Engineered dendritic cells with membrane-bound cytokine and uses thereof

Abstract

Provided herein are dendritic cells comprising a chimeric receptor (e.g, a chimeric antigen receptor (CAR)) and a membrane-bound chimeric interferon (IFN) molecule. Also provided herein are dendritic cells comprising a chimeric receptor (e.g, a chimeric antigen receptor (CAR)) and a membrane-bound chimeric interleukin-2 (IL-2) family cytokine molecule. Also provided are methods of making such engineered dendritic cells and methods of using such engineered dendritic cells for various treatments.

Description

Attorney Docket No. 14857-003-228ENGINEERED DENDRITIC CELLS WITH MEMBRANE-BOUND CYTOKINE AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Application No. 63 / 728,628, filed December 5, 2024, and U. S. Application No. 63 / 728,631, filed December 5, 2024, the disclosure of each of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14857-003-228_SL.xml”, was created on November 25, 2025, and is 84,216 bytes in size.1. FIELD

[0003] The present disclosure relates to dendritic cells comprising a chimeric receptor (e.g., a chimeric antigen receptor (CAR)) and a membrane-bound chimeric interferon (IFN) molecule, and methods of making and using such cells. The present disclosure also relates to dendritic cells comprising a chimeric receptor (e.g., a chimeric antigen receptor (CAR)) and a membrane-bound chimeric interleukin-2 (IL-2) family cytokine molecule, and methods of making and using such cells.2. BACKGROUND

[0004] Chimeric antigen receptor (CAR) therapy, e.g., CAR-T cell therapy and CAR-macrophage therapy, has achieved remarkable results for treating patients in clinical studies. Although promising, antigen escape and associated treatment failure and relapses occur have been observed in existing CAR therapies. In addition, toxicity (e.g., cytokine release syndrome (CRS)) has been reported for CAR T-cell therapy. Therefore, there remain needs of improved immunotherapy, e.g., improved curative systemic anti -turn or immune response. Dendritic cells (DCs) are professional antigen-presenting cells that serve as a crucial bridge between innate and adaptive immunity. DCs specialize in antigen capture, processing, and presentation to T cells. They are not only able to present extracellular antigens on major histocompatibility complex (MHC) class II molecules to CD4+T helper (TH) cells but also to present extracellular antigens on MHC class I molecules to CD8+T cells. This phenomenon- 1 - NAI-5007600029vlis known as cross-presentation and is crucial for efficacious anti-tumor immune responses. (R. S. Laureano etal., Oncolmmunology (2022); 11: 1, 2096363).

[0005] Despite extensive development of DC-based strategies for cancer treatment over recent decades, clinical results have been largely underwhelming. Current approaches typically involve generating vaccines using autologous dendritic cells exposed to tumor-associated or specific antigens (TAAs or TSAs) in the presence of immunostimulatory molecules to induce DC maturation, followed by reinfusion into patients. While these approaches can successfully induce TAA / TSA-specific immune responses, DC vaccination still shows suboptimal anti-tumor efficacy in the clinic. Therefore, there remain needs for improved DC-based immunotherapy.3. SUMMARY

[0006] The present disclosure provides engineered dendritic cells (DCs) or precursors thereof. In certain embodiments, the DC or precursor thereof comprises: a) a chimeric antigen receptor (CAR) that binds to a target antigen, and b) a membrane-bound chimeric interferon (IFN) molecule. In certain embodiments, the IFN is a Type I IFN. In certain embodiments, the IFN is IFN-a or IFN-p. In certain embodiments, the IFN is IFN-y.

[0007] In certain embodiments, the membrane-bound chimeric IFN molecule comprises i) an IFN domain. In certain embodiments, the IFN domain comprises an active form of the IFN. In certain embodiments, the active form of the IFN comprises a native active form of the IFN or a variant thereof. In certain embodiments, the active form of the IFN comprises a native active form of the IFN.

[0008] In certain embodiments, the IFN is IFN-y. In certain embodiments, the native active form of IFN-y comprises the amino acid sequence set forth in SEQ ID NO: 47 or a fragment thereof. In certain embodiments, the native active form of IFN-y comprises the amino acid sequence set forth in SEQ ID NO: 48 or a fragment thereof.

[0009] In certain embodiments, the IFN is IFN-a. In certain embodiments, the IFN is IFN-al. In certain embodiments, the native active form of IFN-al comprises the amino acid sequence set forth in SEQ ID NO: 49 or a fragment thereof. In certain embodiments, the native active form of IFN-al comprises the amino acid sequence set forth in SEQ ID NO: 50 or a fragment thereof.

[0010] In certain embodiments, the IFN is IFN-p. In certain embodiments, the native active form of IFN-P comprises the amino acid sequence set forth in SEQ ID NO: 51 or a fragment thereof. In certain embodiments, the native active form of IFN-P comprises the amino acid - 2 - NAI-5007600029vlsequence set forth in SEQ ID NO: 52 or a fragment thereof.

[0011] In certain embodiments, the membrane-bound IFN molecule further comprises ii) an anchor domain that anchors the IFN domain to the membrane of the dendritic cell or precursor thereof. In certain embodiments, the anchor domain comprises a GPI-anchored polypeptide. In certain embodiments, the GPI-anchored polypeptide is derived from CD59, CD55, CD48, CD24, CD14, Thy-1, or Ly-6. In certain embodiments, the GPI-anchored polypeptide is derived from CD59. In certain embodiments, the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 53 or a fragment thereof. In certain embodiments, the GPI-anchored polypeptide is derived from CD55. In certain embodiments, the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 54 or a fragment thereof. In certain embodiments, the anchor domain comprises a transmembrane domain derived from CD8, a Toll-like receptor, CD40, CD3ζ, a FcR, CD166, 0X40, CD28, 4-1BB, or ICOS. In certain embodiments, the anchor domain is derived from human CD8a. In certain embodiments, the anchor domain comprises a transmembrane domain of human CD8a or a fragment thereof. In certain embodiments, the anchor domain comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

[0012] In certain embodiments, the membrane-bound chimeric IFN molecule further comprises iii) a hinge domain. In certain embodiments, the hinge domain is positioned between i) the IFN domain and ii) the anchor domain. In certain embodiments, the hinge domain has a length of at least about 5 amino acids and / or up to about 30 amino acids. In certain embodiments, the hinge domain has a length of between about 5 and about 30 amino acids. In certain embodiments, the hinge domain has a length of about 30 amino acids. In certain embodiments, the hinge domain is derived from CD8, CD55, a Toll-like receptor (TLR), CD40, CD3ζ a FcR, CD166, IgGl, IgG4, CD28, 4-1BB, or ICOS. In certain embodiments, the hinge domain is derived from CD8. In certain embodiments, the hinge domain comprises a native or modified extracellular domain of CD8a or a fragment thereof. In certain embodiments, the hinge domain comprises amino acids 149 to 176 of SEQ ID NO: 26 or a fragment thereof. In certain embodiments, the hinge domain is derived from CD55. In certain embodiments, the hinge domain comprises a native or modified extracellular domain of CD55 or a fragment thereof. In certain embodiments, the hinge domain comprises amino acids 323 to 340 of SEQ ID NO: 55 or a fragment thereof.

[0013] In certain embodiments, the membrane-bound chimeric IFN molecule further comprises: iv) a signal peptide. In certain embodiments, the signal peptide is covalently connected to the N-terminus of the IFN domain.- 3 - NAI-5007600029vl

[0014] The present disclosure also provides engineered dendritic cells (DCs) or precursors thereof. In certain embodiments, the DC or precursor thereof comprises: a) a chimeric antigen receptor (CAR) that binds to a target antigen, and b) a membrane-bound chimeric interleukin-2 (IL-2) family cytokine molecule. In certain embodiments, the IL-2 family cytokine is selected from the group consisting of IL-2, interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin- 15 (IL-15), and interleukin-21 (IL-21). In certain embodiments, the IL-2 family cytokine is IL-2.

[0015] In certain embodiments, the membrane-bound chimeric IL-2 family cytokine molecule comprises i) a cytokine domain. In certain embodiments, the cytokine domain comprises an active form of the IL-2 family cytokine. In certain embodiments, the active form of the IL-2 family cytokine comprises a native active form of the IL-2 family cytokine or a variant thereof.

[0016] In certain embodiments, the active form of the IL-2 family cytokine comprises a native active form of the IL-2 family cytokine. In certain embodiments, the IL-2 family cytokine is IL-2, and the native active form of IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 67 or a fragment thereof. In certain embodiments, the IL-2 family cytokine is IL-2, and the native active form of IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 68 or a fragment thereof.

[0017] In certain embodiments, the IL-2 family cytokine is IL-15, and the native active form of IL-15 comprises the amino acid sequence set forth in SEQ ID NO: 70 or a fragment thereof. In certain embodiments, the IL-2 family cytokine is IL- 15, and the native active form of IL-15 comprises the amino acid sequence set forth in SEQ ID NO: 71 or a fragment thereof.

[0018] In certain embodiments, the IL-2 family cytokine is IL-21, and the native active form of IL-21 comprises the amino acid sequence set forth in SEQ ID NO: 72 or a fragment thereof. In certain embodiments, the IL-2 family cytokine is IL-21, and the native active form of IL-21 comprises the amino acid sequence set forth in SEQ ID NO: 73 or a fragment thereof.

