method comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the disease is cancer. In some embodiments, the individual is human.

[0032] Further provided are compositions, uses, kits and articles of manufacture comprising any one of the modified immune cells. [Brief explanation of the drawings]

[0033] [Figure 1]

[0023] Figure 1 shows the bicistronic expression design of a CAR fusion construct comprising an anti-CD19-CAR and an anti-CD20 TLR polypeptide (CD19-co-CD20 CAR, SEQ ID NO: 1). The sequence of the CAR backbone follows the following pattern from N- to C-terminus: signal peptide (SEQ ID NO: 5), anti-CD19 scFv (SEQ ID NO: 6), CD8α hinge domain (SEQ ID NO: 7), CD8α transmembrane (TM) domain (SEQ ID NO: 8), the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain (SEQ ID NO: 9), and the CD3ζ primary intracellular signaling domain (SEQ ID NO: 10). The CD3ζ primary intracellular signaling domain is linked via a P2A cleavage site (SEQ ID NO:24) to an inducible costimulatory molecule that includes, from N- to C-terminus: a signal peptide (SEQ ID NO:5), an anti-CD20 scFv (SEQ ID NO:11), a TLR4 transmembrane (TM) region (SEQ ID NO:12), and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain) (SEQ ID NO:13). [Figure 2] Figure 2 shows the in vitro cytotoxic effects of CD19 BM CAR-T cells and TLR4-armed CD19-co-CD20 CAR-T cells. In particular, Figure 2 shows that CD19 BM CAR-T and CD19-co-CD20 CAR T cells induce lysis of Raji target cells in vitro in a dose-dependent manner. In this experiment, untransduced T cells (i.e., "unT") were used as a control. [Figure 3] Figure 1 shows an in vitro IFNγ cytokine profile of CD19 BM CAR-T cells and TLR4-armed CD19-co-CD20 CAR-T cells co-cultured with Raji target cells. Untransduced T cells (i.e., "unT") were used as a control in this experiment. [Figure 4]Figure 1 shows an in vitro TNFα cytokine profile of CD19 BM CAR-T cells and TLR4-armed CD19-co-CD20 CAR-T cells co-cultured with Raji target cells. Untransduced T cells (i.e., "unT") were used as a control in this experiment. [Figure 5] Figure 1 shows the in vitro killing efficacy of CD19 BM CAR-T cells and TLR4-armed CD19-co-CD20 CAR-T cells in a repeated tumor stimulation assay with Raji target cells. Untransduced T cells (i.e., "UNT") were used as a control in this experiment. [Figure 6] Figure 1 shows in vitro expansion (e.g., magnification) of CD19 BM CAR-T cells and TLR4-armed CD19-co-CD20 CAR-T cells after incubation with Raji target cells. [Figure 7] Figure 1 shows the bicistronic expression design of CAR fusion constructs comprising anti-CLL1-CAR and anti-CD33 TLR polypeptides (CLL1-co-CD33 CAR, SEQ ID NO:3 and CLL1-co-CD33-2 CAR-T, SEQ ID NO:71). [Figure 8] Figure 8 shows the in vitro cytotoxic effects of CLL1 BM CAR-T cells, CLL1-co-CD33 CAR-T cells armed with TLR2 and TLR1, and CLL1-co-CD33-2 CAR-T cells armed with TLR4. In particular, Figure 8 shows that CLL1 BM CAR-T, CLL1-co-CD33 CAR-T, and CLL1-co-CD33-2 CAR-T cells induce lysis of U937 target cells in vitro in a dose-dependent manner. In this experiment, untransduced T cells (i.e., "unT") were used as a control. [Figure 9]Figure 1 shows in vitro IFNγ cytokine profiles of CLL1 BM CAR-T cells, TLR2- and TLR1-armed CLL1-co-CD33 CAR-T cells, and TLR4-armed CLL1-co-CD33-2 CAR-T cells co-cultured with U937 target cells. Untransduced T cells (i.e., "unT") were used as a control in this experiment. [Figure 10] Figure 1 shows in vitro TNFα cytokine profiles of CLL1 BM CAR-T cells, TLR2 and TLR1 armed CLL1-co-CD33 CAR-T cells, and TLR4 armed CLL1-co-CD33-2 CAR-T cells co-cultured with U937 target cells. Untransduced T cells (i.e., "unT") were used as a control in this experiment. [Figure 11] Figure 1 shows the in vitro killing efficacy of CLL1 BM CAR-T cells, CLL1-co-CD33 CAR-T cells armed with TLR2 and TLR1, and CLL1-co-CD33-2 CAR-T armed with TLR4 in a repeated tumor stimulation assay with U937 target cells. Untransduced T cells (i.e., "UNT") were used as a control in this experiment. [Figure 12] Figure showing in vitro proliferation (e.g., magnification) of CLL1 BM CAR-T cells, CLL1-co-CD33 CAR-T cells armed with TLR2 and TLR1, and CLL1-co-CD33-2 CAR-T armed with TLR4 after incubation with U937 target cells. [Figure 13] FIG. 1 is a schematic diagram showing in vivo efficacy studies of exemplary CLL1-co-CD33 CAR αβT and CLL1-co-CD33 CAR γδT treatments in the U937-Luc xenograft mouse model. [Figure 14] Figure 1 shows in vivo efficacy of CLL1-co-CD33 CAR αβ T cells in a U937-Luc xenograft mouse model. [Figure 15]Figure 1 shows the in vivo efficacy of CLL1-co-CD33 CAR γδ T cells and CLL1-co-CD33-2 CAR γδ T cells in a U937-Luc xenograft mouse model. [Figure 16A] 16A-16B show exemplary constructs of CARs or TCRs armed with NKG2D or mutant NKG2D TLR chimeric receptors. Figure 16A shows a schematic diagram of a CAR armed with NKG2D or mutant NKG2D TLR chimeric receptors. Figure 16B shows a schematic diagram of a TCR armed with NKG2D or mutant NKG2D TLR chimeric receptors. [Figure 16B] See the legend to Figure 16A. [Figure 17A]

[0013] Figures 17A-17B show exemplary constructs of a CAR or TCR armed with a chimeric receptor comprising a binding domain targeting an NKG2D ligand, a TLR transmembrane domain, and an intracellular effector domain. Figure 17A shows a schematic diagram of a CAR armed with a chimeric receptor comprising a binding domain targeting an NKG2D ligand, a TLR transmembrane domain, and an intracellular effector domain. Figure 17B shows a schematic diagram of a TCR armed with a chimeric receptor comprising a binding domain targeting an NKG2D ligand, a TLR transmembrane domain, and an intracellular effector domain. [Figure 17B] See the legend to Figure 17A. [Figure 18] Figures 18A-18B show exemplary constructs of CARs or TCRs armed with NKG2D or mutant NKG2D TLR4 chimeric receptors. Figure 18A shows a second generation CAR armed with NKG2D or mutant NKG2D TLR4 chimeric receptors. Figure 18B shows a TCR armed with NKG2D or mutant NKG2D TLR4 chimeric receptors. [Figure 19]Figure 1 shows the in vitro killing effect of anti-GPC3 CAR-T and anti-GPC3 CAR-T cells armed with NKG2D-TLR4 or NKG2D-CD8-TLR4 chimeric receptors in a repeated tumor stimulation assay with Huh7 target cells. In this experiment, untransduced T cells (i.e., "UNT") were used as a control. [Figure 20] Figure 1 shows the in vitro proliferation of anti-GPC3 CAR-T and anti-GPC3 CAR-T cells armed with NKG2D-TLR4 or NKG2D-CD8-TLR4 chimeric receptors after incubation with Huh7 target cells. [Figure 21] Figure 1 shows in vitro IFNγ cytokine profiles of anti-GPC3 CAR-T and anti-GPC3 CAR-T cells armed with NKG2D-TLR4 or NKG2D-CD8-TLR4 chimeric receptors co-cultured with Huh7 target cells. In this experiment, untransduced T cells (i.e., "UNT") were used as a control. [Figure 22] Figure 1 shows the in vitro killing efficacy of anti-CD19 CAR-T and anti-CD19 CAR-T cells armed with NKG2D-TLR4 or NKG2D-CD8-TLR4 chimeric receptors in a repeated tumor stimulation assay with Raji target cells. Untransduced T cells (i.e., "UNT") were used as a control in this experiment. [Figure 23] Figure 1 shows the in vitro expansion of anti-CD19 CAR-T and anti-CD19 CAR-T cells armed with NKG2D-TLR4 or NKG2D-CD8-TLR4 chimeric receptors after incubation with Raji target cells. [Figure 24] Figure 1 shows the in vivo efficacy of anti-GPC3 CAR-T cells and anti-GPC3 CAR-T cells armed with NKG2D-CD8-TLR4 chimeric receptors in a Huh7 xenograft model. [Figure 25]Figure 1 shows the bicistronic expression design of CAR fusion constructs including tandem anti-BCMA-co-anti-BCMA CAR (SEQ ID NO:61) or single anti-BCMA-co-anti-BCMA CAR (SEQ ID NO:62), single anti-BCMA-co-anti-BCMA-CD8 CAR (SEQ ID NO:63), single anti-BCMA-co-anti-BCMA-CD28 CAR (SEQ ID NO:64), as well as tandem anti-BCMA-co-anti-GPRC5D CAR (SEQ ID NO:65), tandem anti-BCMA-co-anti-GPRC5D-CD8 CAR (SEQ ID NO:72), tandem anti-BCMA-co-anti-GPRC5D-CD28 CAR (SEQ ID NO:73). [Figure 26] Figure 1 shows the in vitro cytotoxic effects of anti-BCMA-CAR-γδT and anti-BCMA-co-anti-BCMA-γδT cells armed by TLR4 intracellular signaling. In this experiment, untransduced γδT cells (i.e., "Un-γδT") were used as a control. [Figure 27] Figure 1 shows in vitro IFN-γ, TNF-α, and GM-CSF cytokine secretion of anti-BCMA-CAR-γδ T and anti-BCMA-co-anti-BCMA-γδ T cells armed with TLR4 intracellular signaling co-cultured with BCMA-positive NCI-H929 target tumor cells. Untransduced γδ T cells (i.e., "Un-γδ T") were used as a control in this experiment. [Figure 28] Figure 1 shows the in vitro killing efficacy and persistence of tandem anti-BCMA-CAR-γδT, tandem anti-BCMA-co-anti-BCMA-CAR γδT, single anti-BCMA-co-anti-BCMA CAR γδT, single anti-BCMA-co-anti-BCMA-CD8 CAR γδT, single anti-BCMA-co-anti-BCMA-CD28 CAR γδT, tandem anti-BCMA-co-anti-GPRC5D-CAR γδT, and tandem anti-BCMA-co-anti-GPRC5D-CD8 CAR-γδT cells in a repeated tumor stimulation assay with BCMA-positive NCI-H929 target tumor cells. Untransduced γδ T cells (i.e., "Un-γδT") were used as a control in this experiment. [Figure 29] FIG. 1 shows persistence of tandem anti-BCMA-CAR-γδ T and tandem anti-BCMA-co-anti-BCMA-γδ T cells armed with TLR4 intracellular signaling in an allogeneic environment, where γδ T cells, allogeneic PBMCs, and BCMA-positive NCI-H929 target tumor cells are co-incubated at a ratio of 1:60:1. [Figure 30] Figure 1 shows the in vivo killing efficacy and persistence of tandem anti-BCMA-CAR-γδ T, tandem anti-BCMA-co-anti-BCMA-γδ T cells, and tandem anti-BCMA-co-anti-GPRC5D-CD8-γδ T cells armed with TLR4 intracellular signaling in a BCMA-positive RPMI-8226 tumor-burdened xenograft model. Untransduced γδ T cells (i.e., "Un-γδ T") and the vehicle HBSS were used as controls in this experiment. [Figure 31A] Figures 31A-31B show body weight change and IFN-γ, TNF-α, and GM-CSF cytokine secretion of tandem anti-BCMA-CAR-γδ T and tandem anti-BCMA-co-anti-BCMA-γδ T cells armed with TLR4 intracellular signaling in a BCMA-positive RPMI-8226 tumor-burdened xenograft model. Untransduced γδ T cells (i.e., "Un-γδ T") and the vehicle HBSS were used as controls in this experiment. [Figure 31B] See the description of Figure 31A. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description The present application provides engineered immune cells comprising Toll-like receptor (TLR) costimulatory molecules (e.g., TLR polypeptides) and methods of using same to treat cancer. In some embodiments, the engineered immune cells comprise TLR-based multimers containing a first TLR polypeptide and a second TLR polypeptide, and compared to engineered immune cells that do not express the polypeptides, these engineered immune cells have effective and sustained tumor lytic activity and improved exhaustion characteristics. In some embodiments, the first TLR polypeptide comprises i) a first target binding domain (e.g., an antibody portion or fragment thereof), ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain, and the second TLR polypeptide comprises i) a second target binding domain (e.g., an antibody portion or fragment thereof), ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain. When the first target binding domain and the second target binding domain bind to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the modified immune cell is a T cell.

[0035] In some embodiments, the modified cells further express a chimeric antigen receptor (CAR) that specifically recognizes a target antigen of interest. The TLR polypeptides described herein increase the proliferation of CAR-T cells and enhance anti-tumor activity.

[0036] Activation of TLR signaling in TLR polypeptides is target-dependent and is initiated by the formation of dimerized or multimerized TLR polypeptides and TLR signaling moieties after the TLR polypeptides bind to their corresponding target molecules. The present application provides several strategies for inducing the formation of TLR signaling moieties. In the first strategy, a first target binding domain of a first polypeptide and a second binding domain of a second polypeptide each bind to a subunit of a multimeric target molecule. When the target binding domains bind to their homologous target subunits, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety. In the second strategy, the first target binding domain of a first polypeptide and the second binding domain of a second polypeptide each bind to different, non-overlapping target sites on a target molecule. When the target binding domains bind to their homologous target molecules, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety. In a third strategy, when an immune cell contains an engineered receptor containing an extracellular domain, the extracellular domain and the first or second target binding domain each bind to different, non-overlapping target sites on the same target molecule. Without being bound by theory, it is believed that when the extracellular domain of the engineered receptor binds to a target molecule, an immune synapse is formed around the engineered receptor. The first and second polypeptides are recruited to the same immune synapse, allowing the first and second TLR signaling domains to associate with each other to form a TLR signaling moiety. In another strategy, the first and second target binding domains each bind to the same monomeric target molecule (e.g., at the same target site), and binding of the first and second target binding domains to the target molecule allows the first and second TLR signaling domains to associate with each other to form a TLR signaling moiety.

[0037] Thus, in one aspect of the present application, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain (e.g., an antibody portion or fragment thereof), ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain (e.g., an antibody portion or fragment thereof), ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule. In some embodiments, the first target binding domain and the second binding domain each bind to the same target site on the target molecule. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor, an engineered T cell receptor, or a T cell antigen coupler (TAC) receptor.

[0038] In another aspect of the present application, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain (e.g., an antibody portion or fragment thereof), ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain (e.g., an antibody portion or fragment thereof), ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second target binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the modified immune cells further comprise an engineered receptor, e.g., a chimeric antigen receptor (CAR), a modified T cell receptor, or a T cell antigen coupler (TAC) receptor.