[0019] In certain embodiments, the IL-2 family cytokine is IL-7, and the native active form of IL-7 comprises the amino acid sequence set forth in SEQ ID NO: 74 or a fragment thereof. In certain embodiments, the IL-2 family cytokine is IL-7, and the native active form of IL-7 comprises the amino acid sequence set forth in SEQ ID NO: 75 or a fragment thereof.

[0020] In certain embodiments, the IL-2 family cytokine is IL-9, and the native active form of IL-9 comprises the amino acid sequence set forth in SEQ ID NO: 76 or a fragment thereof.- 4 - NAI-5007600029vlIn certain embodiments, the IL-2 family cytokine is IL-9, and the native active form of IL-9 comprises the amino acid sequence set forth in SEQ ID NO: 77 or a fragment thereof.

[0021] In certain embodiments, the IL-2 family cytokine is IL-4, and the native active form of IL-4 comprises the amino acid sequence set forth in SEQ ID NO: 78 or a fragment thereof. In certain embodiments, the IL-2 family cytokine is IL-4, and the native active form of IL-4 comprises the amino acid sequence set forth in SEQ ID NO: 79 or a fragment thereof.In certain embodiments, the cytokine domain comprises a variant of a native active form of the IL-2 family cytokine. In certain embodiments, the variant comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the native active form of the IL-2 family cytokine. In certain embodiments, the variant comprises an amino acid sequence that is at least about 95% identical to the native active form of the IL-2 family cytokine. In certain embodiments, the variant comprises at least one modification that increases the activation of cytotoxic T cells. In certain embodiments, the variant comprises at least one modification that increases the activation of regulatory T cells (Tregs). In certain embodiments, the at least one modification is selected from deletion, insertion, substitutions, and combination thereof. In certain embodiments, the at least one modification comprises one or more substitutions. In certain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises an amino acid sequence that is at least about 95% to the amino acid sequence set forth in SEQ ID NO: 67 or a fragment thereof. In certain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 67 with at least one amino acid substitution. In certain embodiments, the at least one amino acid substitution is selected from the group consisting of F42, L80, R81, L85, 186, 192, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the at least one amino acid substitution is selected from the group consisting of F42A, L80F, R81D, L85V, I86V, I92F, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 69 or a fragment thereof. In certain embodiments, the at least one amino acid substitution comprises N88 of the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the at least one amino acid substitution comprises N88R of the amino acid sequence set forth in SEQ ID NO: 67. In - 5 - NAI-5007600029vlcertain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 80 or a fragment thereof. In certain embodiments, the at least one amino acid substitution comprises N88D of the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 81 or a fragment thereof.

[0022] In certain embodiments, the at least one amino acid substitution is selected from the group consisting of N103, V106, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 68. In certain embodiments, the at least one amino acid substitution is selected from the group consisting of N103R, V106D, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 68. In certain embodiments, the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 82 or a fragment thereof.

[0023] In certain embodiments, the membrane-bound chimeric IL-2 family cytokine molecule further comprises ii) an anchor domain that anchors the i) cytokine domain to the membrane of the dendritic cell or precursor thereof. In certain embodiments, the anchor domain comprises a GPI-anchored polypeptide. In certain embodiments, the GPI-anchored polypeptide is derived from CD59, CD55, CD48, CD24, CD14, Thy-1, or Ly-6. In certain embodiments, the GPI-anchored polypeptide is derived from CD59. In certain embodiments, the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 53 or a fragment thereof. In certain embodiments, the GPI-anchored polypeptide is derived from CD55. In certain embodiments, the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 54 or a fragment thereof. In certain embodiments, the anchor domain comprises a transmembrane domain derived from CD8, a Toll-like receptor (TLR), CD40, CD3ζ, a FcR, CD166, OX40, CD28, 4-1BB, or ICOS. In certain embodiments, the anchor domain is derived from human CD8a. In certain embodiments, the anchor domain comprises a transmembrane domain of human CD8a or a fragment thereof. In certain embodiments, the anchor domain comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

[0024] In certain embodiments, the membrane-bound chimeric IL-2 family cytokine molecule further comprises iii) a hinge domain. In certain embodiments, the hinge domain is positioned between i) the cytokine domain and ii) the anchor domain. In certain embodiments, the hinge domain has a length of at least about 5 amino acids and / or up to about 30 amino acids. In certain embodiments, the hinge domain has a length of between about 5 and about 30 amino acids. In certain embodiments, the hinge domain has a length of - 6 - NAI-5007600029vlabout 30 amino acids. In certain embodiments, the hinge domain is derived from CD8, CD55, a Toll-like receptor (TLR), CD40, CD3ζ, a FcR, CD166, IgG1, IgG4, CD28, 4-1BB, or ICOS. In certain embodiments, the hinge domain is derived from CD8. In certain embodiments, the hinge domain comprises a native or modified extracellular domain of CD8a or a fragment thereof. In certain embodiments, the hinge domain comprises amino acids 149 to 176 of SEQ ID NO: 26 or a fragment thereof. In certain embodiments, the hinge domain is derived from CD55. In certain embodiments, the hinge domain comprises a native or modified extracellular domain of CD55 or a fragment thereof. In certain embodiments, the hinge domain comprises amino acids 323 to 340 of SEQ ID NO: 55 or a fragment thereof.

[0025] In certain embodiments, the membrane-bound chimeric IL-2 family cytokine molecule further comprises: iv) a signal peptide. In certain embodiments, the signal peptide is covalently connected to the N-terminus of the cytokine domain.

[0026] In certain embodiments, the CAR comprises: a) an extracellular antigen-binding domain that binds to the target antigen, b) a transmembrane domain, and c) an intracellular domain.

[0027] In certain embodiments, the intracellular domain is derived from a molecule that is capable of activating a dendritic cell (DC), wherein the DC-activating molecule is selected from the group consisting of CD3ζ, a toll-like receptor (TLR), a Fc receptor (FcR), and CD40. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of the DC-activating molecule or a fragment thereof.

[0028] In certain embodiments, the TLR is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11. In certain embodiments, the TLR is TLR4. In certain embodiments, the intracellular domain of the CAR is derived from TLR4. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27 or a fragment thereof, or amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof.

[0029] In certain embodiments, the intracellular domain of the CAR is derived from CD3ζ. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 33 or a fragment thereof, or amino acids 52 to 164 of SEQ ID NO: 34 or a fragment thereof.

[0030] In certain embodiments, the intracellular domain of the CAR is derived from a FcR. In certain embodiments, the FcR is Fc gamma receptor. In certain embodiments, the Fc gamma receptor is FcεRγ. In certain embodiments, the intracellular domain of the CAR is derived from FcεRγ. In certain embodiments, the intracellular domain of the CAR- 7 - NAI-5007600029vlcomprises amino acids 45 to 86 of SEQ ID NO: 35 or a fragment thereof, or amino acids 45 to 86 of SEQ ID NO: 36 or a fragment thereof.

[0031] In certain embodiments, the intracellular domain of the CAR is derived from CD40. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31 or a fragment thereof, or amino acids 216 to 277 of SEQ ID NO: 32 or a fragment thereof.

[0032] In certain embodiments, the intracellular domain is derived from Flt3. In certain embodiments, the intracellular domain of the CAR comprises amino acids 565 to 992 of SEQ ID NO: 45 or a fragment thereof, or amino acids 564 to 993 of SEQ ID NO: 46 or a fragment thereof.

[0033] In certain embodiments, the intracellular domain of the CAR is derived from 0X40. In certain embodiments, the intracellular domain of the CAR comprises amino acids 237 to 272 of SEQ ID NO: 29 or a fragment thereof, or amino acids 236 to 277 of SEQ ID NO: 30 or a fragment thereof.

[0034] In certain embodiments, the transmembrane domain of the CAR is derived from CD8, a Toll-like receptor, CD40, CD3ζ, a FcR, CD166, OX40, CD28, 4-1BB, or ICOS.

[0035] In certain embodiments, the transmembrane domain of the CAR is derived from CD8a. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof, or amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

[0036] In certain embodiments, the transmembrane domain of the CAR is derived from CD8p. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 176 to 196 of SEQ ID NO: 43 or a fragment thereof, or amino acids 171 to 191 of SEQ ID NO: 44 or a fragment thereof.

[0037] In certain embodiments, the transmembrane domain of the CAR is derived from TLR4. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof, or amino acids 632-652 of SEQ ID NO: 28 or a fragment thereof.

[0038] In certain embodiments, the transmembrane domain of the CAR is derived from 0X40. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 212 to 236 of SEQ ID NO: 29 or a fragment thereof, or amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof.

[0039] In certain embodiments, the transmembrane domain of the CAR is derived from FcεRγ. In certain embodiments, the transmembrane domain of the CAR comprises amino - 8 - NAI-5007600029vlacids 24 to 44 of SEQ ID NO: 35 or a fragment thereof, or amino acids 24 to 44 of SEQ ID NO: 36 or a fragment thereof.

[0040] In certain embodiments, the CAR further comprises a hinge region. In certain embodiments, the hinge region has a length of between about 5 and about 100 amino acids, between about 10 and about 50 amino acids, or between about 50 and about 100 amino acids. In certain embodiments, the hinge region has a length of about 20, about 40, or about 50, or about 70 amino acids. In certain embodiments, the hinge region is positioned between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge region is derived from CD8, a TLR, CD40, CD3ζ, a FcR, CD166, OX40, IgG1, IgG4, CD28, 4-1BB, or ICOS.