[0039] In another aspect of the present application, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain (e.g., an antibody portion or fragment thereof), ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain (e.g., an antibody portion or fragment thereof), ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor (e.g., a CAR), wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes a non-overlapping target site on the same target molecule as the first polypeptide and / or the second polypeptide.

[0040] Additionally, compositions (e.g., pharmaceutical compositions), kits and articles of manufacture comprising the modified immune cells, as well as methods of treating a disease or condition (e.g., cancer) with the modified immune cells described herein are provided.

[0041] I. Definition As used herein, "treatment" or "treating" refers to a method for obtaining beneficial or desired results (including clinical results). For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of: alleviating one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or attenuating the progression of the disease, ameliorating the state of the disease, providing partial or complete relief from the disease, reducing the dosage of one or more other drugs required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. "Treatment" further covers reducing the pathological consequences of a disease (e.g., cancer). The methods of the present application contemplate any one or more of these aspects of treatment.

[0042] "Prevent" and similar terms such as "prevented" or "preventing" refer to a method for preventing, inhibiting, or reducing the likelihood of a disease or condition (e.g., cancer) recurrence. It further refers to delaying the recurrence of the disease or condition or delaying the recurrence of symptoms of the disease or condition. As used herein, "prevention" and similar terms further include reducing the intensity, effects, symptoms, and / or burden of the disease or condition before the disease or condition recurs.

[0043] As used herein, "delaying" cancer progression refers to slowing, inhibiting, slowing, extending, stabilizing, and / or slowing the progression of the disease. Such delay may have different lengths of time depending on the history of the disease and / or the individual being treated. A method that "delays" cancer progression is a method that reduces the probability of disease progression within a given time range and / or reduces the extent of disease within a given time range, compared to not using the method. Such comparisons are usually based on clinical trials using a statistically significant number of individuals. Cancer progression can be detected using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Progression may also refer to the progression of cancer, which may be initially undetectable, and includes occurrence, recurrence, and seizures.

[0044] As used herein, the term "effective amount" refers to the amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, e.g., ameliorate, alleviate, reduce, and / or delay one or more symptoms thereof. For cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay disease progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, slow, slow to some extent, and preferably prevent cancer cell invasion into surrounding organs, (iv) inhibit (i.e., slow to some extent, and preferably prevent) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0045] As used herein, "individual" or "subject" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0046] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0047] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0048] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing heterologous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with heterologous nucleic acid. This cell includes the primary target cell and its progeny.

[0049] "Percent (%) amino acid sequence identity" with respect to a polypeptide sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the polypeptide being compared, after alignment of the sequences (including any conservative substitutions considered part of the sequence identity). For purposes of determining percent amino acid sequence identity, alignment may be achieved by a variety of methods within the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that achieves maximum alignment within the full length of the sequences being compared. However, for purposes of this specification, the sequence comparison computer program MUSCLE is used to generate percent amino acid sequence identity values ​​(Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0050] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that specifically transfers one or more antigens into a cell, such as a T cell. CARs are also called "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises the extracellular variable domain of an antibody with specificity for a tumor antigen and the intracellular signaling domain of a T cell or other receptor, e.g., one or more costimulatory domains. "CAR-T" refers to a T cell expressing a CAR. As used herein, "CLL1 CAR" refers to a CAR that specifically recognizes CLL1, "CD19 CAR" refers to a CAR that specifically recognizes CD19, "GPC3 CAR" refers to a CAR that specifically recognizes GPC3, and "BCMA CAR" refers to a CAR that specifically recognizes BCMA.

[0051] As used herein, "T cell receptor" or "TCR" refers to an endogenous or modified T cell receptor comprising an extracellular antigen-binding domain that binds to a specific antigenic peptide that binds to an MHC molecule. In some embodiments, the TCR comprises a TCR alpha polypeptide chain and a TCR beta polypeptide chain. In some embodiments, the TCR comprises a TCR gamma polypeptide chain and a TCR delta polypeptide chain. In some embodiments, the TCR specifically binds to a tumor antigen. "TCR-T" refers to a T cell that expresses a recombinant TCR.

[0052] As used herein, a "T cell antigen coupler receptor" or "TAC receptor" refers to an engineered receptor that contains an extracellular antigen-binding domain that binds to a specific antigen, a T cell receptor (TCR) binding domain, a transmembrane domain, and an intracellular domain of a co-receptor molecule. The TAC receptor identifies the endogenous TCR of T cells that express the TAC receptor to elicit an antigen-specific T cell response against the target cell.

[0053] As used herein, the term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. The term antibody includes, but is not limited to, fragments capable of binding to antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody includes conventional four-chain antibodies and single-domain antibodies, e.g., antibodies having only a heavy chain or fragments thereof, e.g., V H Contains H.

[0054] As used herein, the terms "binding," "specifically binding," or "specific for" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target, which determines the presence of the target in the presence of heteromolecules (including biomolecules). For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with an affinity, avidity, readiness, and / or duration that is superior to binding to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, an antibody specifically binds to a protein epitope that is conserved among proteins from different species. In another embodiment, specific binding can include, but is not limited to, exclusive binding.

[0055] The term "cell" includes the primary target cell and its progeny.

[0056] It should be understood that embodiments of the present disclosure described herein include embodiments "consisting of" and / or "consisting essentially of."

[0057] As used herein, reference to "about" a value or parameter includes (and describes) variations on the value or parameter itself. For example, a statement about "about X" includes a statement of "X."

[0058] As used herein, reference to a value or parameter that is "not" generally means and describes a "different" value or parameter. For example, a method is not for treating cancer type X means that the method is used to treat a cancer different from type X.

[0059] The term "about X to Y" used in this specification has the same meaning as "about X to about Y."

[0060] As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0061] It should be understood that, for clarity, certain features of the present disclosure that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, different features of the present disclosure that are described in the context of a single embodiment may also be provided alone or in any suitable subcombination. All combinations of embodiments relating to the modified immune cells and therapeutic methods described herein are expressly included in this application and are disclosed herein as if each combination were individually and expressly disclosed. Furthermore, all subcombinations of modified immune cells listed in embodiments describing such variables are also expressly included in this application and are disclosed herein as if each such subcombination of proteins were individually and expressly disclosed herein.

[0062] II. Modified immune cells In one aspect of the present application, modified immune cells are provided that include a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each encode a TLR polypeptide (e.g., a TLR costimulatory polypeptide) that includes a TLR signaling domain. In some embodiments, upon binding of the TLR polypeptides to their corresponding targets, the TLR signaling domains of the first and second polypeptides associate with each other to form a TLR signaling moiety, wherein the TLR signaling moiety induces TLR signaling, thereby resulting in a potent anti-tumor effect. The first polypeptide and the second polypeptide may be the same or different.

[0063] In some embodiments, engineered immune cells comprising first and second TLR polypeptides have increased T cell activation, function, and / or survival induced by T cell receptor (TCR) signaling, compared to engineered immune cells that do not contain the TLR polypeptides. In some embodiments, the TLR polypeptides induce an increased tumor cell killing effect by the engineered immune cells, compared to engineered immune cells that do not contain the TLR polypeptides. In some embodiments, TLR polypeptide induction induces an increased tumor cell killing effect, e.g., an increase of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 12-fold, 14-fold, 16-fold, 20-fold, 25-fold, 30-fold, 40-fold, or more, compared to engineered immune cells that do not contain the TLR polypeptides. In some embodiments, the TLR polypeptide confers sustained efficacy to the engineered immune cells, e.g., at least about any one of a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more increase, compared to engineered immune cells that do not contain the TLR polypeptide. In some embodiments, the engineered immune cells have reduced in vivo toxicity when administered to an individual, compared to engineered immune cells comprising the TLR polypeptide. In some embodiments, the engineered immune cells exhibit reduced in vivo exhaustion when administered to an individual, compared to engineered immune cells comprising the TLR polypeptide. In some embodiments, the TLR polypeptide is a costimulatory molecule. In some embodiments, the engineered immune cells further comprise an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0064] In some embodiments, modified immune cells are provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first TLR transmembrane domain and the first TLR signaling domain are derived from the same TLR molecule. In some embodiments, the first TLR transmembrane domain and the first TLR signaling domain are derived from different TLR molecules. In some embodiments, the second TLR transmembrane domain and the second TLR signaling domain are derived from the same TLR molecule. In some embodiments, the second TLR transmembrane domain and the second TLR signaling domain are derived from different TLR molecules. In some embodiments, the first TLR transmembrane domain and the second TLR transmembrane domain are the same. In some embodiments, the first TLR transmembrane domain and the second TLR transmembrane domain are different. In some embodiments, the first TLR signaling domain and the second TLR signaling domain are different. In some embodiments, the one or more TLR molecules are selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, e.g., TLR1, TLR2, TLR4, or TLR6. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor.In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0065] In some embodiments, modified immune cells are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling, and wherein the first TLR transmembrane domain, the first TLR signaling domain, the second TLR transmembrane domain, and the second TLR signaling domain are derived from TLR4. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cells further comprise an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0066] In some embodiments, modified immune cells are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain bind to their corresponding targets. The TLR signaling domains associate with each other to form a TLR signaling moiety capable of inducing TLR signaling, wherein the first TLR transmembrane domain and the first TLR signaling domain are derived from TLR2, and wherein the second TLR transmembrane domain and the second TLR signaling domain are derived from TLR1, or wherein the first TLR transmembrane domain and the first TLR signaling domain are derived from TLR1, and wherein the second TLR transmembrane domain and the second TLR signaling domain are derived from TLR2. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0067] In some embodiments, modified immune cells are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain bind to their corresponding targets. The TLR signaling domains associate with each other to form a TLR signaling moiety capable of inducing TLR signaling, wherein the first TLR transmembrane domain and the first TLR signaling domain are derived from TLR6, and wherein the second TLR transmembrane domain and the second TLR signaling domain are derived from TLR2, or wherein the first TLR transmembrane domain and the first TLR signaling domain are derived from TLR2, and wherein the second TLR transmembrane domain and the second TLR signaling domain are derived from TLR6. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0068] In some embodiments, modified immune cells are provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling, and wherein the first target binding domain and / or the second target binding domain are antibody moieties or antigen-binding fragments thereof. In some embodiments, the first target binding domain and the second target binding domain are antibody moieties or antigen-binding fragments thereof. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain is an scFv or sdAb. In some embodiments, the first target binding domain and / or the second target binding domain specifically binds to CD33, CLL1, CD123, CD19, CD20, CD22, BCMA, GPRC5D, NKG2D, or GPC3. In some embodiments, the first target binding domain and the second target binding domain are the same. In some embodiments, the first target binding domain and the second target binding domain are different. In some embodiments, the target molecule of the first polypeptide and / or the second polypeptide is an immune checkpoint protein. In some embodiments, the target molecule of the first polypeptide and / or the second polypeptide is selected from the group consisting of PD-1, CD70, CD27, SIRPα, and TIGIT. In some embodiments of any one of the above modified immune cells, the target molecule is a naturally occurring protein expressed on an immune cell. In some embodiments, the target molecule is NKG2D. In some embodiments, the target molecule is a mutant NKG2D.In some embodiments, the mutant NKG2D comprises a truncated sequence and / or amino acid substitutions, mutations, additions, and / or deletions. In some embodiments, the target molecule is the extracellular antigen-binding domain of NKG2D. In some embodiments, the target molecule is the full-length sequence of NKG2D. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen coupler (TAC) receptor. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0069] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling, and wherein the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the subunits of the multimeric target molecule are different. In some embodiments, the first target binding domain specifically recognizes the V subunit of CD33, and the second target binding domain specifically recognizes the C2 subunit of CD33. In some embodiments, the subunits of the multimeric target molecule are the same. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD20. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD33. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of BCMA. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of NKG2D. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of GPRC5D.In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of NKG2D. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes any of CD19, CLL1, BCMA, or GPC3. In some embodiments, the engineered receptor is a CAR, e.g., a CD19 CAR, a CLL1 CAR, a GPC3 CAR, or a BCMA CAR (e.g., a single BCMA CAR or a tandem BCMA CAR). In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0070] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling, and wherein the first target binding domain and the second binding domain each bind to the same target site on a target molecule. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD20. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD33. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of BCMA. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of NKG2D. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of GPRC5D. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of NKG2D. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor.In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes any one of CD19, CLL1, GPC3, and BCMA. In some embodiments, the engineered receptor is a CAR, e.g., a CD19 CAR, a CLL1 CAR, a GPC3 CAR, or a BCMA CAR (e.g., a single BCMA CAR or a tandem BCMA CAR). In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0071] In some embodiments, modified immune cells are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR4 transmembrane domain, and iii) a first TLR4 signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR4 transmembrane domain, and iii) a second TLR4 signaling domain, wherein the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and the second target binding domain are each an extracellular NKG2D-binding domain of NKG2D. In some embodiments, the first target binding domain and / or the second target binding domain are an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain and / or the second target binding domain are an scFv or sdAb. In some embodiments, the first polypeptide and the second polypeptide are the same. In some embodiments, the first target binding domain and the second target binding domain are each an scFv that specifically binds to CD20. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD33. In some embodiments, the first target binding domain and the second target binding domain are each an scFv that specifically binds to GPRC5D. In some embodiments, the first target binding domain and the second target binding domain are each an sdAb that specifically binds to BCMA. In some embodiments, the first polypeptide and the second polypeptide are different.In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0072] In some embodiments, modified immune cells are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR2 transmembrane domain, and iii) a first TLR2 signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR1 transmembrane domain, and iii) a second TLR1 signaling domain, wherein the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR2 signaling domain and the second TLR1 signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain and / or the second target binding domain is an scFv or sdAb. In some embodiments, the first target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain specifically recognizes the V subunit of CD33, and the second target binding domain specifically recognizes the C2 subunit of CD33. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0073] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The scFv comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0074] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D extracellular domain (ECD), a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D extracellular domain (ECD), a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the ... In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0075] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The scFv comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0076] In some embodiments, an engineered immune cell (e.g., T cell) is provided that comprises: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The sdAb comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0077] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The sdAb comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0078] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., a cytoplasmic portion of TLR2), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., a cytoplasmic portion of TLR2). The modified immune cell further comprises a C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1), wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0079] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second target binding domain bind to the same target molecule, wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and the second target binding domain are each an extracellular NKG2D-binding domain of NKG2D. In some embodiments, the first target binding domain and / or the second target binding domain are an antibody portion or an antigen-binding fragment thereof. In some embodiments, the first target binding domain and / or the second target binding domain are an scFv or an sdAb. In some embodiments, the subunits of the multimeric target molecule are the same. In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD20. In some embodiments, the first target binding domain and the second target binding domain are each an scFv that specifically binds to GPRC5D. In some embodiments, the first target binding domain and the second target binding domain are each an sdAb that specifically binds to BCMA. In some embodiments, the first target binding domain and the second target binding domain are each an sdAb that specifically binds to CD33.In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes any one of CD19, CLL1, GPC3, and BCMA. In some embodiments, the engineered receptor is a CAR, e.g., a CD19 CAR, a CLL1 CAR, a GPC3 CAR, or a BCMA CAR (e.g., a single BCMA CAR or a tandem BCMA CAR). In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TCR receptor. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0080] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR4 transmembrane domain, and iii) a first TLR4 signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR4 transmembrane domain, and iii) a second TLR4 signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and the second target binding domain are each an extracellular NKG2D-binding domain of NKG2D. In some embodiments, the first target binding domain and / or the second target binding domain are antibody portions or antigen-binding fragments thereof. In some embodiments, the first target binding domain and / or the second target binding domain are scFvs or sdAbs. In some embodiments, the first polypeptide and the second polypeptide are the same. In some embodiments, the first target binding domain and the second target binding domain are each scFvs that specifically bind to CD20. In some embodiments, the first target binding domain and the second target binding domain are each scFvs that specifically bind to GPRC5D. In some embodiments, the first target binding domain and the second target binding domain are each sdAbs that specifically bind to BCMA. In some embodiments, the first target binding domain and the second target binding domain are each an sdAb that specifically binds to CD33. In some embodiments, the first polypeptide and the second polypeptide are different.In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0081] In some embodiments, modified immune cells are provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR2 transmembrane domain, and iii) a first TLR2 signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR1 transmembrane domain, and iii) a second TLR1 signaling domain, wherein the first target binding domain and the second target binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR2 signaling domain and the second TLR1 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain and / or the second target binding domain is an scFv or sdAb. In some embodiments, the first target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain specifically recognizes the V subunit of CD33, and the second target binding domain specifically recognizes the C2 subunit of CD33. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0082] In some embodiments, an engineered immune cell (e.g., T cell) is provided that comprises: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The scFv comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0083] In some embodiments, an engineered immune cell (e.g., T cell) is provided that comprises: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the ... In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0084] In some embodiments, an engineered immune cell (e.g., T cell) is provided that comprises: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The sdAb comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0085] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The scFv comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0086] In some embodiments, an engineered immune cell (e.g., T cell) is provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The sdAb comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0087] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein the first target binding domain and the second binding domain bind to the same target molecule, and wherein the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., a cytoplasmic portion of TLR2), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., a cytoplasmic portion of TLR2). The modified immune cell further comprises an engineered receptor, e.g., a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the ... In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0088] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and the second polypeptide. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide (different from the target molecule of the second polypeptide). In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the second polypeptide (different from the target molecule of the first polypeptide). In some embodiments, the first polypeptide and the second polypeptide do not comprise the extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain are antibody moieties or antigen-binding fragments thereof. In some embodiments, the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule. In some embodiments, the subunits of the multimeric target molecule are different. In some embodiments, the first target binding domain specifically recognizes the V subunit of CD33, and the second target binding domain specifically recognizes the C2 subunit of CD33. In some embodiments, the subunits of the multimeric target molecule are the same.In some embodiments, the first target binding domain and the second target binding domain specifically recognize a subunit of CD20. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes a non-overlapping target site on the same target molecule as the first polypeptide and / or the second polypeptide. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor comprises an extracellular domain that specifically recognizes any one of CD19, CLL1, GPC3, and BCMA. In some embodiments, the engineered receptor is a CAR, such as a CD19 CAR, a CLL1 CAR, a GPC3 CAR, or a BCMA CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0089] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR4 transmembrane domain, and iii) a first TLR4 signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR4 transmembrane domain, and iii) a second TLR4 signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and the second target binding domain are each an extracellular NKG2D-binding domain of NKG2D. In some embodiments, the first target binding domain and / or the second target binding domain are antibody portions or antigen-binding fragments thereof. In some embodiments, the first target binding domain and / or the second target binding domain are scFvs or sdAbs. In some embodiments, the first polypeptide and the second polypeptide are the same. In some embodiments, the first target binding domain and the second target binding domain are each scFvs that specifically bind to CD20. In some embodiments, the first target binding domain and the second target binding domain are each scFvs that specifically bind to GPRC5D. In some embodiments, the first target binding domain and the second target binding domain are each sdAbs that specifically bind to BCMA.In some embodiments, the first target binding domain and the second target binding domain are each an sdAb that specifically binds to CD33. In some embodiments, the first polypeptide and the second polypeptide are different. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen coupler (TAC) receptor. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0090] In some embodiments, modified immune cells are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR2 transmembrane domain, and iii) a first TLR2 signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR1 transmembrane domain, and iii) a second TLR1 signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR2 signaling domain and the second TLR1 signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first target binding domain and / or the second target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain and / or the second target binding domain is an scFv or sdAb. In some embodiments, the first target binding domain is an antibody portion or antigen-binding fragment thereof. In some embodiments, the first target binding domain specifically recognizes the V subunit of CD33, and the second target binding domain specifically recognizes the C2 subunit of CD33. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor.In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0091] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The scFv comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0092] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The engineered receptor comprises an ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0093] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The scFv comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0094] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes a target molecule that is the same as the first polypeptide and / or the second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The sdAb comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0095] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). The sdAb comprises a TLR4 transmembrane domain and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ-binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0096] In some embodiments, modified immune cells (e.g., T cells) are provided that comprise: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain; and c) an engineered receptor, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide, wherein the engineered receptor comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., a cytoplasmic portion of TLR2), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., a cytoplasmic portion of TLR2). The engineered receptor comprises a C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1), and wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and the second polypeptide do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen coupler (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0097] In some embodiments, immune cells (e.g., CAR-T cells) expressing a CAR are provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule. In some embodiments, the subunits of the multimeric target molecule are different. In some embodiments, the subunits of the multimeric target molecule are the same. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule. In some embodiments, the first target binding domain and the second binding domain each bind to the same target site on the target molecule. In some embodiments, the first target binding domain and the second target binding domain are the same. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule, and the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule. In some embodiments, the CAR comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide. In some embodiments, the CAR comprises an extracellular domain that specifically recognizes a non-overlapping target site on the target molecule that is the same as the first polypeptide and / or the second polypeptide. In some embodiments, the CAR comprises an extracellular domain that specifically recognizes any one of CD19, CLL1, GPC3, and BCMA (e.g., a single BCMA CAR or a tandem BCMA CAR).In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, and a γδ T cell.