[0041] In certain embodiments, the hinge region is derived from CD8. In certain embodiments, the hinge region comprises a native or modified extracellular domain of CD8a or a fragment thereof. In certain embodiments, the native extracellular domain of CD8a or fragment thereof comprises amino acids 149 to 196 of SEQ ID NO: 25 or a fragment thereof, amino acids 159 to 196 of SEQ ID NO: 25 or a fragment thereof, or amino acids 179 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the modified extracellular domain of CD8a or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 37 or a fragment thereof. In certain embodiments, the modified extracellular domain of CD8a or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 38 or a fragment thereof.

[0042] In certain embodiments, the hinge region is derived from a TLR. In certain embodiments, the hinge region comprises a native or modified extracellular domain of a TLR. In certain embodiments, the hinge region comprises a native or modified extracellular domain of TLR4 or a fragment thereof. In certain embodiments, the native extracellular domain of TLR4 or fragment thereof comprises amino acids 619 to 638 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the hinge region is derived from TLR4 and comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof.

[0043] In certain embodiments, the hinge region is derived from 0X40. In certain embodiments, the hinge region comprises a native or modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the native extracellular domain of 0X40 or fragment thereof comprises amino acids 166 to 211 of SEQ ID NO: 29 or a fragment thereof. In certain embodiments, the native extracellular domain of 0X40 or fragment thereof comprises amino acids 167 to 214 of SEQ ID NO: 30 or a fragment thereof. In certain embodiments, the modified extracellular domain of 0X40 or fragment thereof comprises the - 9 - NAI-5007600029vlamino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 84.

[0044] In certain embodiments, the hinge region comprises a native or modified extracellular domain of TLR or a fragment thereof, and a native or modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the native extracellular domain of TLR or fragment thereof comprises amino acids 619 to 638 of SEQ ID NO: 27 or a fragment thereof, and the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the native extracellular domain of TLR or fragment thereof comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof, and the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 84. In certain embodiments, the intracellular domain of the CAR is derived from a TLR. In certain embodiments, the intracellular domain of the CAR is derived from TLR4. In certain embodiments, the transmembrane domain of the CAR is derived from a TLR. In certain embodiments, the transmembrane domain of the CAR is derived from TLR4.

[0045] In certain embodiments, the target antigen is a tumor antigen. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the target antigen is selected from the group consisting of epidermal growth factor receptor (EGFR), Prostatespecific membrane antigen (PSMA), IL13Ra2 (Interleukin- 13 Receptor Alpha 2), MUC1 (Mucin 1), Claudin 18.2, Mesothelin, GD2, CEA, FAP, R0R1 (Receptor Tyrosine Kinase-Like Orphan Receptor 1), GPC3 (Glypican-3), MAGE-A4 (Melanoma-Associated Antigen A4), B7-H4, and Axl. In certain embodiments, the tumor is a liquid tumor. In certain embodiments, the target antigen is selected from the group consisting of CD 19, CD20, B-cell maturation antigen (BCMA), G-protein-coupled receptor class 5 member D (GPRC5D), Fc receptor-like 5 (FCRL5), CD22, CD33, CD123, and CD30. In certain embodiments, the target antigen is overexpressed in one or more types of tumor or cancer tissues but has no or a limited expression in essential normal tissues. In certain embodiments, the target antigen is selected from the group consisting of B7H3, EphA2, and Her2. In certain embodiments, the target antigen is a cancer antigen. In certain embodiments, the target antigen is selected from the group consisting of B7H3, PSMA, CD19, CD20, EGFR, Her2, and EphA2. In certain embodiments, the target antigen is associated with an autoimmune disease. In certain embodiments, the autoimmune disease is a T-cell mediated autoimmune disease. In certain embodiments, the autoimmune disease is selected from the group consisting of type 1- 10 - NAI-5007600029vldiabetes (T1D), multiple sclerosis (MS), primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune myositis, psoriasis / psoriatic arthritis, and autoimmune vasculitis. In certain embodiments, the target antigen is selected from the group consisting of CD19, CD20, Sulfonylurea Receptor 1 (SURI), Myelin Basic Protein (MBP), Proteolipid Protein (PLP), Myelin Oligodendrocyte Glycoprotein (MOG), Myelin-Associated Glycoprotein (MAG), Oligodendrocyte Myelin Glycoprotein (OMgp), Galactocerebroside (GalC), cytokeratins, Aquaporin-1 (AQP1), CD55, VCAM-1 (Vascular Cell Adhesion Molecule 1), Desmin, Dystrophin, CD56, myeloperoxidase (MPO), proteinase 3 (PR3), CD 177, and keratin.

[0046] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a variable heavy domain of heavy chain (VHH) domain. In certain embodiments, the target antigen is B7H3 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 4. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0047] In certain embodiments, the target antigen is PSMA and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 8. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0048] In certain embodiments, the target antigen is CD 19 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 12. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0049] In certain embodiments, the target antigen is CD20 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 16. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, and the CDR3 comprises the amino- 11 - NAI-5007600029vlacid sequence set forth in SEQ ID NO: 15. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0050] In certain embodiments, the target antigen is EGFR and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 20. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0051] In certain embodiments, the target antigen is beta amyloid and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 64. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 62, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 64.

[0052] In certain embodiments, the CDR1, CDR2, and / or CDR3 are determined according to the IMGT numbering scheme, Kabat numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof.

[0053] In certain embodiments, the extracellular antigen-binding domain comprises a monobody. In certain embodiments, the monobody is based on a fibronectin type III (FN3) domain. In certain embodiments, the monobody comprises a BC loop, a DE loop, and an FG loop. In certain embodiments, the target antigen is EphA2, and the monobody comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO: 21, a DE loop comprising the amino acid sequence set forth in SEQ ID NO: 22, and an FG loop comprising the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the monobody comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0054] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an antigen-binding fragment. In certain embodiments, the extracellular antigenbinding domain of the CAR comprises a single chain variable fragment (scFv).

[0055] In certain embodiments, the CAR further comprises a signal peptide. In certain embodiments, the signal peptide is covalently connected to the N-terminus of the extracellular antigen-binding domain.

[0056] In certain embodiments, the CAR and / or the membrane-bound chimeric IFN molecule is recombinantly expressed. In certain embodiments, the CAR is expressed from a vector. In certain embodiments, the membrane-bound chimeric IFN molecule is expressed from a - 12 - NAI-5007600029vlvector. In certain embodiments, the CAR is expressed from a mRNA. In certain embodiments, the membrane-bound chimeric IL-2 family cytokine molecule is expressed from a mRNA.

[0057] In certain embodiments, the cell is a conventional dendritic cell (cDC). In certain embodiments, the cell is a conventional type 1 dendritic cell (cDCl). In certain embodiments, the cell is a murine cDCl or a human cDCl.

[0058] In certain embodiments, the cell is a monocyte-derived dendritic cell (MoDC). In certain embodiments, the cell is a murine MoDC or a human MoDC.

[0059] The present disclosure further provides compositions comprising an effective amount of the cells described herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0060] The present disclosure further provides various methods of using the presently disclosed cells or compositions. In one aspect, the present disclosure further provides various methods of treating a disease or a disorder in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells described herein, or the composition described herein. In certain embodiments, the disease or disorder is tumor. In certain embodiments, the administration results in elimination of a tumor cell expressing the target antigen. In certain embodiments, the administration results in elimination of a tumor cell that does not express the target antigen through epitope spreading. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cells are capable of cross priming a T cell. In certain embodiments, the T cell is reactive to a target cell expressing the target antigen. In certain embodiments, the T cell is a CD8+T cell. In certain embodiments, the T cell is a regulatory T cell (Treg). In certain embodiments, the disease or disorder is an autoimmune disease. In certain embodiments, the autoimmune disease is a T-cell mediated autoimmune disease. In certain embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes (T1D), multiple sclerosis (MS), primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune myositis, psoriasis / psoriatic arthritis, and autoimmune vasculitis. In certain embodiments, the disease or disorder is a neurodegenerative disease. In certain embodiments, the disease or disorder is associated with beta amyloid accumulation. In certain embodiments, the disease or disorder associated with beta amyloid accumulation is selected from the group consisting of Alzheimer’s disease, Down’s Syndrome, Cerebral Amyloid Angiopathy (CAA), Dementia with Lewy Bodies (DLB), Parkinson’s Disease Dementia (PDD), and Traumatic Brain Injury (TBI). In certain embodiments, the disease or - 13 - NAI-5007600029vldisorder is an infectious disease.

[0061] In another aspect, the present disclosure provides methods of inducing an immune response in a subject, comprising administering to the subject an effective amount of the dendritic cells or precursors thereof described herein, or the composition described herein. In certain embodiments, the immune response is a T cell-mediated immune response. In certain embodiments, the T cell is reactive to a target cell expressing the target antigen. In certain embodiments, the T cell is reactive to a tumor cell that does not express the target antigen through epitope spreading. In certain embodiments, the T cell is a CD8+T cell.

[0062] In another aspect, the method further comprises administering to the subject a second therapeutic agent. In certain embodiments, the second therapeutic agent is a soluble programmed-death 1 (PD-1) inhibitor. In certain embodiments, the soluble PD-1 inhibitor is an anti -PD-1 antibody or an antigen-binding fragment thereof. In certain embodiments, the second therapeutic agent is a soluble programmed death ligand 1 (PD-L1) inhibitor. In certain embodiments, the soluble PD-L1 inhibitor is an anti-PD-Ll antibody or an antigenbinding fragment thereof.

[0063] The present disclosure further provides nucleic acid compositions comprising (a) a first nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a target antigen, and (b) a second nucleic acid encoding a membrane-bound chimeric IFN molecule. In certain embodiments, the first nucleic acid is operably linked to a promoter. In certain embodiments, the second nucleic acid is operably linked to a promoter. In certain embodiments, the first nucleic acid is comprised in a vector. In certain embodiments, the second nucleic acid is comprised in a vector. In certain embodiments, first nucleic acid is a mRNA. In certain embodiments, the second nucleic acid is a mRNA.

[0064] The present disclosure further provides vectors comprising the nucleic acid compositions described herein.

[0065] The present disclosure further provides methods of producing an antigen-specific dendritic cell or a precursor thereof. In certain embodiments, the method comprises introducing into a dendritic cell or a precursor thereof (a) a first nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a target antigen, and (b) a second nucleic acid encoding a membrane-bound chimeric IFN molecule. In certain embodiments, the first nucleic acid is operably linked to a promoter. In certain embodiments, the second nucleic acid is operably linked to a promoter. In certain embodiments, the first nucleic acid is comprised in a vector. In certain embodiments, the second nucleic acid is comprised in a vector. In certain embodiments, the first nucleic acid is a mRNA. In certain embodiments, the second - 14 - NAI-5007600029vlnucleic acid is a mRNA. In certain embodiments, the method comprises introducing into the dendritic cell or precursor thereof the nucleic acid composition described herein.4. BRIEF DESCRIPTION OF THE FIGURES

[0066] FIGs. 1A and IB show that membrane-bound IFNy improved the priming capacity of the DCs. Schematic illustration is shown in FIG. 1 A. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF (Ou, Ferris et al. 2023, Eur J Immunol 53(9): e2250201) were electroporated with the indicated CAR + / - membrane bound IFNy. These cells were then cocultured with CellTrace Violet (CTV) labeled OTI T-cells (ova-antigen specific T cells) and B2M- / - 1956 sarcoma tumor cells that had been loaded with full length ovalbumin protein and express B7H3 on the surface (by transduction and sorting using FACS). 5k tumor cells, 25k OTI T-cells, and 25k DCs were plated per well. After three days of coculture, the cells were analyzed by flow cytometry, and proliferated OTI T-cell numbers were quantified as the number of CD44+ CTV diluted CD8+ T-cells in each well. Results are shown in FIG. IB. aCDI 9 CAR tested here is a CD 19 targeting CAR with a CD40 intracellular signaling domain and a CD8 extracellular linker (38 amino acids) plus a CD8 transmembrane domain. <zB7H3 CAR (<zB7H3 CD40(m)) is a B7H3 targeting CAR with a CD40 intracellular signaling domain and a CD8 extracellular linker (38 amino acids) plus a CD8 transmembrane domain.

[0067] FIGs. 2A and 2B show that more mIFNy mRNA electroporated into the DC led to more surface mIFNy expression by that DC. Schematic illustration is shown in FIG. 2A. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF (Ou, Ferris et al.2023, Eur J Immunol 53(9): e2250201) were electroporated with a B7H3 targeting CAR (<zB7H3 CD40(m)) plus varying amounts of mRNA encoding membrane bound IFNy (0, 10 ug, 30 ug, or 50 ug). These cells were then analyzed for surface IFNy expression by flow cytometry with an anti-IFNy antibody (FIG. 2B). They were also analyzed for CAR expression using an anti-camelid antibody (recognizing the VHH domain of the CAR, and for the ability to bind B7H3 using AF647-labeled soluble B7H4 protein) (FIG. 2B).

[0068] FIGs. 3A-3C show that CAR DCs expressing membrane-bound IFNy significantly induced MHC-I expression on tumor cells. Schematic illustration is shown in FIG. 3B.Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF were electroporated with a B7H3 targeting CAR (<zB7H3 CD40(m)) plus varying amounts of mRNA encoding membrane bound IFNy (0, 10 ug, 30 ug, or 50 ug; as shown in FIG. 2B). These cells were then cocultured with 1956 sarcoma tumor cells that express B7H3 on the- 15 - NAI-5007600029vlsurface (by transduction and sorting using FACS). After one day of coculture, the tumor cells were analyzed for MHC-I expression by flow cytometry. Representative flow plots shown in FIG. 3A, quantified shown in FIG. 3C.

[0069] FIG. 4 shows that mlFNy expressing CAR DCs induced MHC-I expression on both B7H3+ and B7H3- tumor cells, in a mlFNy-dependent manner. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF were electroporated with a B7H3 targeting CAR (<zB7H3 CD40(m)) plus varying amounts of mRNA encoding membrane bound IFNy (0, 10 ug, 30 ug, or 50 ug; as shown in FIG. 2B). These cells were then cocultured with 1956 sarcoma tumor cells that either express B7H3 on the surface (by transduction and sorting using FACS) or do not express B7H3 on the surface(indicated by Tumor B7H3 + or -). After one day of coculture, the tumor cells were analyzed for MHC-I expression by flow cytometry. mlFNy expressing CAR DCs induced MHC-I expression on both B7H3+ and B7H3- tumor cells, in a mlFNy-dependent manner.

[0070] FIG. 5 shows that mlFNy induced PD-L1 expression on tumor cells. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF were electroporated with a B7H3 targeting CAR CAR (<zB7H3 CD40(m)) plus varying amounts of mRNA encoding membrane bound IFNy (0, 10 pg, 30 pg, or 50 pg; as shown in FIG. 2B). These cells were then cocultured with 1956 sarcoma tumor cells that either express B7H3 on the surface (by transduction and sorting using FACS) or do not (indicated by Tumor B7H3 + or -). After one day of coculture, the tumor cells were analyzed for PD-L1 expression by flow cytometry.

[0071] FIGs. 6A-6D show the in vivo activity of the presently disclosed mlFNy expressing CAR DC (aB7 TLR CAR + mlFNy) (FIG. 6D) compared with control aB7(t) CAR DC ((t) denotes CAR lacking the intracellular domain) (FIG. 6B), aB7 TLR CAR DC (FIG. 6C), PBS treated groups (FIG. 6A). A 75:25 mixture of B7H3 expressing (B7+) and nonexpressing (B7-) 1956 sarcoma was injected orthotopically into the flank of WT B6 mice. On day 8 and 15 after tumor implantation, mice were treated with either 1.5 million aB7 TLR CAR expressing DCs, 1.5 million aB7 TLR CAR + mlFNy expressing DCs, 1.5 million control aB7(t) CAR (with no intracellular signaling domain) expressing DCs, or PBS, intratum orally. Tumor size was monitored over time.

[0072] FIGs 7A and 7B show co-expression of membrane-bound IL-2 and CAR. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF (Ou, Ferris et al. 2023, Eur J Immunol 53(9): e2250201) were electroporated with membrane bound IL-2, and stained with two different anti-IL-2 antibodies (clones indicated on X and Y axis on left), then- 16 - NAI-5007600029vlanalyzed by flow cytometry to assess for surface expression (FIG. 7A). DCs were coelectroporated with both membrane-bound IL-2 and the aB7 CD40(l) CAR, then were analyzed by flow cytometry with an anti-IL-2 antibody (Y axis) and anti-FLAG antibody (X axis, recognizing the FLAG tag in the CAR EC linker) (FIG. 7B).

[0073] FIGs. 8A-8C show that m-IL-2-expressing CAR DCs (CAR DC +mIL-2) significantly outperformed CAR DCs alone (CAR DC), CAR DCs with a soluble high-dose IL-2 (100 U / ml) (CAR DC + IL2), and mIL-2 expressing DCs without CAR (mIL-2) in T-cell cross-priming. FIG. 8A shows the schematic for cross-presentation assay. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF were electroporated with a CAR + / - membrane bound IL-2. These cells were then cocultured with CellTrace Violet (CTV) labeled OTI T-cells and B2M- / - 1956 sarcoma tumor cells that have been loaded with full length ovalbumin protein and express B7H3 on the surface (by transduction and sorting using FACS) + / - soluble IL2 (100 lU / ml). 5k tumor cells, 25k OTI, and 25k DCs were plated per well. After three days of coculture, the cells were analyzed by flow cytometry, and proliferated OTI T-cell numbers were quantified as the number of CD44+ CTV diluted T-cells in each well. FIG. 8B shows representative flow plot of CD8 OTI T-cells in a crosspresentation assay of CAR DCs with CAR DCs in combination with a soluble IL-2 or CAR DCs comprising a membrane-bound IL-2 (CAR DC + mIL-2). FIG. 8C shows quantification of CTV diluted CD44+ CD8 OTI T-cells in a cross-presentation assay of CAR DCs with soluble IL2 (CAR DC + IL-2) or membrane bound IL2 (CAR DC + mIL-2).