[0098] In some embodiments, a TCR-expressing immune cell (e.g., a TCR-T cell) is provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule. In some embodiments, the subunits of the multimeric target molecule are different. In some embodiments, the subunits of the multimeric target molecule are the same. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule. In some embodiments, the first target binding domain and the second binding domain each bind to the same target site on a target molecule. In some embodiments, the first target binding domain and the second target binding domain are the same. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule, and the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule. In some embodiments, the TCR comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide. In some embodiments, the TCR comprises an extracellular domain that specifically recognizes a non-overlapping target site on a target molecule that is the same as the first polypeptide and / or the second polypeptide. In some embodiments, the TCR comprises an extracellular domain that specifically recognizes any one of CD19, CLL1, GPC3, and BCMA (e.g., a single BCMA TCR or a tandem BCMA TCR).In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, and a γδ T cell.

[0099] In some embodiments, a TAC-expressing immune cell (e.g., a TAC-T cell) is provided, comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule. In some embodiments, the subunits of the multimeric target molecule are different. In some embodiments, the subunits of the multimeric target molecule are the same. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule. In some embodiments, the first target binding domain and the second binding domain each bind to the same target site on a target molecule. In some embodiments, the first target binding domain and the second target binding domain are the same. In some embodiments, the first target binding domain and the second binding domain bind to the same target molecule, and the first target binding domain and the second target binding domain each bind to different, non-overlapping target sites on a single target molecule. In some embodiments, the TAC comprises an extracellular domain that specifically recognizes the same target molecule as the first polypeptide and / or the second polypeptide. In some embodiments, the TAC comprises an extracellular domain that specifically recognizes a non-overlapping target site on a target molecule that is the same as the first polypeptide and / or the second polypeptide. In some embodiments, the TAC comprises an extracellular domain that specifically recognizes any one of CD19, CLL1, GPC3, and BCMA (e.g., a single BCMA TAC or a tandem BCMA TAC).In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, and a γδ T cell.

[0100] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-T cell) is provided, comprising: a) a first polypeptide comprising i) a first target binding domain (e.g., an antibody portion or antigen-binding fragment thereof), ii) a first TLR4 transmembrane domain, and iii) a first TLR4 signaling domain; and b) a second polypeptide comprising i) a second target binding domain (e.g., an antibody portion or antigen-binding fragment thereof), ii) a second TLR4 transmembrane domain, and iii) a second TLR4 signaling domain, wherein, upon binding of the target binding domain and the second target binding domain to their corresponding targets, the first TLR4 signaling domain and the second TLR4 signaling domain associate with each other to form a TLR4 signaling moiety capable of inducing TLR4 signaling. In some embodiments, the first polypeptide further comprises a signal peptide (e.g., a leader sequence). In some embodiments, the first polypeptide and the second polypeptide are the same. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, a signal peptide, an anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the first polypeptide and the second polypeptide each comprise, from N-terminus to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4).In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the engineered immune cells express an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CD19 CAR comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:2. In some embodiments, the anti-CD19 CAR comprises SEQ ID NO:2. In some embodiments, the engineered immune cells express an anti-CLL1 CAR. In some embodiments, the anti-CLL1 CAR comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD28α hinge domain, a CD28α transmembrane (TM) domain, a cytoplasmic portion of a CD28 costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CLL1 CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:4. In some embodiments, the anti-CLL1 CAR comprises SEQ ID NO:4. In some embodiments, the engineered immune cells express an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR).In some embodiments, the anti-BCMA CAR comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-BCMA CAR comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:67 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the anti-BCMA CAR comprises SEQ ID NO:67. In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO: 40. In some embodiments, the anti-BCMA CAR comprises, from N-terminus to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-BCMA CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 66. In some embodiments, the anti-BCMA CAR comprises SEQ ID NO: 66. In some embodiments, the modified immune cells express an anti-GPC3 CAR.In some embodiments, the anti-GPC3 CAR comprises, from N- to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-GPC3 CAR comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:56. In some embodiments, the anti-GPC3 CAR comprises SEQ ID NO: 56. In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO: 40. In some embodiments, the engineered immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CD19 CAR and an anti-CD20 TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 1 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO: 1. In some embodiments, the modified immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR and an anti-CD33 TLR polypeptide.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:71 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:71. In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CD19 CAR and an anti-NKG2D TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 59. In some embodiments, the CAR fusion construct comprises SEQ ID NO: 59.In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:60 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:60. In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-GPC3 CAR and an anti-NKG2D TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 57. In some embodiments, the CAR fusion construct comprises SEQ ID NO: 57.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:58 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:58. In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-BCMA TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:62. In some embodiments, the CAR fusion construct comprises SEQ ID NO:62.In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:63 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO: 63. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a CD28α hinge domain, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:64. In some embodiments, the CAR fusion construct comprises SEQ ID NO:64.In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a tandem anti-BCMA sdAb, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:61 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:61. In some embodiments, the engineered immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-GPRC5D TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:65. In some embodiments, the CAR fusion construct comprises SEQ ID NO:65. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:72 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).In some embodiments, the CAR fusion construct comprises SEQ ID NO: 72. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a CD28α hinge domain, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:73 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:73. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, and a γδ T cell.

[0101] In some embodiments, an immune cell (e.g., a CAR-T cell) expressing a CAR is provided, comprising: a) a first polypeptide comprising i) a first target binding domain (e.g., an antibody portion or antigen-binding fragment thereof), ii) a TLR2 transmembrane domain, and iii) a first TLR2 signaling domain; and b) a second polypeptide comprising i) a second target binding domain (e.g., an antibody portion or antigen-binding fragment thereof), ii) a TLR1 transmembrane domain, and iii) a TLR1 signaling domain, wherein, upon binding of the first target binding domain and the second target binding domain to their corresponding targets, the TLR1 signaling domain and the TLR2 signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the first polypeptide and / or the second polypeptide further comprise a signal peptide (e.g., a leader sequence). In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., the cytoplasmic portion of TLR2), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1). In some embodiments, the engineered immune cells express an anti-CLL1 CAR. In some embodiments, the engineered immune cells express an anti-CLL1 / CD33 dual CAR. In some embodiments, the anti-CLL1 / CD33 dual CAR comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, an anti-CD33 V domain sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain.In some embodiments, the anti-CLL1 / CD33 dual CAR comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:43 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the anti-CLL1 / CD33 dual CAR comprises SEQ ID NO:43. In some embodiments, the first polypeptide comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:41 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the first polypeptide comprises SEQ ID NO:41. In some embodiments, the second polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 42. In some embodiments, the second polypeptide comprises SEQ ID NO: 42. In some embodiments, the engineered immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR and an anti-CD33 (e.g., an anti-CD33 V domain and / or an anti-CD33 C2 domain) TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD28α hinge domain, a CD28α transmembrane (TM) domain, the cytoplasmic portion of the CD28 costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, a TLR2 signaling domain (e.g., the cytoplasmic portion of TLR2), a P2A cleavage site, a signal peptide, an anti-CD33 C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of the TLR).In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:3. In some embodiments, the CAR fusion construct comprises SEQ ID NO:3. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and / or the second polypeptide further comprises an intracellular domain of a second cytokine receptor. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, and a γδ T cell.

[0102] immune cells The engineered immune cells can be derived from a variety of cell types and cell sources. Cells from any mammalian species are contemplated herein, including, but not limited to, mouse, rat, guinea pig, rabbit, dog, monkey, and human. In some embodiments, the engineered immune cells are human cells. In some embodiments, the engineered immune cells and the recipient individual are allogeneic (i.e., from the same species but from different donors). In some embodiments, the engineered immune cells are autologous (i.e., the donor and recipient are the same). In some embodiments, the engineered immune cells are isogenic (i.e., the donor and recipient are different individuals but are identical twins).

[0103] In some embodiments, the modified immune cells are derived from primary cells. In some embodiments, the modified immune cells are primary cells isolated from an individual. In some embodiments, the modified immune cells are propagated (e.g., expanded and / or differentiated) from primary cells isolated from an individual. In some embodiments, the primary cells are hematopoietic cells. In some embodiments, the primary cells are obtained from the thymus. In some embodiments, the primary cells are obtained from lymph or lymph nodes (e.g., tumor-draining lymph nodes). In some embodiments, the primary cells are obtained from the spleen. In some embodiments, the primary cells are obtained from bone marrow. In some embodiments, the primary cells are obtained from blood, e.g., peripheral blood. In some embodiments, the primary cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the primary cells are derived from plasma. In some embodiments, the primary cells are derived from a tumor. In some embodiments, the primary cells are obtained from the mucosal immune system. In some embodiments, the primary cells are obtained from a biopsy sample.

[0104] In some embodiments, the engineered immune cells are derived from a cell line. In some embodiments, the engineered immune cells are obtained from a commercial cell line. In some embodiments, the engineered immune cells are a cell line established from primary cells isolated from an individual. In some embodiments, the engineered immune cells are propagated (e.g., expanded and / or differentiated) from a cell line. In some embodiments, the cell line is a non-immortalized cell line. In some embodiments, the cell line is an immortalized cell line. In some embodiments, the cell line is a tumor cell line, e.g., a leukemia or lymphoma cell line. In some embodiments, the cell line is a cell line derived from PBMCs. In some embodiments, the cell line is a stem cell line. In some embodiments, the cell line is selected from the group consisting of HEK293-6E cells, NK-92 cells, and Jurkat cells.

[0105] Exemplary immune cells that may be used in the present application include, but are not limited to, dendritic cells (including immature and mature dendritic cells), T lymphocytes (e.g., early T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, helper T cells, natural killer T cells, Treg cells, tumor-infiltrating lymphocytes (TILs), and lymphokine-activated killer (LAK) cells), B cells, natural killer (NK) cells, monocytes, macrophages, neutrophils, granulocytes, and combinations thereof. Immune cell subgroups may be defined by the presence or absence of one or more cell surface markers known in the art (e.g., CD3, CD4, CD8, CD19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc.). When the pharmaceutical composition comprises multiple modified immune cells, these modified immune cells may be a specific subgroup of immune cell types, a combination of subgroups of immune cell types, or a combination of two or more immune cell types. In some embodiments, the immune cells are present in a homogeneous cell population. In some embodiments, the immune cells are present in an enriched heterogeneous cell population of immune cells. In some embodiments, the modified immune cells are lymphocytes. In some embodiments, the modified immune cells are not lymphocytes. In some embodiments, the modified immune cells are suitable for adoptive immunotherapy. In some embodiments, the modified immune cells are PBMCs. In some embodiments, the modified immune cells are immune cells derived from PBMCs. In some embodiments, the modified immune cells are T cells. In some embodiments, the modified immune cells are CD4 + In some embodiments, the modified immune cells are CD8 T cells. + In some embodiments, the modified immune cells are T cells. In some embodiments, the modified immune cells are B cells. In some embodiments, the modified immune cells are NK cells.

[0106] In some embodiments, the modified immune cells are derived from stem cells. In some embodiments, the stem cells are totipotent stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are unipotent stem cells. In some embodiments, the stem cells are progenitor cells. In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are hematopoietic stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0107] The engineered immune cells may comprise any number (e.g., any one of 1, 2, 3, 4, 5, 10, 50, 100, 1000, or more) heterologous nucleic acid sequences (including first and second nucleic acid sequences). In some embodiments, the engineered immune cells comprise a single copy of the first and / or second heterologous nucleic acid sequence. In some embodiments, the engineered immune cells comprise multiple copies of the first and / or second heterologous nucleic acid sequence. In some embodiments, the engineered immune cells further comprise at least one additional heterologous nucleic acid sequence, e.g., a heterologous nucleic acid sequence encoding an immunomodulator such as a cytokine, a chemokine, and / or an immune checkpoint inhibitor.

[0108] One or more nucleic acids, including one or more heterologous nucleic acid sequences described herein, may be instantaneously or stably incorporated into the modified immune cells. In some embodiments, the one or more nucleic acids are instantaneously expressed in the modified immune cells. For example, the one or more nucleic acids can be present in the nucleus of the modified immune cells in the form of an extrachromosomal array. The one or more nucleic acids can be introduced into the modified immune cells using any transfection or transduction method known in the art, including viral or non-viral methods. Exemplary non-viral transfection methods include, but are not limited to, chemical-based transfection, such as the use of calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, hydrodynamic delivery, or transposons; particle-based methods, such as the use of gene guns, magnetofection, or magnet-assisted transfection, particle bombardment, and mixing methods, such as nucleofection.

[0109] In some embodiments, one or more heterologous nucleic acid sequences are present in the genome of the modified immune cell. For example, one or more nucleic acids comprising one or more heterologous nucleic acid sequences can be integrated into the genome of the modified immune cell by any method known in the art, including, but not limited to, viral-mediated integration, random integration, homologous recombination methods, and site-specific integration methods, such as the use of site-specific recombinases or integrases, transposases, transcription activator-like effector nucleases (TALEN®), CRISPR / Cas9, and zinc finger nucleases. In some embodiments, one or more heterologous nucleic acid sequences are integrated into specifically designed loci in the modified immune cell genome. In some embodiments, one or more heterologous nucleic acid sequences are integrated into integration hotspots in the modified immune cell genome. In some embodiments, heterologous nucleic acids (sequences) are integrated into random loci in the modified immune cell genome. When one or more multicopy heterologous nucleic acid sequences are present in a single modified immune cell, the heterologous nucleic acid sequences may be integrated into multiple loci in the modified immune cell genome.

[0110] TLR polypeptides The modified immune cells described herein express toll-like receptor (TLR) polypeptides (e.g., a first TLR polypeptide and a second TLR polypeptide, also referred to herein as a first polypeptide and a second polypeptide, respectively). In some embodiments, the modified immune cells express at least one TLR polypeptide. In some embodiments, the modified immune cells express two TLR polypeptides. The present application further provides TLR polypeptides and compositions thereof. In some embodiments, the TLR polypeptides described herein provide a strong costimulatory anti-tumor effect by inducing TLR signaling after antigen stimulation.

[0111] TLR polypeptides are derived from TLR molecules. TLRs are type I transmembrane glycoproteins characterized by the presence of an extracellular domain containing leucine-rich repeats (LRRs), which mediates ligand recognition. The extracellular domain is followed by a single transmembrane helix and an intracellular Toll-like / interleukin-1 (IL-1) receptor (TIR) ​​domain. In some embodiments, the TIR domain is responsible for downstream signaling (e.g., inducing TLR signaling). The domains and functions of TLRs are well known in the art. See, for example, Jin et al., 2008 29(2):182-91.

[0112] In some embodiments, these TLR molecules are TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10. In some embodiments, the TLR polypeptide is derived from TLR1. In some embodiments, the TLR polypeptide is derived from TLR2. In some embodiments, the TLR polypeptide is derived from TLR4. In some embodiments, the TLR polypeptide is derived from TLR6.

[0113] In some embodiments, the TLR polypeptides dimerize when the first target binding domain and the second target binding domain bind to their corresponding targets, such that the first TLR signaling domain and the second TLR signaling domain associate to form a TLR signaling moiety capable of inducing TLR signaling. In some embodiments, the TLR polypeptides form homodimers (e.g., dimerization of two identical TLR molecules). Homodimers can be formed with TLR polypeptides derived from TLR3, TLR4, TLR5, or TLR9. In some embodiments, the first and second TLR polypeptides each comprise a TLR3 transmembrane domain and a TLR3 signaling domain. In some embodiments, the first and second TLR polypeptides each comprise a TLR4 transmembrane domain and a TLR4 signaling domain. In some embodiments, the first and second TLR polypeptides each comprise a TLR5 transmembrane domain and a TLR5 signaling domain. In some embodiments, the first and second TLR polypeptides each comprise a TLR9 transmembrane domain and a TLR9 signaling domain.

[0114] In some embodiments, the TLR polypeptides form heterodimers (e.g., dimerization of two different TLR polypeptides). Heterodimers can be formed with TLR polypeptides from TLR1 / TLR2, TLR1 / TLR4, TLR2 / TLR6, TLR2 / TLR10, or TLR4 / TLR5. In some embodiments, a first TLR polypeptide comprises a TLR1 transmembrane domain and a TLR1 signaling domain, and a second TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain, or a first TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain, and a second TLR polypeptide comprises a TLR1 transmembrane domain and a TLR1 signaling domain. In some embodiments, the first TLR polypeptide comprises a TLR1 transmembrane domain and a TLR1 signaling domain, and the second TLR polypeptide comprises a TLR4 transmembrane domain and a TLR4 signaling domain, or the first TLR polypeptide comprises a TLR4 transmembrane domain and a TLR4 signaling domain, and the second TLR polypeptide comprises a TLR1 transmembrane domain and a TLR1 signaling domain. In some embodiments, the first TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain, and the second TLR polypeptide comprises a TLR6 transmembrane domain and a TLR6 signaling domain, or the first TLR polypeptide comprises a TLR6 transmembrane domain and a TLR6 signaling domain, and the second TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain. In some embodiments, the first TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain, and the second TLR polypeptide comprises a TLR10 transmembrane domain and a TLR10 signaling domain, or the first TLR polypeptide comprises a TLR10 transmembrane domain and a TLR10 signaling domain, and the second TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain.In some embodiments, the first TLR polypeptide comprises a TLR4 transmembrane domain and a TLR4 signaling domain, and the second TLR polypeptide comprises a TLR5 transmembrane domain and a TLR5 signaling domain, or the first TLR polypeptide comprises a TLR5 transmembrane domain and a TLR5 signaling domain, and the second TLR polypeptide comprises a TLR4 transmembrane domain and a TLR4 signaling domain.

[0115] Exemplary TLR molecules are listed in Table 1 below.

[0116] Table 1. Exemplary Toll-like receptor sequences TIFF2024534783000001.tif81157TIFF2024534783000002.tif206157TIFF20245347830 00003.tif206157TIFF2024534783000004.tif206157TIFF2024534783000005.tif218157

[0117] In some embodiments, the TLR polypeptide induces an increase in cytotoxicity against target cells by the engineered immune cells, e.g., at least about any one of a 10%, 20%, 30%, 40%, 2-fold, 4-fold, 6-fold, or more increase, compared to engineered immune cells that do not contain the TLR receptor domain. In some embodiments, the TLR receptor domain induces an increase in cytotoxicity against target cells by the engineered immune cells, e.g., about no more than 6-fold, no more than 4-fold, no more than 2-fold, no more than 40%, no more than 30%, no more than 20%, no more than 10%, or no more increase, compared to engineered immune cells that do not contain the TLR receptor domain. In some embodiments, cytotoxicity is measured by lactate dehydrogenase (LDH) cytotoxicity assay and / or co-culture assay with target tumor cells. In some embodiments, cytotoxicity and tumor cell killing capacity are measured in cell-based assays. In some embodiments, cytotoxicity and tumor cell killing capacity are measured in vivo.

[0118] In some embodiments, the TLR polypeptide induces a reduction in the level of inflammatory cytokine secretion by the engineered immune cells, e.g., at least about any one of a 10%, 20%, 30%, 40%, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more reduction, compared to engineered immune cells that do not contain the TLR receptor domain. In some embodiments, the TLR polypeptide induces a reduction in the level of inflammatory cytokine secretion by the engineered immune cells, e.g., by about 1000-fold or less, 500-fold or less, 200-fold or less, 100-fold or less, 50-fold or less, 30-fold or less, 20-fold or less, 18-fold or less, 16-fold or less, 14-fold or less, 12-fold or less, 10-fold or less, 8-fold or less, 6-fold or less, 4-fold or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or less than or equal to a decrease, compared to engineered immune cells that do not contain the TLR polypeptide. In some embodiments, the TLR polypeptide induces a reduction in inflammatory cytokine secretion levels by the engineered immune cells, e.g., within about any one of the following: 10% to 50%, 2 to 1000-fold, 2 to 50-fold, 50 to 100-fold, 100 to 1000-fold, 50 to 500-fold, 10 to 100-fold, 10 to 50-fold, or 50 to 200-fold, compared to engineered immune cells that do not contain the TLR polypeptide. Exemplary inflammatory cytokines include, but are not limited to, IFN-γ, TNF-α, and GM-CSF. In some embodiments, the inflammatory cytokine secretion levels are measured in a serum immunoassay (e.g., enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), or flow cytometry). In some embodiments, the inflammatory cytokine secretion levels are measured in a cell-based assay. In some embodiments, the inflammatory cytokine secretion levels are measured in vivo.

[0119] In some embodiments, the TLR polypeptide is a costimulatory molecule. In some embodiments, the TLR polypeptide costimulatory molecule is an inducible costimulatory molecule, where the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety that can induce TLR signaling when the first target binding domain and the second target binding domain bind to their corresponding targets.

[0120] In some embodiments, the inducible costimulatory molecule is linked to an engineered receptor (e.g., any one of the engineered receptors described herein) via a self-cleaving peptide (e.g., P2A). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the TLR polypeptide comprises a transmembrane domain and a signaling domain. In some embodiments, the TLR polypeptide is a costimulatory molecule and comprises a TLR transmembrane domain and a TLR signaling domain. In some embodiments, the signaling domain is an intracellular domain, e.g., an intracellular signaling domain. In some embodiments, the TLR polypeptide comprises (a) a target binding domain, (b) a transmembrane domain, and (c) a signaling domain (e.g., an intracellular signaling domain). Optionally, the TLR polypeptide further comprises a signaling peptide.

[0121] In some embodiments, the TLR polypeptide comprises a transmembrane domain fused directly or indirectly to a TLRd signaling domain. In some embodiments, the TLR polypeptide comprises a transmembrane domain fused directly or indirectly to a target binding domain (e.g., a first target binding domain and / or a second target binding domain). In some embodiments, the TLR polypeptide comprises a transmembrane domain fused directly or indirectly to a target binding domain and a TLR signaling domain simultaneously. In some embodiments, the TLR polypeptide comprises a TLR transmembrane domain from a TLR molecule. In some embodiments, the TLR molecule is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10. In some embodiments, the TLR transmembrane domain is derived from TLR1. In some embodiments, the TLR transmembrane domain is derived from TLR2. In some embodiments, the TLR transmembrane domain is derived from TLR4. In some embodiments, the TLR transmembrane domain is derived from TLR6.

[0122] In some embodiments, the modified immune cells comprise a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide comprises a first target binding domain, a first TLR transmembrane domain, and a first TLR signaling domain. In some embodiments, the second polypeptide comprises a second target binding domain, a second TLR transmembrane domain, and a second TLR signaling domain.

[0123] In some embodiments, the first TLR transmembrane domain and the first TLR signaling domain are derived from the same TLR molecule. In some embodiments, the first TLR transmembrane domain and / or the first TLR signaling domain are derived from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10. In some embodiments, the first TLR transmembrane domain and / or the first TLR signaling domain are derived from the group consisting of TLR1, TLR2, TLR4, and TLR6. In some embodiments, the first TLR transmembrane domain and / or the first TLR signaling domain are derived from TLR1. In some embodiments, the first TLR transmembrane domain and / or the first TLR signaling domain are derived from TLR4.

[0124] In some embodiments, the second TLR transmembrane domain and the second TLR signaling domain are derived from the same TLR molecule. In some embodiments, the second TLR transmembrane domain and / or the second TLR signaling domain are derived from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10. In some embodiments, the second TLR transmembrane domain and / or the second TLR signaling domain are derived from TLR1, TLR2, TLR4, and TLR6. In some embodiments, the second TLR transmembrane domain and / or the first TLR signaling domain are derived from TLR2.

[0125] In some embodiments, the first TLR transmembrane domain and the second TLR transmembrane domain are the same. In some embodiments, the first TLR transmembrane domain is derived from TLR1 and the second TLR transmembrane domain is derived from TLR1. In some embodiments, the first TLR transmembrane domain is derived from TLR2 and the second TLR transmembrane domain is derived from TLR2. In some embodiments, the first TLR transmembrane domain is derived from TLR4 and the second TLR transmembrane domain is derived from TLR4. In some embodiments, the first TLR transmembrane domain is derived from TLR6 and the second TLR transmembrane domain is derived from TLR6.