[0074] FIGs. 9A-9C show that CAR DCs expressing mIL-2 (CAR DC +mIL-2) significantly enhanced antigen-specific T-cell not the adjacent WT T-cells. FIG. 9A shows the schematic of a cross-presentation assay of CAR DCs with soluble IL2 (CAR DC + IL-2) or membrane bound IL2 (CAR DC + mIL-2) and a mixture of tumor reactive (OTI) and nonspecific (WT) CD8 T-cells to assess specificity of tumor-reactive T-cell priming and activation. Primary bone marrow derived DCs differentiated with Flt3L and GM-CSF were electroporated with a CAR + / - membrane bound IL-2. These cells were then cocultured with CellTrace Violet (CTV) labeled CD45.1 CD8 OTI T-cells plus CD45.2 WT CD8 T-cells and B2M- / - 1956 sarcoma tumor cells that have been loaded with full length ovalbumin protein and express B7H3 on the surface (by transduction and sorting using FACS), + / - soluble IL2 (100 lU / ml).5k tumor cells, 12.5k OTI CD8 T-cells, 12.5k WT CD8 T-cells, and 25k DCs were plated per well. After three days of coculture, the cells were analyzed by flow cytometry, and proliferated CD45.1 OTI T-cell as well as CD45.2 WT T-cells numbers were quantified as the number of CD44+ CTV diluted T-cells in each well. FIG. 9B shows quantification of - 17 - NAI-5007600029vlCTV diluted CD44+ CD8 OTI T-cells in cross-presentation assays of CAR DCs with soluble IL-2 (CAR DC + IL-2) or membrane bound IL2 (CAR DC + mIL-2) and a mixture of tumor reactive (OTI) and nonspecific (WT) CD8 T-cells to assess specificity of tumor-reactive T-cell priming and activation. FIG. 9C shows representative flow plot of CTV diluted CD44+ CD8 T-cells in cross-presentation assays of CAR DCs with soluble IL-2 (CAR DC + IL-2) or membrane bound IL2 (CAR DC + mIL-2) and a mixture of tumor reactive (OTI) and nonspecific (WT) CD8 T-cells to assess specificity of tumor-reactive T-cell priming and activation. CD8+ CD45.2 (WT T-cell) gate is shown on left and CD8+ CD45.1 (OTI T-cell) gate is shown on right.5. DETAILED DESCRIPTION

[0075] The present disclosure provides dendritic cells (DCs) and precursors thereof comprising a chimeric receptor (e.g., a chimeric antigen receptor (CAR)) that binds to a target antigen, and a membrane-bound chimeric interferon (IFN) molecule. Further provided are methods of making such engineered cells and using such engineered cells for treatments, e.g., for treating a disease or a disorder (e.g., a tumor or an autoimmune disease).

[0076] The present disclosure is based, at least in part, on the discovery that the presently disclosed engineered DCs comprising a membrane-bound chimeric IFN molecule surprisingly possess increased ability to cross-prime T cells (especially antigen reactive T cells) as compared to non-engineered DCs, CAR-DCs lacking a membrane bound chimeric IFN molecule. A membrane-bound chimeric IFN molecule increases the DCs’s ability to cross prime T-cells, especially T cells reactive to a neoantigen within the tumor cells, wherein the neoantigen is cross-presented by the engineered DC after the engineered DC recognizes the tumor (based on expression of the target antigen to which the chimeric receptor (e.g., CAR) targets), endocytoses tumor cells, cross-presents endogenous tumor antigens and neoantigens present within the tumor, and cross-primes T-cells reactive to those antigens. Furthermore, the membrane-bound chimeric IFN molecule significantly induces the expression of Major histocompatibility complex (MHC) class I (MHC-I) on tumor cells including Ag+tumor cells (tumor cells expressing the target antigen to which the CAR binds), and Ag’ tumor cells (tumor cells that do not express the target antigen to which the CAR binds). Remarkably, even tumor cells that express very low MHC-I at baseline (<1% of tumor cells) can be induced such that the majority (>60% of tumor cells) express MHC-I after co-culturing with the presently disclosed engineered DCs comprising a membranebound chimeric IFN molecule. This is significant because MHC-I expression on the tumor is important for CD8+T-cells to recognize and kill the tumor cells. MHC-I downregulation is - 18 - NAI-5007600029vlone common mechanism by which tumor cells evade the immune system, and become resistant to immune checkpoint inhibitor therapy. In addition, the membrane-bound chimeric IFN molecule induces the expression of PD-L1 on tumor cells including Ag+tumor cells and Ag’ tumor. This supports for combining PD-1 or PD-L1 blocking therapy with the presently disclosed engineered DCs comprising a membrane-bound chimeric IFN molecules. Given that a significant number of tumor cells have heterogeneous expressions of tumor-associated antigens, the surprising features possessed by the presently disclosed engineered DCs or precursors make them suitable candidates for immunotherapy.

[0077] The present disclosure is also based, at least in part, on the discovery that the presently disclosed engineered DCs comprising a membrane-bound chimeric IL-2 family cytokine molecule surprisingly possess increased ability to cross-prime T cells (especially antigen reactive T cells) as compared to non-engineered DCs, CAR-DCs lacking a membrane bound chimeric IL-2 like cytokine molecule, or CAR-DCs in combination with a soluble IL-2 family cytokine. In addition, the presently disclosed engineered DCs comprising a membrane-bound IL-2 family cytokine molecule exhibit high co-expression of the CAR and the membrane bound chimeric IL-2 family cytokine molecule on the cells. All the superior features possessed by the presently disclosed engineered DCs or precursors make them suitable candidates for immunotherapy.

[0078] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:5.1. Definitions;5.2. Chimeric Receptors;5.3. Cells;5.4. Nucleic Acid Compositions and Methods of Producing;5.5. Formulations and Administration; and5.6. Methods of Uses5.1. Definitions

[0079] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed.2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2d ed. 2010).- 19 - NAI-5007600029vlUnless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0080] The term “antibody” is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies), nanobody, monobody as described below. An antibody can be human, humanized, chimeric and / or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy -terminal portion of each chain includes a constant region. See, e.g, Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti -Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (e.g., antigen binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc., Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one - 20 - NAI-5007600029vlor more CDRs of an antibody). Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pliickthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0081] The term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the aminoterminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH”. The variable region of the light chain may be referred to as “VL”. The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the P sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. In specific embodiments, the variable region is a human variable region.

[0082] As used herein, the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably. A “CDR” refers to one of three - 21 - NAI-5007600029vlhypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework. CDR1, CDR2 and CDR3 in VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively. CDR1, CDR2 and CDR3 in VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.

[0083] CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra, Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g, Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates.Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-70 (2001). Correspondence between the numbering system, including, for example, the Kabat - 22 - NAI-5007600029vlnumbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra, Chothia and Lesk, supra, Martin, supra, Lefranc etal., supra).

[0084] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, AbM or Contact method. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR1 as set forth in a specific VH or VHH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VHH, VH or VL) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

[0085] By “substantially identical” is meant a polypeptide exhibiting at least about 50% identical to a reference amino acid sequence (for example, any one of the amino acid sequences described herein). In certain embodiments, such a sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence used for comparison.

[0086] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.- 23 - NAI-5007600029vl

[0087] Sequence identity can be measured by using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0088] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology or identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the amino acids sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the specified sequences (e.g., heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900) disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0089] The term “isolated cell” refers to a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0090] As used herein, the term “operatively linked,” and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional - 24 - NAI-5007600029vlrelationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0091] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome.Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0092] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.- 25 - NAI-5007600029vl

[0093] “Allogeneic” refers to a graft derived from a different individual of the same species.

[0094] The term “transfected”, “transfection”, “transfecting”, “transformed”, “transformation”, “transforming”, “transduced”, “transduction”, or “transducing” as used herein refers to a process by which an exogenous nucleic acid is transferred or introduced into a host cell. A “transfected” or “transformed” or “transduced” cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0095] “Excipient” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant, or vehicle.

[0096] The term “effective amount” or “therapeutically effective amount” as used herein refers to an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In certain embodiments, an effective amount can be an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount can be determined by a physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

[0097] As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., human). In certain embodiments, the subject is a human. In certain embodiments, the subject is a mammal (e.g., a human) diagnosed with a disease or disorder. In certain embodiments, the subject is a mammal (e.g., a human) who is at risk of developing a disease or disorder.

[0098] As used herein, “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing progression or recurrence of a disease or disorder, - 26 - NAI-5007600029vlalleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0099] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., diabetes or a cancer).

[0100] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0101] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0102] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0103] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0104] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).5.2. Chimeric Receptors

[0105] The present disclosure provides various chimeric receptors. In certain embodiments, the chimeric receptors are chimeric antigen receptors (CARs). A CAR is a chimeric molecule, which usually comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the extracellular antigen-binding domain is fused to the transmembrane domain, which is fused to the intracellular domain. The intracellular domain is capable of activating an immune cell - 27 - NAI-5007600029vl(e.g., dendritic cell). In certain embodiments, the intracellular domain of the CAR is capable of activating a dendritic cell (DC). Different from a conventional CAR, whose extracellular antigen-binding domain comprises a single chain variable fragment (scFv), the presently disclosed CAR comprises a nanobody- or a monobody-based extracellular antigen-binding domain. For example, in certain embodiments, the extracellular antigen-binding domain of the presently disclosed CAR comprises a nanobody. In certain embodiments, the extracellular antigen-binding domain of the presently disclosed CAR comprises a variable heavy domain of heavy chain (VHH). In certain embodiments, the extracellular antigenbinding domain of the presently disclosed CAR comprises a monobody.