[0126] In some embodiments, the first TLR signaling domain and the second TLR signaling domain are the same. In some embodiments, the first TLR signaling domain is derived from TLR1 and the second TLR signaling domain is derived from TLR1. In some embodiments, the first TLR signaling domain is derived from TLR2 and the second TLR signaling domain is derived from TLR2. In some embodiments, the first TLR signaling domain is derived from TLR4 and the second TLR signaling domain is derived from TLR4. In some embodiments, the first TLR signaling domain is derived from TLR6 and the second TLR signaling domain is derived from TLR6.

[0127] In some embodiments, the first TLR transmembrane domain and the second TLR transmembrane domain are different. In some embodiments, the first TLR transmembrane domain is derived from TLR2, and the second TLR transmembrane domain is derived from TLR1. In some embodiments, the first TLR signaling domain and the second TLR signaling domain are different. In some embodiments, the first TLR signaling domain is derived from TLR2, and the second TLR signaling domain is derived from TLR1. In some embodiments, the first TLR transmembrane domain is derived from TLR2, the first TLR signaling domain is derived from TLR2, the second TLR transmembrane domain is derived from TLR1, and the second TLR signaling domain is derived from TLR1.

[0128] In some embodiments, the TLR polypeptides described herein comprise a target binding domain. In some embodiments, the target binding domain is not derived from a receptor or a ligand. In some embodiments, the first and second polypeptides are not derived from TGFβ. In some embodiments, the first and second polypeptides are not TGFβ. In some embodiments, the first and second polypeptides do not comprise a TGFβ extracellular domain. In some embodiments, the first and second polypeptides do not comprise an extracellular TGFβ binding domain of TGFβ (e.g., TGFβR1 and / or TGFβR2). In some embodiments, the target binding domain does not comprise an extracellular TGFβ binding domain of TGFβR1. In some embodiments, the target binding domain does not comprise an extracellular TGFβ binding domain of TGFβR2. In some embodiments, the target binding domain is not an extracellular TGFβ binding domain. In some embodiments, the target binding domain is not an extracellular TGFβ binding domain of TGFβR2. In some embodiments, the target binding domain is not an extracellular TGFβ binding domain of TGFβR1. In some embodiments, the TLR polypeptide (e.g., the first polypeptide and / or the second polypeptide) is not a chimeric TGFβ receptor-CTBR signal converter as described in WO 2018 / 094244 A1, which is incorporated herein by reference in its entirety.

[0129] In some embodiments, the modified immune cells provided herein comprise a first and a second TLR polypeptide, wherein the first TLR polypeptide comprises a first target binding domain and the second TLR polypeptide comprises a second target binding domain. In some embodiments, the first target binding domain and the second target binding domain are the same. In some embodiments, the first target binding domain and the second target binding domain are different.

[0130] In some embodiments, the target binding domain of the TLR polypeptide is an antibody or antibody fragment, e.g., an scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or V H In some embodiments, the target binding domain of the TLR polypeptide specifically binds to a single tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CLL1, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other clinically important tumor antigens, and combinations thereof. In some embodiments, the TLR polypeptide specifically binds to a target antigen selected from the group consisting of NKG2D, GPRC5D, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, AFP, CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CEA, EGFR (e.g., EGFRvIII), GD2, GPC-2, GPC3, CLDN18.2, HER2, LILRB4, IL-13Rα2, IGF1R, mesothelin, PSMA, ROR1, WT1, NKG2D, CLL1, TGFaRII, TGFbRII, CCR5, CXCR4, CCR4, an HPV-associated antigen, and an EBV-associated antigen (e.g., LMP1 or LMP2). In some embodiments, the target binding domain specifically binds to CD33, CLL1, CD123, CD19, CD20, CD22, BCMA, NKG2D, GPRC5D, or GPC3.

[0131] In some embodiments, the target molecule is an immune checkpoint protein, hi some embodiments, the target molecule is selected from the group consisting of PD-1, CD70, CD27, SIRPα, and TIGIT.

[0132] In some embodiments, the target molecule is a naturally occurring protein expressed on immune cells. In some embodiments, the target molecule is NKG2D. In some embodiments, the target molecule is a mutant NKG2D. In some embodiments, the mutant NKG2D comprises a truncated sequence and / or amino acid substitutions, mutations, additions, and / or deletions. In some embodiments, the target molecule is the extracellular antigen-binding domain of NKG2D (also referred to herein as "NKG2D ECD"). In some embodiments, the target molecule is the full-length sequence of NKG2D. In some embodiments, the NKG2D comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:54. In some embodiments, the second polypeptide comprises SEQ ID NO:54. In some embodiments, the NKG2D comprises amino acids 81 to 216 of the full-length ECD sequence of NKG2D (SEQ ID NO:54). In some embodiments, the NKG2D comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:51 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the second polypeptide comprises SEQ ID NO:51. In some embodiments, the NKG2D comprises amino acids 89 to 216 of the full-length ECD sequence of NKG2D (SEQ ID NO:54). In some embodiments, the NKG2D comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO: 52. In some embodiments, the second polypeptide comprises SEQ ID NO: 52. In some embodiments, the NKG2D comprises amino acids 98 to 216 of the full-length ECD sequence of NKG2D (SEQ ID NO: 54).In some embodiments, the NKG2D comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 53. In some embodiments, the second polypeptide comprises SEQ ID NO: 53.

[0133] In some embodiments, the TLR polypeptide further comprises a signal peptide that targets the TLR polypeptide to the secretory pathway of the cell (e.g., the ER) and enables incorporation and anchoring of the TLR polypeptide into the lipid bilayer of the host cell. Suitable signal peptides for the TLR polypeptides described herein, including signal sequences of naturally occurring proteins or non-naturally occurring synthetic signal sequences, will be apparent to those of skill in the art. In some embodiments, the signal peptide is from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, IL-3, and IgG1 heavy chain. In some embodiments, the signal peptide is from CD8α.

[0134] A TLR polypeptide may include one or more peptide linkers located between different domains. For example, the target binding domain and the TLR transmembrane domain and / or the TLR transmembrane domain and the TLR signaling domain can be fused to each other via a peptide bond or peptide linker. The peptide linkers connecting different domains can be the same or different. Each peptide linker can be individually optimized. The peptide linker may have any suitable length. In some embodiments, the length of the peptide linker is at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, or more amino acids. In some embodiments, the length of the peptide linker is any one of about 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. In some embodiments, the length of the peptide linker is any one of about 1 to about 10 amino acids, about 1 to about 20 amino acids, about 1 to about 30 amino acids, about 5 to about 15 amino acids, about 10 to about 25 amino acids, about 5 to about 30 amino acids, about 10 to about 30 amino acids, about 30 to about 50 amino acids, or about 1 to about 50 amino acids.

[0135] The peptide linker may have a naturally occurring or non-naturally occurring sequence. In some embodiments, the peptide linker is a flexible linker. An exemplary flexible linker is a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n (SEQ ID NO:27), (GSGGS) n (SEQ ID NO: 28) and (GGGS) n(SEQ ID NO:29), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker has the amino acid sequence of SEQ ID NO:25 or 26.

[0136] In some embodiments, the TLR polypeptide comprises a transmembrane domain and a signaling domain derived from the same TLR molecule. In some embodiments, the TLR polypeptide comprises a TLR1 transmembrane domain and a TLR1 signaling domain. In some embodiments, the TLR polypeptide comprises a TLR2 transmembrane domain and a TLR2 signaling domain. In some embodiments, the TLR polypeptide comprises a TLR4 transmembrane domain and a TLR4 signaling domain. In some embodiments, the TLR polypeptide comprises a TLR6 transmembrane domain and a TLR6 signaling domain. In some embodiments, the TLR polypeptide further comprises a signal peptide (e.g., a leader sequence). In some embodiments, the TLR polypeptide further comprises the intracellular domain of a cytokine receptor.

[0137] In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain. In some embodiments, the target binding domain is an scFv or sdAb that specifically binds to NKG2D, CD33, CLL1, CD123, CD19, CD20, CD22, BCMA, GPRC5D, or GPC3. In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain that specifically binds to CD33. In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain that specifically binds to the V domain of CD33. In some embodiments, TLR polypeptides comprise a TLR2 transmembrane domain, an induced TLR2 signaling domain, and a target binding domain that specifically binds to the V domain of CD33. In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain that specifically binds to the C2 domain of CD33. In some embodiments, the TLR polypeptide comprises a TLR1 transmembrane domain, an induced TLR1 signaling domain, and a target binding domain that specifically binds to the C2 domain of CD33. In some embodiments, a TLR polypeptide comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprises a target binding domain that specifically binds to CD20. In some embodiments, a TLR polypeptide comprises a TLR4 transmembrane domain, an induced TLR4 signaling domain, and a target binding domain that specifically binds to CD20. In some embodiments, a TLR polypeptide comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprises a target binding domain that specifically binds to CD33. In some embodiments, a TLR polypeptide comprises a TLR4 transmembrane domain, an induced TLR4 signaling domain, and a target binding domain that specifically binds to CD33.In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain that specifically binds to NKG2D. In some embodiments, TLR polypeptides comprise a TLR4 transmembrane domain, an induced TLR4 signaling domain, and a target binding domain that specifically binds to NKG2D. In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain that specifically binds to BCMA. In some embodiments, TLR polypeptides comprise a TLR4 transmembrane domain, an induced TLR4 signaling domain, and a target binding domain that specifically binds to BCMA. In some embodiments, TLR polypeptides comprising a transmembrane domain and a signaling domain derived from the same TLR molecule further comprise a target binding domain that specifically binds to GPRC5D. In some embodiments, TLR polypeptides comprise a TLR4 transmembrane domain, an induced TLR4 signaling domain, and a target binding domain that specifically binds to GPRC5D. In some embodiments, TLR polypeptides further comprise a signal peptide (e.g., a leader sequence).

[0138] In some embodiments, the TLR polypeptide further comprises an intracellular domain of a cytokine receptor. In some embodiments, the TLR polypeptide comprises two or more intracellular domains of one or more cytokine receptors. In some embodiments, the C-terminus of the intracellular domain of the cytokine receptor is fused to the N-terminus of the TLR signaling domain. In some embodiments, the C-terminus of the intracellular domain of the cytokine receptor is fused directly to the N-terminus of the TLR domain. In some embodiments, the C-terminus of the intracellular domain of the cytokine receptor is fused indirectly to the N-terminus of the TLR domain, e.g., via a linker (e.g., a flexible peptide linker) or another domain. In some embodiments, the TLR polypeptide comprises, from N-terminus to C-terminus: i) a target binding domain; ii) an intracellular domain of the cytokine receptor; iii) a TLR transmembrane domain; and iv) a TLR signaling domain.

[0139] In some embodiments, the N-terminus of the cytokine receptor intracellular domain is fused to the C-terminus of the TLR signaling domain. In some embodiments, the N-terminus of the cytokine receptor intracellular domain is fused directly to the C-terminus of the TLR signaling domain. In some embodiments, the N-terminus of the cytokine receptor intracellular domain is fused indirectly to the C-terminus of the TLR signaling domain, e.g., via a linker (e.g., a flexible peptide linker) or another domain. In some embodiments, the TLR polypeptide comprises, from N-terminus to C-terminus: i) a target binding domain; ii) a TLR transmembrane domain; iii) a TLR signaling domain; and iv) a cytokine receptor intracellular domain.

[0140] In some embodiments, the intracellular domain from a cytokine receptor confers improved TLR signaling (e.g., anti-tumor activity) to one or more TLR polypeptides. In some embodiments, the intracellular domain from a cytokine receptor is selected from the group consisting of GM-CSF receptor, IL-18 receptor, IL-21 receptor, IL-15 receptor, and IL-23 receptor. In some embodiments, the intracellular domain from a cytokine receptor comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0141] The modified immune cells described herein comprise a first TLR polypeptide and a second TLR polypeptide. In some embodiments, the first TLR polypeptide further comprises a first intracellular domain of a first cytokine receptor. In some embodiments, the second polypeptide further comprises a second intracellular domain of a second cytokine receptor. In some embodiments, the first polypeptide further comprises a first intracellular domain of a first cytokine receptor, and the second polypeptide further comprises a second intracellular domain of a second cytokine receptor. In some embodiments, the first intracellular domain of the first cytokine receptor and the second intracellular domain of the second cytokine receptor are the same. In some embodiments, the first intracellular domain of the first cytokine receptor and the second intracellular domain of the second cytokine receptor are different. In some embodiments, the first cytokine receptor is selected from the group consisting of GM-CSF receptor, IL-18 receptor, IL-21 receptor, IL-15 receptor, and IL-23 receptor. In some embodiments, the second cytokine receptor is selected from the group consisting of GM-CSF receptor, IL-18 receptor, IL-21 receptor, IL-15 receptor, and IL-23 receptor. In some embodiments, the first cytokine receptor and the second cytokine receptor are each selected from the group consisting of GM-CSF receptor, IL-18 receptor, IL-21 receptor, IL-15 receptor, and IL-23 receptor. In some embodiments, the first intracellular domain of the first cytokine receptor comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the second intracellular domain of the second cytokine receptor comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the first intracellular domain of the first cytokine receptor and the second intracellular domain of the second cytokine receptor comprise an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the C-terminus of the first intracellular domain of the first cytokine receptor is fused to the N-terminus of the first TLR signaling domain.In some embodiments, the C-terminus of the second intracellular domain of the second cytokine receptor is fused to the N-terminus of the second TLR signaling domain. In some embodiments, the C-terminus of the first intracellular domain of the first cytokine receptor is fused to the N-terminus of the first TLR signaling domain and the C-terminus of the second intracellular domain of the second cytokine receptor is fused to the N-terminus of the second TLR signaling domain. In some embodiments, the N-terminus of the first intracellular domain of the first cytokine receptor is fused to the C-terminus of the TLR signaling domain. In some embodiments, the N-terminus of the second intracellular domain of the second cytokine receptor is fused to the C-terminus of the TLR signaling domain. In some embodiments, the N-terminus of the first intracellular domain of the first cytokine receptor is fused to the C-terminus of the TLR signaling domain and the N-terminus of the second intracellular domain of the second cytokine receptor is fused to the C-terminus of the TLR signaling domain.

[0142] In some embodiments, the TLR polypeptides described herein are comprised in engineered immune cells. In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CD19 CAR molecule and an anti-CD20 TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the engineered immune cells express an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CD19 CAR comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:2. In some embodiments, the anti-CD19 CAR comprises SEQ ID NO:2. In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO:40.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 1 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO: 1. In some embodiments, the TLR polypeptide coding sequence lacks some or all of the sequence of the predicted upstream start codon. In some embodiments, the TLR polypeptide may contain several amino acid mutations that do not affect the induction of TLR signaling without affecting the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain.

[0143] In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CD19 CAR molecule and an anti-NKG2D TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., a cytoplasmic portion of TLR4). In some embodiments, the engineered immune cells express an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CD19 CAR comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:2. In some embodiments, the anti-CD19 CAR comprises SEQ ID NO:2. In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO:40.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:59 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:59. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:60 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:60. In some embodiments, the TLR polypeptide coding sequence lacks the sequence of some or all of the predicted upstream start codon.In some embodiments, the TLR polypeptide may contain several amino acid mutations so as not to affect induction of TLR signaling, without affecting the formation of the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain.

[0144] In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-GPC3 CAR molecule and an anti-NKG2D TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the engineered immune cells express an anti-GPC3 CAR. In some embodiments, the anti-GPC3 CAR comprises, from N- to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-GPC3 CAR comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:56. In some embodiments, the anti-GPC3 CAR comprises SEQ ID NO:56. In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO:40.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:57 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:57. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:58 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:58. In some embodiments, the TLR polypeptide coding sequence lacks the sequence of some or all of the predicted upstream start codon.In some embodiments, the TLR polypeptide may contain several amino acid mutations so as not to affect induction of TLR signaling, without affecting the formation of the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain.

[0145] In some embodiments, the TLR polypeptide described herein is comprised in an engineered immune cell. In some embodiments, the engineered immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR molecule and an anti-CD33 TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the engineered immune cell expresses an anti-CLL1 CAR. In some embodiments, the anti-CLL1 CAR comprises, from N- to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:71 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:71. In some embodiments, the TLR polypeptide coding sequence lacks the sequence of some or all of the predicted upstream start codon.In some embodiments, the TLR polypeptide may contain several amino acid mutations so as not to affect induction of TLR signaling, without affecting the formation of the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain.

[0146] In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR molecule (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-BCMA TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the anti-BCMA CAR comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-BCMA CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 67. In some embodiments, the anti-BCMA CAR comprises SEQ ID NO: 67. In some embodiments, the anti-BCMA CAR comprises, from N-terminus to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-BCMA CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:66. In some embodiments, the anti-BCMA CAR comprises SEQ ID NO:66.In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO: 40. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:62 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:62. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a TLR4 transmembrane (TM) region and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain).In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:63. In some embodiments, the CAR fusion construct comprises SEQ ID NO:63. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a CD28α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:64 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO: 64. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a tandem anti-BCMA sdAb, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain).In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:61. In some embodiments, the CAR fusion construct comprises SEQ ID NO:61. In some embodiments, the TLR polypeptide coding sequence lacks the sequence of some or all of the predicted upstream start codon. In some embodiments, the TLR polypeptide may contain several amino acid mutations so as not to affect the induction of TLR signaling, without affecting the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain. In some embodiments, the modified immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR molecule (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-GPRC5D TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the anti-BCMA CAR comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-BCMA CAR comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:66 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the anti-BCMA CAR comprises SEQ ID NO:66. In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:40.In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO: 40. In some embodiments, the engineered immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-GPRC5D TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:65. In some embodiments, the CAR fusion construct comprises SEQ ID NO:65. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:72 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).In some embodiments, the CAR fusion construct comprises SEQ ID NO: 72. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a CD28α hinge domain, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:73. In some embodiments, the CAR fusion construct comprises SEQ ID NO:73. In some embodiments, the TLR polypeptide coding sequence lacks some or all of the sequence of the predicted upstream start codon. In some embodiments, the TLR polypeptide may include several amino acid mutations such that induction of TLR signaling is unaffected without affecting the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain.

[0147] In some embodiments, the engineered immune cells comprise a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR and an anti-CD33 (e.g., anti-CD33 V domain and / or anti-CD33 C2 domain) TLR polypeptide. In some embodiments, the first polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., the cytoplasmic portion of TLR2), and the second polypeptide comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD33 C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1). In some embodiments, the engineered immune cells express an anti-CLL1 CAR. In some embodiments, the anti-CLL1 CAR comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD28α hinge domain, a CD28α transmembrane (TM) domain, a cytoplasmic portion of a CD28 costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CLL1 CAR comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:4. In some embodiments, the anti-CLL1 CAR comprises SEQ ID NO:4. In some embodiments, the engineered immune cells express an anti-CLL1 / CD33 dual CAR. In some embodiments, the anti-CLL1 / CD33 dual CAR comprises, from N- to C-terminus, a signal peptide, an anti-CLL1 sdAb, an anti-CD33 V-domain sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain.In some embodiments, the anti-CLL1 / CD33 dual CAR comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:43 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the anti-CLL1 / CD33 dual CAR comprises SEQ ID NO:43. In some embodiments, the first polypeptide comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:41 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the first polypeptide comprises SEQ ID NO:41. In some embodiments, the second polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 42. In some embodiments, the second polypeptide comprises SEQ ID NO: 42. In some embodiments, the engineered immune cell comprises a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR and an anti-CD33 (e.g., an anti-CD33 V domain and / or an anti-CD33 C2 domain) TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD28α hinge domain, a CD28α transmembrane (TM) domain, the cytoplasmic portion of the CD28 costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, a TLR2 signaling domain (e.g., the cytoplasmic portion of TLR2), a P2A cleavage site, a signal peptide, an anti-CD33 C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1).In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:3. In some embodiments, the TLR polypeptide coding sequence lacks the sequence of some or all of the predicted upstream start codon. In some embodiments, the TLR polypeptide may comprise several amino acid mutations such that induction of TLR signaling is unaffected without affecting the association of the TLR signaling moieties between the first TLR signaling domain and the second TLR signaling domain.

[0148] Variants of any of the TLR domains (e.g., TLR transmembrane domains and / or TLR signaling domains) described herein are also within the scope of the present disclosure, whereby the TLR domains can modulate the immune response of immune cells. In some embodiments, the costimulatory signaling domain contains up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue changes compared to a wild-type counterpart. Such TLR domains containing one or more amino acid changes may be referred to as variants. Compared to a costimulatory signaling domain that does not contain the mutation, mutations of amino acid residues in the TLR signaling domain can result in increased signal transduction and enhanced stimulation of the immune response. Compared to a costimulatory signaling domain that does not contain the mutation, mutations of amino acid residues in the TLR signaling domain can result in decreased signal transduction and decreased stimulation of the immune response.

[0149] In some embodiments, the TLR polypeptide comprises an amino acid sequence variant of a TLR domain (e.g., a TLR transmembrane domain and / or a TLR signaling domain) described herein. In some embodiments, the TLR polypeptide comprises an amino acid sequence variant of a TLR molecule (e.g., a TLR transmembrane domain and / or a TLR signaling domain of a TLR molecule) described herein. For example, it may be desirable to modulate the biological properties of a TLR polypeptide. Amino acid sequence variants of the TLR molecule (e.g., a transmembrane domain and / or a signaling domain of the TLR molecule) can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the TLR molecule or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the TLR molecule. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., TLR binding and / or proinflammatory activity.

[0150] In some embodiments, the TLR molecule contains one or more (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or more amino acid) conservative substitutions compared to the sequence of any one of the TLR molecules described herein. In some embodiments, the TLR molecule has at least about 80% sequence identity, e.g., at least about 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, to the sequence of any one of the TLR molecules described herein. Similar to the TLR polypeptides described herein, TLR polypeptide variants, including modified TLR molecules, have similar anti-tumor activity and reduced toxicity.

[0151] Conservative substitutions are shown in Table A below.

[0152] (Table A) Conservative substitutions TIFF2024534783000006.tif103153Amino acids can be grouped into different classes according to common side chain properties. a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, c. Acidic: Asp, Glu, d. Basic: His, Lys, Arg, e. Residues that affect chain orientation: Gly, Pro, f. Aromatics: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0153] Those skilled in the art will recognize that mutations can be generated in a gene of interest using any suitable method, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to those skilled in the art. Mutations can involve single nucleotides (e.g., point mutations involving the removal, addition, or substitution of a single nucleotide base within a DNA sequence) or can involve the insertion or deletion of large numbers of nucleotides. Mutations can occur spontaneously in events such as errors in fidelity, such as DNA replication, or can be induced after exposure to chemical or physical mutagens. Site-specific mutagenesis can also be performed using specific targeting methods well known to those skilled in the art.

[0154] As described in Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying residues or regions of a polypeptide that can be targeted for mutagenesis is called "alanine scanning mutagenesis." In this method, a target residue or group of residues (e.g., charged residues, e.g., arg, asp, his, lys, and glu) is identified and substituted with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the polypeptide reagent with its target (e.g., a first TLR signaling domain and a second TLR signaling domain) is affected. Additional substitutions can be introduced at amino acid positions to demonstrate functional sensitivity to the initial substitution. Variants may be screened to determine whether they contain the desired properties.

[0155] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0156] In some embodiments, a peptide tag (usually a short peptide sequence that can be recognized by available antisera or chemical compounds) may be included for subsequent expression and transport of the TLR polypeptide. Various tag peptides may be used with the TLR polypeptides described herein, including, but not limited to, the PK tag, the FLAG octapeptide, the MYC tag, the HIS tag (usually a segment of 4-10 histidine residues), and the e-tag (U.S. Pat. No. 6,686,152). When multiple tags are used, one or more tag peptides may be independently located at the N-terminus of the protein, its C-terminus, internally, or at any one of these locations. Tag peptides may be detected by immunodetection assays using anti-tag antibodies.

[0157] Engineered receptors Any of the above modified immune cells can further express an engineered receptor. Exemplary engineered receptors include, but are not limited to, CAR, engineered TCR, and TAC receptors. In some embodiments, the engineered receptor comprises an extracellular domain that specifically binds to an antigen (e.g., a tumor antigen), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain and / or a costimulatory domain. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of a TCR co-receptor. In some embodiments, the engineered receptor is encoded by a third nucleic acid operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). In some embodiments, the engineered receptor is introduced into the modified immune cell by inserting the protein into the cell membrane and passing the cell through a microfluidic system (e.g., CELL SQUEEZE®) (see, e.g., U.S. Patent Application Publication No. 20140287509). The engineered receptor can enhance the function of the engineered immune cell, for example, by targeting the engineered immune cell, transducing a signal, and / or enhancing the cytotoxicity of the engineered immune cell. In some embodiments, the engineered immune cell does not express an engineered receptor, e.g., a CAR, TCR, or TAC receptor.

[0158] In some embodiments, the engineered receptor comprises one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, e.g., one or more primary intracellular signaling domains and / or costimulatory domains.

[0159] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and can be applied to the modified immune cells of the present application. CARs can be constructed to have specificity for any cell surface marker, for example, by using antigen-binding fragments of antibody molecules or antibody variable domains. Any method for producing CARs can be used herein. See, e.g., US 6,410,319, US 7,446,191, US 7,514,537, US 9765342 B2, WO 2002 / 077029, WO 2015 / 142675, US 2010 / 065818, US 2010 / 025177, US 2007 / 059298, WO 2017025038 A1, and Berger C. et al., J. Clinical Investigation 118:1 294-308 (2008), which are incorporated by reference. In some embodiments, the modified immune cells are CAR-T cells.

[0160] The CAR of the present application comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises at least one targeting domain that specifically binds to at least one tumor antigen. In some embodiments, the intracellular signaling domain generates a signal that promotes the immune effector function of the CAR-containing cell (e.g., a CAR-T cell). An "immune effector function or immune effector response" refers, for example, to a function or response of an immune effector cell that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to the property of a T or NK cell that kills or inhibits the growth or proliferation of a target cell. Examples of immune effector functions include, for example, cytolytic activity (e.g., antibody-dependent cellular cytotoxicity, or ADCC) and helper activity (e.g., cytokine secretion) in a CAR-T cell. In some embodiments, a CAR has an intracellular signaling domain with reduced immune effector function. In some embodiments, the CAR has an intracellular signaling domain that has about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of an immune effector function (e.g., cytolytic function against target cells) compared to a CAR having full-length and wild-type CD3ζ and, optionally, one or more costimulatory domains. In some embodiments, the intracellular signaling domain produces a signal that promotes the proliferation and / or survival of the CAR-containing cell. In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule may comprise the entire intracellular (i.e., cytoplasmic) portion of the molecule or a fragment or derivative thereof, or the entire native intracellular signaling domain.

[0161] In some embodiments, the intracellular signaling domain of the CAR comprises a primary intracellular signaling domain. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts stimulatory to induce immune effectors to function. In some embodiments, the primary intracellular signaling domain contains a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. In some embodiments, the primary intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP 12. In some embodiments, the primary intracellular signaling domain comprises a non-functional or reduced signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP 12. The non-functional or reduced signaling domain may be a mutated signaling domain having a point mutation, insertion, or deletion that reduces or eliminates one or more immune effector functions (e.g., cytolytic activity or helper activity, including antibody-dependent cellular cytotoxicity (ADCC)). In some embodiments, the CAR comprises a non-functional or reduced CD3ζ (i.e., CD3ζ or CD3z) signaling domain. In some embodiments, the intracellular signaling domain does not comprise a primary intracellular signaling domain. Compared to a CAR having the same construct but with a wild-type primary intracellular signaling domain, the reduced primary intracellular signaling domain can induce no more than about any one of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of immune effector function (e.g., cytolytic function against target cells).

[0162] In some embodiments, the intracellular signaling domain of the CAR comprises one or more (e.g., any one of 1, 2, 3, or more) costimulatory domains. A "costimulatory domain" may be the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a homologous binding partner in an immune cell (e.g., T cell) that mediates the costimulatory response of the immune cell, such as, but not limited to, proliferation and survival, by specifically binding to a costimulatory ligand. A costimulatory molecule refers to a cell surface molecule, other than an antigen receptor or its ligand, that is instrumental in an effective immune response. Costimulatory molecules can be represented by the protein families TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, 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, These include ligands that specifically bind to ITGB7, NKG2D, NKG2C, TGFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0163] In some embodiments, the CAR comprises a single costimulatory domain. In some embodiments, the CAR comprises two or more costimulatory domains. In some embodiments, the intracellular signaling domain comprises a functional primary intracellular signaling domain and one or more costimulatory domains. In some embodiments, the CAR does not comprise a functional primary intracellular signaling domain (e.g., CD3ζ). In some embodiments, the CAR comprises an intracellular signaling domain that consists of, or consists essentially of, one or more costimulatory domains. In some embodiments, the CAR comprises an intracellular signaling domain that consists of, or consists essentially of, a non-functional or reduced primary intracellular signaling domain (e.g., mutated CD3ζ) and one or more costimulatory domains. After the targeting domain binds to the tumor antigen, the costimulatory domain of the CAR can transduce a signal to enhance proliferation, survival, and differentiation of the engineered immune cells (e.g., T cells) bearing the CAR, and inhibit activation-induced cell death. In some embodiments, the one or more costimulatory signaling domains are derived from one or more molecules selected from the group consisting of CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0164] In some embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a costimulatory signaling domain derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of 4-1BB.

[0165] In some embodiments, the intracellular signaling domain of the CAR comprises the costimulatory signaling domain of CD28 and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ, the costimulatory signaling domain of CD28, and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising, from N-terminus to C-terminus, the costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, and the cytoplasmic signaling domain of CD3ζ.