[0106] In certain embodiments, the presently disclosed CAR comprises an extracellular antigen-binding domain described in Section 5.2.1 below, a transmembrane domain described in Section 5.2.2 below, and an intracellular domain described in Section 5.2.3 below. In certain embodiments, the CAR further comprises a hinge region described in Section 5.2.4 below.

[0107] In certain embodiments, the chimeric receptor is a non-signaling CAR, namely a CAR lacking an intracellular domain. In certain embodiments, the non-signaling CAR comprises an extracellular antigen-binding domain (described in Section 5.2.1 below, and a transmembrane domain (any one of the transmembrane domains described in Section 5.2.2 below). In certain embodiments, the non-signaling CAR further comprises a hinge region described in Section 5.2.4 below.5.2.1. Extracellular Antigen-Binding Domain

[0108] In certain embodiments, the extracellular antigen-binding domain of the presently disclosed chimeric receptor (e.g., CAR) comprises an antigen-binding fragment. Non-limiting examples of antigen-binding fragments include a single chain variable fragment (scFv), a Fab, and a F(ab)2. In certain embodiments, the extracellular antigen-binding domain comprises an scFv. In certain embodiments, the scFv is a human scFv, a humanized scFv, or a murine scFv. In certain embodiments, the extracellular antigen-binding domain comprises a Fab, which is optionally crosslinked. In certain embodiments, the extracellular antigen binding domain is a F(ab)2. In certain embodiments, any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

[0109] In certain embodiments, the extracellular antigen-binding domain of the presently disclosed chimeric receptor (e.g., CAR) comprises a nanobody. In certain embodiments, the extracellular antigen-binding domain comprises a single domain antibody.- 28 - NAI-5007600029vlIn specific embodiments, the extracellular antigen-binding domain does not comprise a light chain (VL). A single domain antibody (sdAb) is a single monomeric variable antibody that is capable of binding to a target antigen. Such single domain antibodies are also called Nanobody®. sdAbs include VHH domains as described herein. In certain embodiments, the VHH domains described herein have a hydrophilic Framework 2 region (FW2). In certain embodiments, the VHH domains described herein have a soluble FW2. In certain embodiments, the VHH domains described herein comprise a FW2 with one or more of the following amino acid residues: 37 (F / Y), 44 (E), 45 (R) and 47 (G). Non-limiting examples of sdAbs include antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), sdAbs derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. sdAbs can be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, and bovine. For example, a sdAb can be derived from antibodies raised in Camelidae species, including, but not limited to, camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure. In certain embodiments, the sdAb has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In specific embodiments, a sdAb is not a single VH domain.

[0110] In certain embodiments, the extracellular antigen-binding domain of the presently disclosed chimeric receptor (e.g., CAR) comprises a monobody. A monobody is a polypeptide having a sequence and structure related to the tenth module of the fibronectin type III domain (FN3) that includes a beta-strand domain lacking in disulfide bonds and containing a plurality of beta-strands, two or more loop regions each connecting one betastrand to another beta-strand, and optionally an N-terminal tail, a C-terminal tail, or both, wherein at least one of the two or more loop regions, the N-terminal tail, and or the C-terminal tail is characterized by activity in binding a target protein or molecule. In certain embodiments, a monobody comprises three or more loop regions. In certain embodiments, the monobody comprises a BC loop, a DE loop, and an FG loop.

[0111] In some embodiments, the extracellular antigen-binding domain is monovalent. In some embodiments, the extracellular antigen-binding domain comprises a single VHH domain. In some embodiments, the extracellular antigen-binding domain comprises a single monobody domain. In some embodiments, the extracellular antigenbinding domain is bivalent. In some embodiments, the extracellular antigen-binding domain comprises two VHH domains. In some embodiments, the extracellular antigen-binding - 29 - NAI-5007600029vldomain comprises two monobody domains. In some embodiments, the extracellular antigenbinding domain is multivalent. In some embodiments, the extracellular antigen-binding domain comprises three or more VHH domains. In some embodiments, the extracellular antigen-binding domain comprises three or more monobody domains.

[0112] In some embodiments, a CAR comprising a VHH extracellular antigenbinding domain exhibits enhanced expression of the CAR on the cell surface of dendritic cells (DCs) compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 2-fold to about a 10-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 5-fold to about a 10-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 5-fold to about a 50-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 10-fold to about a 1000-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 2-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 5-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 10-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 20-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 30-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 40-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 50-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 100-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs.- 30 - NAI-5007600029vl

[0113] In some embodiments, a CAR comprising a VHH extracellular antigenbinding domain exhibits enhanced expression of the CAR on the cell surface of differentiated dendritic cells (e.g. cDCls) compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 2-fold to about a 10-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 5-fold to about a 10-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 5-fold to about a 50-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigenbinding domain exhibits about a 10-fold to about a 1000-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 2-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 5-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 10-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 20-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 30-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 40-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 50-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a VHH extracellular antigen-binding domain exhibits about a 100-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs.

[0114] In some embodiments, a CAR comprising a monobody extracellular antigenbinding domain exhibits enhanced expression of the CAR on the cell surface of dendritic cells (DCs) compared to a traditional scFv-based CARs. In some embodiments, a CAR - 31 - NAI-5007600029vlcomprising a monobody extracellular antigen-binding domain exhibits about a 2-fold to about a 10-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 5-fold to about a 10-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 5-fold to about a 50-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 10-fold to about a 1000-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 2-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 5-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 10-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 20-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 30-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 40-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigenbinding domain exhibits about a 50-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 100-fold greater expression on the cell surface of DCs compared to a traditional scFv-based CARs.

[0115] In some embodiments, a CAR comprising a monobody extracellular antigenbinding domain exhibits enhanced expression of the CAR on the cell surface of differentiated dendritic cells (e.g. cDCls) compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 2-fold to about a 10-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody - 32 - NAI-5007600029vlextracellular antigen-binding domain exhibits about a 5-fold to about a 10-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 5-fold to about a 50-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 10-fold to about a 1000-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 2-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigenbinding domain exhibits about a 5-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 10-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 20-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 30-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 40-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 50-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs. In some embodiments, a CAR comprising a monobody extracellular antigen-binding domain exhibits about a 100-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs.

[0116] In various embodiments of any of the aspects delineated herein, the extracellular antigen-binding domain of the presently disclosed chimeric receptor (e.g., CAR) binds to a target antigen. In certain embodiments, the target antigen is associated with a disease or disorder. Non-limiting examples of diseases or disorders include tumors, cancer, autoimmune diseases, neurodegenerative diseases, and infectious diseases. The target antigen can be a tumor antigen or a pathogen antigen.

[0117] In certain embodiments, the target antigen is a tumor antigen. In certain embodiments, the target antigen is a tumor-specific antigen (TSA). In certain embodiments,- 33 - NAI-5007600029vlthe target antigen is a tumor-associated antigen (TAA). In certain embodiments, the target antigen is overexpressed in one or more types of tumor or cancer tissues but has no or a limited expression in essential normal tissues, e.g., B7H3, EphA2, and Her2. In certain embodiments, the target antigen is a solid tumor antigen. In certain embodiments, the target antigen is a liquid tumor antigen. In certain embodiments, the target antigen is a cancer antigen. In certain embodiments, the target antigen is an antigen associated with cancer. In certain embodiments, the target antigen is a liquid tumor antigen, e.g., CD 19, CD20, B-cell maturation antigen (BCMA), G-protein-coupled receptor class 5 member D (GPRC5D), Fc receptor-like 5 (FCRL5), CD22, CD33, CD123, and CD30. In certain embodiments, the target antigen is an antigen associated with solid tumor, e.g., epidermal growth factor receptor (EGFR), Prostate-specific membrane antigen (PSMA), IL13Ra2 (Interleukin- 13 Receptor Alpha 2), MUC1 (Mucin 1), Claudin 18.2, Mesothelin, GD2, CEA, FAP, R0R1 (Receptor Tyrosine Kinase-Like Orphan Receptor 1), GPC3 (Glypican-3), MAGE-A4 (Melanoma-Associated Antigen A4), B7-H4, and Axl.

[0118] In certain embodiments, the target antigen is associated with an autoimmune disease. In certain embodiments, the autoimmune disease is a T-cell mediated autoimmune disease. In certain embodiments, the autoimmune disease is selected from the group consisting of type 1 diabetes (T1D), multiple sclerosis (MS), primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune myositis, psoriasis / psoriatic arthritis, and autoimmune vasculitis. Non-limiting examples of target antigens associated with an autoimmune disease include CD 19, CD20, Sulfonylurea Receptor 1 (SURI), Myelin Basic Protein (MBP), Proteolipid Protein (PLP), Myelin Oligodendrocyte Glycoprotein (MOG), Myelin-Associated Glycoprotein (MAG), Oligodendrocyte Myelin Glycoprotein (OMgp), Galactocerebroside (GalC), cytokeratins, Aquaporin-1 (AQP1), CD55, VCAM-1 (Vascular Cell Adhesion Molecule 1), Desmin, Dystrophin, CD56, myeloperoxidase (MPO), proteinase 3 (PR3), CD 177, and keratin.

[0119] In certain embodiments, the target antigen is selected from the group consisting of B7H3, CD19, CD20, PSMA, EGFR, Her2, and EphA2.

[0120] In certain embodiments, the target antigen is B7H3. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a single domain antibody that binds to B7H3. In certain embodiments, the anti-B7H3 single domain antibody is camelid. In certain embodiments, the anti-B7H3 single domain antibody is humanized. In certain embodiments, the anti-B7H3 single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus - 34 - NAI-5007600029vlframework. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a VHH domain that binds to B7H3. B7H3 (also known as CD276), is a member of the B7 family of immune checkpoint proteins, is highly expressed in cancer cells.

[0121] In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain sequence as set forth in SEQ ID NO: 4. CDR sequences can be determined or identified according to any well-known CDR numbering systems, including, but not limited to IMGT numbering system, Kabat numbering system, AbM numbering system, Chothia numbering system, and Contact numbering system. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NOs: 1-4 are provided below. In certain embodiments, the CDR1, CDR2, and CDR3 are identified according to the IMGT numbering system.GFTYNSYS (SEQ ID NO: 1)INSGGSST (SEQ ID NO: 2)AARSPSPLTFQTRTLREDSYNY (SEQ ID NO: 3) QVQLVESGGGSVQVGGSLRLSCAASGFTYNSYSVGWFRQAPGKEREGVAAINSGGS STYYAASVKGRFTISRDNAKNTVYLQMNSLKPEDTAMYYCAARSPSPLTFQTRTLRE DSYNYWGQGTQVTVSS (SEQ ID NO: 4)

[0122] In certain embodiments, the target antigen is PSMA. Prostate-specific membrane antigen (PSMA) is a protein that is overexpressed on prostate cancer cells and tumor associated neovasculature. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a single domain antibody that binds to PSMA. In certain embodiments, the anti-PSMA single domain antibody is camelid. In certain embodiments, the anti-PSMA single domain antibody is humanized. In certain embodiments, the anti-PSMA single domain antibody comprises an acceptor human framework, e.g, a human immunoglobulin framework or a human consensus framework. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a VHH domain that binds to PSMA.

[0123] In certain embodiments, the anti-PSMA VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain sequence as set forth in SEQ ID NO: 8. The CDR sequences can be determined or identified according to any well-known CDR numbering systems,- 35 - NAI-5007600029vlincluding, but not limited to IMGT numbering system, Kabat numbering system, AbM numbering system, Chothia numbering system, and Contact numbering system. In certain embodiments, the CDR1, CDR2, and CDR3 are identified according to the IMGT numbering system. In certain embodiments, the anti-PSMA VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the anti-PSMA VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NOs: 5-8 are provided below.RFMISEYS (SEQ ID NO: 5)INPAGTT (SEQ ID NO: 6)DGYGY (SEQ ID NO: 7) EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGT TDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSS(SEQ ID NO: 8)

[0124] In certain embodiments, the target antigen is CD 19. CD 19 is expressed on B-cells. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a single domain antibody that binds to CD 19. In certain embodiments, the anti-CD19 single domain antibody is camelid. In certain embodiments, the anti-CD19 single domain antibody is humanized. In certain embodiments, the anti-CD19 single domain antibody comprises an acceptor human framework, e.g, a human immunoglobulin framework or a human consensus framework. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor e.g., CAR) comprises a VHH domain that binds to CD 19.

[0125] In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain sequence as set forth in SEQ ID NO: 12. The CDR1, CDR2 and CDR3 sequences can be determined or identified according to any well-known CDR numbering systems, including, but not limited to IMGT numbering system, Kabat numbering system, AbM numbering system, Chothia numbering system, and Contact numbering system. In certain embodiments, the CDR1, CDR2, and CDR3 are identified according to the IMGT numbering system. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain - 36 - NAI-5007600029vlcomprises the amino acid sequence set forth in SEQ ID NO: 12. SEQ ID NOs: 9-12 are provided below.RPTDSRNC (SEQ ID NO: 9)IDIYKTT (SEQ ID NO: 10)AAARPCKYGSEWRRSASDFLY (SEQ ID NO: 11) QVQLQESGGGSVQAGGSLRLSCAVRRPTDSRNCMAWFRQAPGEQREAVAGIDIYKT TGYAESVKGRFTISQDNAKNTLFLQMNSLKPEDSGTYYCAAARPCKYGSEWRRSAS DFLYWGQGTQVTVSS (SEQ ID NO: 12)

[0126] In certain embodiments, the target antigen is CD20. CD20 is expressed on B-cells. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a single domain antibody that binds to CD20. In certain embodiments, the anti-CD20 single domain antibody is camelid. In certain embodiments, the anti-CD20 single domain antibody is humanized. In certain embodiments, the anti-CD20 single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a VHH domain that binds to CD20.

[0127] In certain embodiments, the anti-CD20 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain sequence as set forth in SEQ ID NO: 16. The CDR1, CDR2 and CDR3 sequences can be determined or identified according to any well-known CDR numbering systems, including, but not limited to IMGT numbering system, Kabat numbering system, AbM numbering system, Chothia numbering system, and Contact numbering system. In certain embodiments, the CDR1, CDR2, and CDR3 are identified according to the IMGT numbering system. In certain embodiments, the anti-CD20 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the anti-CD20 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 16. SEQ ID NOs: 13-16 are provided below.RYGVTLPY (SEQ ID NO: 13)ITLRGST (SEQ ID NO: 14)AAGTSARSLSPSDYGY (SEQ ID NO: 15)- 37 - NAI-5007600029vlQVQLQESGGGSVQAGGSLRLRCIISRYGVTLPYMAWFRQGPGEEREGVAAITLRGST LYADNVKGRFTLSQDPPKRALFLQMNNLQPEDSGMYYCAAGTSARSLSPSDYGYRG RGTQVTVSS (SEQ ID NO: 16)

[0128] In certain embodiments, the target antigen is EGFR. Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein and a member of the ErbB family of receptor tyrosine kinases. EGFR is frequently overexpressed in various types of cancers. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a single domain antibody that binds to EGFR. In certain embodiments, the anti-EGFR single domain antibody is camelid. In certain embodiments, the anti-EGFR single domain antibody is humanized. In certain embodiments, the anti-EGFR single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a VHH domain that binds to EGFR.

[0129] In certain embodiments, the anti-EGFR VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain sequence as set forth in SEQ ID NO: 20. The CDR1, CDR2 and CDR3 sequences can be determined or identified according to any well-known CDR numbering systems, including, but not limited to IMGT numbering system, Kabat numbering system, AbM numbering system, Chothia numbering system, and Contact numbering system. In certain embodiments, the CDR1, CDR2, and CDR3 are identified according to the IMGT numbering system. In certain embodiments, the anti-EGFR VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the anti-EGFR VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 20. SEQ ID NOs: 17-20 are provided below.GRTFSSYA (SEQ ID NO: 17)IRWSGGYT (SEQ ID NO: 18)AATYLSSDYSRYALPQRPLDYDY (SEQ ID NO: 19) QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGG YTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQ RPLDYDYWGQGTQVTVSS (SEQ ID NO: 20)

[0130] In certain embodiments, the extracellular antigen-binding domain of the presently disclosed chimeric receptor (e.g., CAR) comprises a monobody. In certain embodiments, the - 38 - NAI-5007600029vltarget antigen is EphA2. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a monobody that binds to EphA2. In certain embodiments, the anti-EphA2 monobody is based on a fibronectin type III (FN3) domain. In certain embodiments, the anti-EphA2 monobody comprises a BC loop, a DE loop, and an FG loop of a monobody comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the anti-EphA2 monobody comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO: 21; a DE loop comprising the amino acid sequence set forth in SEQ ID NO: 22; and a FG loop comprising the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the anti-EphA2 monobody comprises the amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NOs: 21-24 are provided below.YYPFCAF (SEQ ID NO: 21)RPSD (SEQ ID NO: 22)CLGSYSR (SEQ ID NO: 23) VSDVPRDLEVVAATPTSLLISWYYPFCAFYYRITYGETGGNSPVQEFTVPRPSDTATIS GLKPGVDYTITVYAVTCLGSYSRPISINYRT (SEQ ID NO: 24)

[0131] In certain embodiments, the target antigen is a pathogen antigen, e.g., for use in treating a pathogen infection or other infectious disease. In certain embodiments, the target antigen is an antigen associated with a pathogen. Non-limiting examples of pathogens include bacteria, viruses, fungi, parasites and protozoa.

[0132] In certain embodiments, the target antigen is an antigen associated with a virus. Non-limiting examples of viruses include Coronaviridae (e.g., coronaviruses, e.g, SARS-CoV-2 virus), Hepadnaviridae (e.g., hepatitis A virus, and hepatitis B virus), Adenoviridae (most adenoviruses), Orthomyxoviridae (e.g., influenza viruses), Retroviridae (e.g., human immunodeficiency virus (HIV)), Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, and respiratory syncytial virus), Rhabdoviridae (e.g., vesicular stomatitis viruses, and rabies viruses), Filoviridae (e.g., ebola viruses), Parvoviridae (parvoviruses), and Herpesviridae (e.g., herpes simplex virus (HSV) 1, HSV-2, varicella zoster virus, cytomegalovirus (CMV), and herpes virus).