[0166] In some embodiments, the targeting domain of the CAR is an antibody or antibody fragment, e.g., an scFv, Fv, Fab, (Fab'), single domain antibody (sdAb), or V HIn some embodiments, the targeting domain of the CAR is a ligand or extracellular portion that specifically binds to a receptor for the tumor antigen. In some embodiments, one or more targeting domains of the CAR specifically bind to a single tumor antigen. In some embodiments, the CAR is a bispecific or multispecific CAR having targeting domains that bind to two or more tumor antigens. In some embodiments, the tumor antigen is selected from the group consisting of CD19, NKG2D, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other clinically important tumor antigens, and combinations thereof. In some embodiments, the CAR specifically binds to a target antigen selected from the group consisting of BCMA, NY-ESO-1, VEGFR2, MAGE-A3, AFP, CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CEA, EGFR (e.g., EGFRvIII), GD2, GPC-2, GPC3, CLDN18.2, HER2, LILRB4, IL-13Rα2, IGF1R, mesothelin, PSMA, ROR1, WT1, NKG2D, CLL1, TGFaRII, TGFbRII, CCR5, CXCR4, CCR4, an HPV-associated antigen, and an EBV-associated antigen (e.g., LMP1 or LMP2).

[0167] In some embodiments, the CAR is an anti-CD19 CAR. Various antigen-binding domain sequences can be used as the targeting domain of a CAR. See, for example, WO 2012 / 079000, which is incorporated herein in its entirety. In some embodiments, the anti-CD19 CAR comprises, from N- to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-CD19 CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:2. In some embodiments, the anti-CD19 CAR comprises SEQ ID NO:2.

[0168] In some embodiments, the anti-CD19 CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CD19 CAR molecule and an anti-CD20 TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO:40. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD20 scFv, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 1 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO: 1.

[0169] In some embodiments, the anti-CD19 CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CD19 CAR molecule and an anti-NKG2D TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO:40. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:59 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:59.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CD19 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:60 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:60.

[0170] In some embodiments, the CAR is an anti-CLL1 CAR. Various antigen binding domain sequences can be used as the targeting domain of the CAR. See, for example, WO 2012 / 079000, which is incorporated herein in its entirety. In some embodiments, the anti-CLL1 CAR comprises, from N- to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD28α hinge domain, a CD28α transmembrane (TM) domain, the cytoplasmic portion of the CD28 costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CLL1 sdAb comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CLL1 CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:4. In some embodiments, the anti-CLL1 CAR comprises SEQ ID NO:4.

[0171] In some embodiments, the CAR is an anti-CLL1 / CD33 dual CAR. Various antigen binding domain sequences can be used as the targeting domain of the CAR. See, e.g., WO 2012 / 079000, which is incorporated herein in its entirety. In some embodiments, the anti-CLL1 / CD33 dual CAR comprises, from N- to C-terminus, a signal peptide, an anti-CLL1 sdAb, an anti-CD33 V-domain sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-CLL1 sdAb comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD33 V-domain sdAb comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-CLL1 / CD33 dual CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 43. In some embodiments, the anti-CLL1 / CD33 dual CAR comprises SEQ ID NO: 43.

[0172] In some embodiments, the anti-CLL1 CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR and an anti-CD33 TLR polypeptide. In some embodiments, the first polypeptide and the second polypeptide each comprise, from N- to C-terminus, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane domain, and a TLR4 signaling domain (e.g., the cytoplasmic portion of TLR4). In some embodiments, the first polypeptide and the second polypeptide each comprise an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:40. In some embodiments, the first polypeptide and the second polypeptide each comprise SEQ ID NO:40. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD33 sdAb, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:71 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:71.

[0173] In some embodiments, the anti-CLL1 CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-CLL1 CAR and an anti-CD33 (e.g., anti-CD33 V domain and / or anti-CD33 C2 domain) TLR polypeptide. In some embodiments, the first polypeptide comprises, from N- to C-terminus, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, and a TLR2 signaling domain (e.g., the cytoplasmic portion of TLR2), and the second polypeptide comprises, from N- to C-terminus, a signal peptide, an anti-CD33 C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1). In some embodiments, the first polypeptide comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:41. In some embodiments, the first polypeptide comprises SEQ ID NO:41. In some embodiments, the second polypeptide comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO:42. In some embodiments, the second polypeptide comprises SEQ ID NO:42.In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-CLL1 sdAb, a CD28α hinge domain, a CD28α transmembrane (TM) domain, the cytoplasmic portion of the CD28 costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-CD33 V domain sdAb, a TLR2 transmembrane domain, a TLR2 signaling domain (e.g., the cytoplasmic portion of TLR2), a P2A cleavage site, a signal peptide, an anti-CD33 C2 domain sdAb, a TLR1 transmembrane domain, and a TLR1 signaling domain (e.g., the cytoplasmic portion of TLR1). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:3.

[0174] In some embodiments, the CAR is an anti-GPC3 CAR. Various antigen-binding domain sequences can be used as the targeting domain of a CAR. See, for example, WO2012 / 079000, which is incorporated herein in its entirety. In some embodiments, the anti-GPC3 CAR comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-GPC3 CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 56. In some embodiments, the anti-GPC3 CAR comprises SEQ ID NO: 56.

[0175] In some embodiments, the anti-GPC3 CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-GPC3 CAR and an anti-NKG2D TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO: 57. In some embodiments, the CAR fusion construct comprises SEQ ID NO: 57. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, an anti-GPC3 scFv, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-NKG2D ECD, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 58. In some embodiments, the CAR fusion construct comprises SEQ ID NO: 58.

[0176] In some embodiments, the CAR is an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR). In some embodiments, the anti-BCMA CAR is a single anti-BCMA CAR. In some embodiments, the anti-BCMA CAR is a tandem anti-BCMA CAR. A variety of antigen binding domain sequences can be used as the targeting domain of the CAR. See, for example, WO 2012 / 079000, which is incorporated herein in its entirety. In some embodiments, the single anti-BCMA CAR comprises, from N-terminus to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the single anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, the single anti-BCMA CAR comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:67 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the single anti-BCMA CAR comprises SEQ ID NO:67. In some embodiments, the tandem anti-BCMA CAR comprises, from N-terminus to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain, and a CD3ζ primary intracellular signaling domain. In some embodiments, the tandem anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the tandem anti-BCMA CAR comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:66.In some embodiments, the tandem anti-BCMA CAR comprises SEQ ID NO:66.

[0177] In some embodiments, the anti-BCMA CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-BCMA TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:62. In some embodiments, the CAR fusion construct comprises SEQ ID NO:62. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:63 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:63.In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a single anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, a single anti-BCMA sdAb, a CD28α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:64 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:64. In some embodiments, the CAR fusion construct comprises, from N-terminus to C-terminus, a signal peptide, tandem anti-BCMA sdAbs, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, tandem anti-BCMA sdAbs, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:61 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:61.

[0178] In some embodiments, the anti-BCMA CAR is part of a CAR system (e.g., a CAR fusion construct), wherein the CAR fusion construct comprises an anti-BCMA CAR (e.g., a single anti-BCMA CAR or a tandem anti-BCMA CAR) and an anti-GPRC5D TLR polypeptide. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, the cytoplasmic portion of the 4-1BB (CD137) costimulatory signaling domain and the CD3ζ primary intracellular signaling domain, a P2A cleavage site, the signal peptide, an anti-GPRC5D scFv, a TLR4 transmembrane (TM) region, and the cytoplasmic portion of TLR4 (e.g., the TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:65. In some embodiments, the CAR fusion construct comprises SEQ ID NO:65. In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a CD8α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:72 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:72.In some embodiments, the CAR fusion construct comprises, from N- to C-terminus, a signal peptide, a tandem anti-BCMA sdAb, a CD8α hinge domain, a CD8α transmembrane (TM) domain, a cytoplasmic portion of a 4-1BB (CD137) costimulatory signaling domain and a CD3ζ primary intracellular signaling domain, a P2A cleavage site, a signal peptide, an anti-GPRC5D scFv, a CD28α hinge domain, a TLR4 transmembrane (TM) region, and a cytoplasmic portion of TLR4 (e.g., a TLR4 primary intracellular signaling domain). In some embodiments, the CAR fusion construct comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:73 (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the CAR fusion construct comprises SEQ ID NO:73.

[0179] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS ( CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRFl), CD160, CD19, IL-2 Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGA E, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TGFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY 55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain of the CAR is a CD4, CD3, CD8α, or CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of CD8α.

[0180] In some embodiments, the extracellular domain is linked to the transmembrane domain via a hinge region. In one embodiment, the hinge region comprises the hinge region of CD8α.

[0181] In some embodiments, the CAR comprises a signal peptide, for example, CD8αSP.

[0182] In some embodiments, the engineered receptor is a modified T cell receptor. In some embodiments, the engineered TCR has specificity for a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CLL1, GPC3, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other clinically important tumor antigens. In some embodiments, the tumor antigen is derived from an intracellular protein of a tumor cell. Many TCRs with specificity for tumor antigens (including tumor-associated antigens) have been described, including, for example, TCRs for the NY-ESO-1 cancer-testis antigen, p53 tumor inhibitor antigen, melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art can be used in the present application. In some embodiments, the TCR has enhanced affinity for the tumor antigen. For example, US 5,830,755 and Kessels et al., Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001) describe exemplary TCRs and methods for introducing these TCRs into immune cells. In some embodiments, the modified immune cells are TCR-T cells.

[0183] The TCR receptor complex is an octameric complex formed from the variable TCR receptor α and β chains (γ and δ chains in the case of γδ T cells) and three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247 (T cell surface glycoprotein CD3ζ chain) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domains of each subunit form a polarized network of interactions that hold the complex together. The TCR complex functions to activate signaling cascades in T cells.

[0184] In some embodiments, the engineered receptor is an engineered TCR comprising one or more T cell receptor (TCR) fusion proteins (TFPs). For example, exemplary TFPs are described in US 20170166622 A1, which is incorporated herein by reference. In some embodiments, the TFP comprises the extracellular domain of a TCR subunit, wherein the extracellular domain comprises the extracellular domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the TFP comprises a transmembrane domain, wherein the transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the TFP comprises a transmembrane domain, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0185] In some embodiments, the TFP comprises a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of CD3ε, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein the TFP incorporates into the TCR when expressed in a T cell.

[0186] In some embodiments, the TFP comprises a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of CD3γ, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein the TFP incorporates into the TCR when expressed in a T cell.

[0187] In some embodiments, the TFP comprises a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of CD3δ, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein the TFP incorporates into the TCR when expressed in a T cell.

[0188] In some embodiments, the TFP comprises a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of TCRα, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein the TFP incorporates into the TCR when expressed in a T cell.

[0189] In some embodiments, the TFP comprises a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of TCRβ, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein the TFP incorporates into the TCR when expressed in a T cell.

[0190] In some embodiments, the engineered receptor is a T cell antigen coupler (TAC) receptor. For example, exemplary TAC receptors are described in US 20160368964 A1, which is incorporated herein by reference. In some embodiments, the TAC comprises a targeting domain, a TCR binding domain that specifically binds to a protein associated with the TCR complex, and a T cell receptor signaling domain. In some embodiments, the targeting domain is an antibody fragment, e.g., an scFv or V, that specifically binds to a tumor antigen. H H. In some embodiments, the targeting domain is a designed ankyrin repeat (DARPin) polypeptide. In some embodiments, the tumor antigen is selected from the group consisting of CD19, GPC3, CLL1, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other clinically important tumor antigens. In some embodiments, the protein associated with the TCR complex is CD3, e.g., CD3ε. In some embodiments, the TCR binding domain is a single chain antibody, e.g., scFv or V H H. In some embodiments, the TCR binding domain is derived from UCHT1. In some embodiments, the TAC receptor comprises a cytoplasmic domain and a transmembrane domain. In some embodiments, the T cell receptor signaling domain comprises a cytoplasmic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytoplasmic domain derived from CD4. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytoplasmic domain derived from CD8 (e.g., CD8α).

[0191] T cell coreceptors are expressed on T cells as membrane proteins. They can provide stability to the TCR:peptide:MHC complex and promote signal transduction. Two subtypes of T cell coreceptors, CD4 and CD8, show strong specificity for specific MHC types. The CD4 coreceptor can stabilize only the TCR:MHC II complex, while the CD8 coreceptor can stabilize only the TCR:MHC I complex. Differential expression of CD4 and CD8 on different T cell types results in different functional subgroups of T cells. CD8+ T cells are cytotoxic T cells.

[0192] CD4 is a glycoprotein expressed on the surface of immune cells (e.g., helper T cells, monocytes, macrophages, and dendritic cells). CD4 has four immunoglobulin domains (D1-D4) exposed on the extracellular cell surface. CD4 contains a specific amino acid sequence in its short cytoplasmic / intracellular tail, which allows the CD4 tail to recruit and interact with the tyrosine kinase Lck. When the TCR complex and CD4 each bind to different regions of the MHC II molecule, the close proximity between the TCR complex and CD4 allows Lck, which binds to the cytoplasmic tail of CD4, to tyrosine phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domain of CD3, thereby amplifying the signal generated by the TCR.

[0193] CD8 is a glycoprotein that is either a homodimer (uncommon) consisting of two α chains or a heterodimer (common) consisting of one α chain and one β chain, each of which contains an immunoglobulin variable (IgV)-like extracellular domain and an intracellular tail connected to the membrane via a thin stalk. CD8 is primarily expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 cytoplasmic tail interacts with Lck, which phosphorylates the cytoplasmic CD3 and ζ chains of the TCR complex upon binding of the TCR to its specific antigen. Tyrosine phosphorylation of the cytoplasmic CD3 and ζ chains triggers a phosphorylation cascade that ultimately leads to gene transcription.

[0194] In some embodiments, the modified immune cells express more than one engineered receptor, e.g., any combination of CAR, TCR, TAC receptors.

[0195] In some embodiments, the engineered receptors (e.g., CARs, TCRs, or TACs) expressed by the modified immune cells target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that are involved in an immune response, particularly a T cell-mediated immune response. The choice of target antigens of the present disclosure depends on the specific type of cancer being treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroid globulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, CLL1, BCMA, GPC3, CD19, prostaglandins, PSMA, HER2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0196] In some embodiments, the tumor antigen is one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used as target antigens in immune attacks. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of molecules associated with transformation, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens (e.g., CD19, CD20, and CD37) are other candidate target antigens in B-cell lymphomas.

[0197] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present in other cells in the body. TAA-associated antigens are not unique to tumor cells and can instead be expressed on normal cells under conditions that do not allow for immune tolerance to the antigen to be induced. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or it can be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0198] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15, overexpressed embryonic antigens such as CEA, overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu, unique tumor antigens produced by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other protein-based large antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0199] nucleic acid The modified immune cells described herein comprise one or more heterologous nucleic acid sequences encoding any one of the TLR polypeptides (e.g., first polypeptide and / or second polypeptide) and / or engineered receptors described herein.

[0200] In some embodiments, an isolated nucleic acid is provided, comprising a nucleic acid sequence encoding any one of the polypeptides (e.g., TLR polypeptides) described herein. In some embodiments, an isolated nucleic acid is provided, comprising a nucleic acid sequence encoding any one of the engineered receptors described herein. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is linear. In some embodiments, the nucleic acid is circular.

[0201] The nucleic acid sequence encoding the first polypeptide, the second polypeptide, and / or the nucleic acid encoding the engineered receptor can be operably linked to one or more regulatory sequences. Exemplary regulatory sequences that control transcription and / or translation of coding sequences are known in the art and may include, but are not limited to, promoters and additional elements for proper initiation, regulation, and / or termination of transcription (e.g., polyA transcription termination sequences), mRNA translocation (e.g., nuclear localization signal sequences), processing (e.g., splicing signals), stability (e.g., introns and non-coding 5' and 3' sequences), translation (e.g., initiation Met, triplet leader sequences, IRES ribosome binding sites, signal peptides, etc.), and insertion sites for introducing inserts into viral vectors. In some embodiments, the regulatory sequences are promoters, transcriptional enhancers, and / or sequences that enable correct expression of the TLR polypeptide and / or engineered receptor.

[0202] The terms "regulatory sequence" or "control sequence" refer to DNA sequences that influence the expression of coding sequences operably linked to them. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences usually include promoters, ribosomal binding sites, and terminators. In eukaryotes, regulatory sequences include promoters, terminators, and occasionally enhancers, transactivators, or transcription factors.

[0203] The term "operably linked" refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that it effects expression of the coding sequence under conditions compatible with the regulatory sequences.

[0204] As used herein, "promoter" or "promoter region" refers to a DNA or RNA segment that controls transcription of DNA or RNA operably linked thereto. A promoter region contains specific sequences for RNA polymerase recognition, binding, and transcription initiation. A promoter also contains sequences that regulate RNA polymerase recognition, binding, and transcription initiation activity (i.e., binding of one or more transcription factors). These sequences may act in cis or may respond to trans-acting factors. Depending on the nature of regulation, a promoter may be a constitutive promoter or a regulatable promoter. Regulatable promoters may be inducible or responsive to environmental factors (e.g., pH, anaerobic conditions, osmotic agents, temperature, light, or cell density). Many such promoter sequences are known in the art. See, e.g., U.S. Patent Nos. 4,980,285, 5,631,150, 5,707,928, 5,759,828, 5,888,783, 5,919,670 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989).

[0205] In some embodiments, the nucleic acid sequence encoding the first polypeptide is operably linked to a first promoter. In some embodiments, the nucleic acid sequence encoding the second polypeptide is operably linked to a second promoter. In some embodiments, the first polypeptide and the second polypeptide are the same, and the first nucleic acid simultaneously encodes the first polypeptide and the second polypeptide. In some embodiments, the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the second polypeptide are operably linked to the same promoter. In some embodiments, the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the second polypeptide are operably linked to separate promoters.

[0206] In some embodiments, the modified immune cells comprise a third nucleic acid encoding an engineered receptor. In some embodiments, the first nucleic acid and the third nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid and the third nucleic acid are operably linked to separate promoters. In some embodiments, the second nucleic acid and the third nucleic acid are operably linked to the same promoter. In some embodiments, the second nucleic acid and the third nucleic acid are operably linked to separate promoters. In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are operably linked to separate promoters.

[0207] In some embodiments, the promoter is an endogenous promoter. For example, nucleic acids encoding the first polypeptide, the second polypeptide, and / or the engineered receptor can be knocked into the genome of the modified immune cell downstream of an endogenous promoter using any method known in the art (e.g., CRISPR / Cas9 methodology). In some embodiments, the endogenous promoter is a promoter for an abundant protein such as β-actin. In some embodiments, the endogenous promoter is an inducible promoter, e.g., capable of being induced by an endogenous activation signal of the modified immune cell. In some embodiments, wherein the modified immune cell is a T cell, the promoter is a T cell activation-dependent promoter (e.g., an IL-2 promoter, an NFAT promoter, or an NFκB promoter). In some embodiments, the promoter is a heterologous promoter.

[0208] Various promoters have been explored for gene expression in mammalian cells, and any promoter known in the art may be used in the present application. Promoters can be broadly classified as constitutive promoters or regulatable promoters, e.g., inducible promoters. In some embodiments, a heterologous nucleic acid sequence encoding a first polypeptide, a second polypeptide, and / or an engineered receptor is operably linked to a constitutive promoter. In some embodiments, a heterologous nucleic acid sequence encoding a first polypeptide, a second polypeptide, and / or an engineered receptor is operably linked to an inducible promoter. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and an inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and a third constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and an inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and an inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, a second constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and an inducible promoter is operably linked to a nucleic acid sequence encoding a first polypeptide.In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a first inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and a second inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, a first inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and a second inducible promoter is operably linked to a nucleic acid sequence encoding the first polypeptide. In some embodiments, a first constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, a first inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and a second inducible promoter is operably linked to a nucleic acid sequence encoding the first polypeptide.

[0209] In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and a third inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor. In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and a constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor. In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a second inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and a constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide. In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, a second inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and a constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide. In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding a first polypeptide, a first constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and a second constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide. In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, a first constitutive promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, and a second constitutive promoter is operably linked to a nucleic acid sequence encoding a first polypeptide.In some embodiments, a first inducible promoter is operably linked to a nucleic acid sequence encoding an engineered receptor, a first constitutive promoter is operably linked to a nucleic acid sequence encoding a second polypeptide, and a second constitutive promoter is operably linked to a nucleic acid sequence encoding the first polypeptide.

[0210] In some embodiments, the first inducible promoter can be induced by a first induction condition, the second inducible promoter can be induced by a second induction condition, and the third inducible promoter can be induced by a third induction condition. In some embodiments, the first induction condition and the second induction condition are the same. In some embodiments, the first induction condition and the third induction condition are the same. In some embodiments, the second induction condition and the third induction condition are the same. In some embodiments, the first induction condition, the second induction condition, and the third induction condition are all the same. In some embodiments, the first inducible promoter and the second inducible promoter are induced simultaneously. In some embodiments, the first inducible promoter and the third inducible promoter are induced simultaneously. In some embodiments, the second inducible promoter and the third inducible promoter are induced simultaneously. In some embodiments, the first inducible promoter, the second inducible promoter, and the third inducible promoter are induced simultaneously. In some embodiments, the first inducible promoter, the second inducible promoter, and the third inducible promoter are induced in sequence, e.g., the first inducible promoter is induced before the second inducible promoter and the second inducible promoter is induced before the third inducible promoter, the first inducible promoter is induced after the second inducible promoter and the second inducible promoter is induced before the third inducible promoter, or the first inducible promoter is induced after the second inducible promoter and the second inducible promoter is induced after the third inducible promoter.

[0211] Constitutive promoters allow for the constitutive expression of heterologous genes (also called transgenic) in host cells. Exemplary constitutive promoters considered herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) coupled to the CMV early enhancer. The efficiency of such constitutive promoters for driving transgenic expression has been widely compared in many studies. In some embodiments, the promoter is the hEF1α promoter.

[0212] In some embodiments, the promoter is an inducible promoter. Inducible promoters belong to the class of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the modified immune cell or the physiological state of the modified immune cell, an inducer (i.e., an inducer), or a combination thereof. In some embodiments, the inducing condition does not induce expression of an endogenous gene in the modified immune cell and / or the subject receiving the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of an inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and the activation state of the modified immune cell.

[0213] In some embodiments, the promoter can be induced by an inducer. In some embodiments, the inducer is a small molecule, e.g., a chemical compound. In some embodiments, the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metals, or steroids. Chemically induced promoters have been the most widely explored. Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small molecule chemicals (e.g., doxycycline, tetracycline, alcohol, steroids, metals, and other compounds). The doxycycline-inducible system with a reverse tetracycline-regulated transactivator (rtTA) and a tetracycline-responsive element promoter (TRE) is currently the most mature system. Tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters is described in WO 9429442. Tetracycline-regulated transcriptional regulators are disclosed in WO 9601313. Tet technology (e.g., the Tet-On system) is also described, for example, on the TetSystems.com website. Any known chemically regulated promoter can be used to drive expression of the therapeutic proteins of the present application.

[0214] In some embodiments, the inducer is a polypeptide such as a growth factor, hormone, or cell surface receptor ligand, e.g., a polypeptide that specifically binds to a tumor antigen. In some embodiments, the polypeptide is expressed by the modified immune cell. In some embodiments, the polypeptide is encoded by a nucleic acid in a heterologous nucleic acid. Many polypeptide inducers are known in the art and can be applied to the present application. For example, ecdysone receptor-based gene switches, progesterone receptor-based gene switches, and estrogen receptor-based gene switches belong to gene switches using transactivators derived from steroid receptors (e.g., WO 9637609 and WO 9738117).

[0215] In some embodiments, the inducer simultaneously comprises a small molecule component and one or more polypeptides. For example, inducible promoters dependent on polypeptide dimerization are known in the art and may be applied to the present application. The first small molecule CID system developed in 1993 uses FK1012 (a derivative of the drug FK506) to induce FKBP homodimerization. Using a similar strategy, Wu et al. successfully made CAR-T cells titrable in an ON-switch manner using a Rapalog / FKPB-FRB* and Gibberellin / GID1-GAI dimerization-dependent gene switch (C.-Y. Wu et al., Science 350, aab4077 (2015)). Other dimerization-dependent switch systems include coumermycin / GyrB-GyrB (Nature 383(6596):178-81) and HaXS / Snap-tag-HaloTag (Chemistry and Biology 20(4):549-57).

[0216] In some embodiments, the promoter is a light-inducible promoter, and the inducing condition is light. Light-inducible promoters for regulating gene expression in mammalian cells are also well known in the art (see, for example, Science 332,1565-1568(2011); Nat. Methods 9,266-269(2012); Nature 500:472-476(2013); Nature Neuroscience 18:1202-1212(2015)). Such gene regulation systems can be broadly divided into two categories: (1) DNA binding or (2) regulation of DNA-binding proteins by recruiting transcription activation domains. For example, a synthetic mammalian blue light-controlled transcription system based on melanopsin has been developed and tested in mammalian cells, which induces an increase in intracellular calcium in response to blue light (480 nm), thereby resulting in the recruitment of NFAT mediated by calcineurin. Recently, Motta-Mena et al. described a novel inducible gene expression system developed from the naturally occurring EL222 transcription factor that confers high-level blue-light-sensitive control of transcription initiation in human cell lines and zebrafish embryos ( Nat. Chem. Biol. 10(3):196-202 (2014)). They also exploited the red-light-induced interaction between the Arabidopsis thaliana photoreceptor phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to regulate red-light-induced gene expression. They also developed an ultraviolet B (UVB)-inducible gene expression system and demonstrated its effectiveness in target gene transcription in mammalian cells ( Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Chapter 25, 4th Edition, CRC Press, January 20, 2015). Any of the light-inducible promoters described herein can be used to drive the expression of the therapeutic proteins of the present application.

[0217] In some embodiments, the promoter is a light-inducible promoter that is induced by the combination of a light-inducible molecule and light. For example, a photocleavable photocaged group in a chemical inducer keeps the inducer inactive unless removed by irradiation or other means. Such light-inducible molecules include small molecules, oligonucleotides, and proteins. For example, caged ecdysone, caged IPTG used with the lac operon, caged toyocamycin for ribozyme-mediated gene expression, caged doxycycline used with the Tet-on system, and cage...

Claims

1. 1. A modified immune cell comprising: a) a first polypeptide comprising i) a first target binding domain, ii) a first TLR transmembrane domain, and iii) a first TLR signaling domain; and b) a second polypeptide comprising i) a second target binding domain, ii) a second TLR transmembrane domain, and iii) a second TLR signaling domain. Including, wherein, when the first target binding domain and the second target binding domain bind to their corresponding targets, the first TLR signaling domain and the second TLR signaling domain associate with each other to form a TLR signaling moiety capable of inducing TLR signaling. Modified immune cells.

2. 2. The modified immune cell of claim 1, wherein the first target binding domain and the second target binding domain each bind to a subunit of a multimeric target molecule.

3. 2. The engineered immune cell of claim 1, wherein the first target binding domain and the second target binding domain bind to the same target molecule, and optionally, the first target binding domain and the second target binding domain each bind to different non-overlapping target sites on a single target molecule.

4. 2. The modified immune cell of claim 1, wherein the first TLR transmembrane domain and the first TLR signaling domain are derived from the same TLR molecule, and / or the second TLR transmembrane domain and the second TLR signaling domain are derived from the same TLR molecule.

5. 2. The modified immune cell of claim 1, wherein the first TLR transmembrane domain and the second TLR transmembrane domain are the same and / or the first TLR signaling domain and the second TLR signaling domain are the same.

6. 6. The modified immune cell of claim 4 or claim 5, wherein the first TLR transmembrane domain and / or the first TLR signaling domain is derived from TLR4.

7. 2. The modified immune cell of claim 1, wherein the first TLR transmembrane domain and the second TLR transmembrane domain are different and / or the first TLR signaling domain and the second TLR signaling domain are different.

8. 8. The modified immune cell of claim 7, wherein the first TLR transmembrane domain and / or the first TLR signaling domain is derived from TLR2.

9. 8. The modified immune cell of claim 7, wherein the second TLR transmembrane domain and / or the second TLR signaling domain is derived from TLR6 or TLR1.

10. 2. The modified immune cell of claim 1, wherein the first target binding domain and / or the second target binding domain is an antibody portion or an antigen-binding fragment thereof, and optionally the first target binding domain and / or the second target binding domain is an scFv or an sdAb.

11. The modified immune cell of claim 10, wherein the first target binding domain and / or the second target binding domain specifically binds to CD33, CLL1, CD123, CD19, CD20, CD22, BCMA, GPRC5D, and GPC3.

12. 4. The modified immune cell of claim 3, wherein the target molecule is a natural protein expressed on the immune cell.

13. The modified immune cell of claim 12, wherein the target molecule is NKG2D.

14. 14. The modified immune cell of claim 13, wherein the target molecule is the extracellular antigen-binding domain of NKG2D.

15. 2. The modified immune cell of claim 1, wherein the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

16. The modified immune cell of claim 1 , comprising an engineered receptor.

17. 17. The modified immune cell of claim 16, wherein the engineered receptor is a chimeric antigen receptor (CAR), a modified T cell receptor (TCR), or a T cell antigen coupler (TAC) receptor.

18. 18. The modified immune cell of claim 17, wherein the engineered receptor comprises an extracellular domain that specifically recognizes any of CD19, CLL1, BCMA, and GPC3.

19. 2. The modified immune cell of claim 1, comprising a first nucleic acid encoding the first polypeptide and a second nucleic acid encoding the second polypeptide.

20. 20. The modified immune cell of claim 19, wherein the first polypeptide and the second polypeptide are the same, and wherein the modified immune cell comprises a first nucleic acid encoding the first polypeptide and the second polypeptide.

21. 20. The modified immune cell of claim 19, comprising a third nucleic acid encoding the engineered receptor.

22. The modified immune cell described in claim 19, comprising a nucleic acid encoding a polypeptide having at least about 95% sequence identity to any one of the amino acid sequences of SEQ ID NO: 1, 3, 57-60, 61-65, and 71-73.

23. A pharmaceutical composition comprising the modified immune cells of claim 1 and a pharmaceutically acceptable carrier.

24. A drug for treating a disease in an individual, comprising an effective amount of the pharmaceutical composition of claim 23.

25. The drug of claim 24, wherein the disease is cancer.

Images