[0133] In certain embodiments, the target antigen is a capsid protein. Non-limiting examples of capsid proteins include viral protein 1 (VP1), viral protein 2 (VP2), viral protein 3 (VP3), viral protein 4 (VP4), hexon, penton proteins, major capsid protein (LI), and Core Protein (HBcAg). In certain embodiments, the target protein is a viral envelope glycoprotein. Non-limiting examples of viral envelope glycoproteins include hemagglutinin (HA) (e.g., HA presented on the surface of an influenza virus), neuraminidase (NA) (e.g., NA presented on - 39 - NAI-5007600029vlthe surface of an influenza virus), spike protein (e.g., spike protein of a SARS-CoV-2 virus), glycoprotein 120 (gpl20), and gp41. In certain embodiments, the target protein is a viral fusion protein.

[0134] In certain embodiments, the target antigen is an antigen associated with a bacterium. Non-limiting examples of bacteria include Staphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Bacillus anthracis, Haemophilus influenzae, Mycobacteria, and Legionella pneumophila. In certain embodiments, the target antigen is selected from the group consisting of surface proteins (e.g., Protein A in Staphylococcus aureus, and M protein in Streptococcus pyogenes, capsular polysaccharides (e.g., polysaccharide capsule), lipopolysaccharides (LPS), teichoic acid, flagellar proteins (e.g., H antigen in Escherichia coli), Pili / Fimbriae Proteins e.g., Pilin in Neisseria gonorrhoeae), exotoxins e.g., Tetanus toxin, and Diphtheria toxin), and Endospores e.g., Spore coat proteins in Bacillus anthracis).

[0135] In certain embodiments, the target antigen is associated with neurodegenerative disease. Non-limiting examples of neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (ALS), Multiple sclerosis (MS), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), and prion diseases. In certain embodiments, the target antigen is associated with Alzheimer’s disease. In certain embodiments, the target antigen is beta amyloid. Beta amyloid is the main component of the amyloid plaques found in the brains of people with Alzheimer’s disease. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a single domain antibody that binds to beta amyloid. In certain embodiments, the anti-beta amyloid single domain antibody is camelid. In certain embodiments, the anti-beta amyloid single domain antibody is humanized. In certain embodiments, the anti-beta amyloid single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In certain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a VHH domain that binds to beta amyloid.

[0136] In certain embodiments, the anti -beta amyloid VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain sequence as set forth in SEQ ID NO: 64. The CDR1, CDR2 and CDR3 sequences can be determined or identified according to any well-known CDR numbering systems, including, but not limited to IMGT numbering system, Kabat numbering system, AbM numbering system, Chothia numbering system, and Contact - 40 - NAI-5007600029vlnumbering system. In certain embodiments, the CDR1, CDR2, and CDR3 are identified according to the IMGT numbering system. In certain embodiments, the anti-beta amyloid VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the anti-beta amyloid VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 64. SEQ ID NOs: 61-64 are provided below.GSTFRNYN (SEQ ID NO: 61)VSRTGIST (SEQ ID NO: 62)AAGRPGVGAVNRAMDYDY (SEQ ID NO: 63) QVQLQASGGGLVQTGDSLRLSCADSGSTFRNYNIGWFRQTPGQAREFVAAVSRTGIS THVADSLQGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAGRPGVGAVNRAMDY DYWGQGTQVTVSS (SEQ ID NO: 64)5.2.2. Transmembrane Domain

[0137] The presently disclosed chimeric receptor (e.g., CAR) comprises a transmembrane domain. The transmembrane domain can be directly or indirectly fused to the extracellular antigen-binding domain (e.g., any of extracellular antigen-binding domains disclosed in Section 5.2.1). The transmembrane domain can be derived from a naturally occurring protein. Non-limiting examples of naturally occurring proteins from which the transmembrane domain can be derived from include CD8, a Toll-like receptor (including, but not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11), CD40, CD3 a FcR, CD166, 0X40, CD28, 4-1BB, and ICOS. Alternatively, the transmembrane domain can be derived from a synthetic, non-naturally occurring protein, e.g, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0138] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from CD8. In certain embodiments, the CD8 is CD8a. In certain embodiments, the CD8 is CD8p.

[0139] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from CD8a. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of CD8a or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from mouse CD8a. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of mouse CD8a or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric - 41 - NAI-5007600029vlreceptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P01731 (SEQ ID NO: 25). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor e.g., CAR) comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 197 to 217 of SEQ ID NO: 25. SEQ ID NO: 25 is provided below (amino acids 197 to 217 underlined).MASPLTRFLSLNLLLLGESIILGSGEAKPQAPELRIFPKKMDAELGQKVDLVCEVLGS VSQGC S WLFQNS S SKLPQPTF VVYM AS SHNKITWDEKLNS SKLF S AMRDTNNK YVL TLNKF SKENEGYYFCS VISNSVMYF S S VVP VLQKVNSTTTKPVLRTPSPVHPTGTSQP QRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSRKRVCKCPR PLVRQEGKPRPSEKIV (SEQ ID NO: 25)

[0140] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from human CD8a. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of human CD8a or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P01732 (SEQ ID NO: 26). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 183 to 203 of SEQ ID NO: 26. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 183 to 203 of SEQ ID NO: 26. SEQ ID NO: 26 is provided below (amino acids 183 to 203 underlined).MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWL FQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYF CSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH- 42 - NAI-5007600029vlTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSL SARYV (SEQ ID NO: 26)

[0141] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from CD8p. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of CD8P or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from mouse CD8p. In certain embodiments, the transmembrane domain of the chimeric receptor e.g., CAR) comprises a transmembrane domain of mouse CD8P or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P10300 (SEQ ID NO: 43). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 176 to 196 of SEQ ID NO: 43. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids.. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 176 to 196 of SEQ ID NO: 43 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 176 to 196 of SEQ ID NO: 43. SEQ ID NO: 43 is provided below (amino acids 176 to 196 underlined).MQPWLWLVFSMKLAALWSSSALIQTPSSLLVQTNHTAKMSCEVKSISKLTSIYWLRE RQDPKDKYFEFLASWSSSKGVLYGESVDKKRNIILESSDSRRPFLSIMNVKPEDSDFY FCATVGSPKMVFGTGTKLTVVDVLPTTAPTKKTTLKMKKKKQCPFPHPETQKGLTC SLTTLSLLVVCILLLLAFLGVAVYFYCVRRRARIHFMKQFHK (SEQ ID NO: 43)

[0142] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from human CD8p. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of human CD8P or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P10966 (SEQ ID NO: 44). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 171 to 191 of SEQ ID NO: 44. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to - 43 - NAI-5007600029vlabout 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 171 to 191 of SEQ ID NO: 44 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 171 to 191 of SEQ ID NO: 44. SEQ ID NO: 44 is provided below (amino acids 171 to 191 underlined).MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLR QRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIY FCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITL GLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK (SEQ ID NO: 44)

[0143] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from a Toll-like receptor (TLR). Non-limiting examples of TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or TLR11. In certain embodiments, the TLR is TLR4. In certain embodiments, the transmembrane domain of the chimeric receptor e.g., CAR) is derived from mouse TLR4. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of mouse TLR4 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: Q9QUK6 (SEQ ID NO: 27). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 639 to 659 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below (amino acids 639 to 659 underlined).MMPPWLLARTLIMALFFSCLTPGSLNPCIEVVPNITYQCMDQKLSKVPDDIPSSTKNI DLSFNPLKILKSYSFSNFSELQWLDLSRCEIETIEDKAWHGLHHLSNLILTGNPIQSFSP GSFSGLTSLENLVAVETKLASLESFPIGQLITLKKLNVAHNFIHSCKLPAYFSNLTNLV HVDLSYNYIQTITVNDLQFLRENPQVNLSLDMSLNPIDFIQDQAFQGIKLHELTLRGN FNSSNIMKTCLQNLAGLHVHRLILGEFKDERNLEIFEPSIMEGLCDVTIDEFRLTYTND FSDDIVKFHCLANVSAMSLAGVSIKYLEDVPKHFKWQSLSIIRCQLKQFPTLDLPFLK- 44 - NAI-5007600029vlSLTLTMNKGSISFKKVALPSLSYLDLSRNALSFSGCCSYSDLGTNSLRHLDLSFNGAII MSANFMGLEELQHLDFQHSTLKRVTEFSAFLSLEKLLYLDISYTNTKIDFDGIFLGLTS LNTLKMAGNSFKDNTLSNVFANTTNLTFLDLSKCQLEQISWGVFDTLHRLQLLNMS HNNLLFLDSSHYNQLYSLSTLDCSFNRIETSKGILQHFPKSLAFFNLTNNSVACICEHQ KFLQWVKEQKQFLVNVEQMTCATPVEMNTSLVLDFNNSTCYMYKTIISVSVVSVIV VSTVAFLIYHFYFHLILIAGCKKYSRGESIYDAFVIYSSONEDWVRNELVKNLEEGVP RFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAQTWQFL SSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDG KASNPEQTAEEEQETATWT (SEQ ID NO: 27)

[0144] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from human TLR4. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of human TLR4 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: 000206-1 (SEQ ID NO: 28). In certain embodiments, the transmembrane domain of the chimeric receptor e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor e.g., CAR) comprises amino acids 632 to 652 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor e.g., CAR) comprises amino acids 632 to 652 of SEQ ID NO: 28. SEQ ID NO: 28 is provided below (amino acids 632 to 652 underlined).MMSASRLAGTLIPAMAFLSCVRPESWEPCVEVVPNITYQCMELNFYKIPDNLPFSTK NLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSL ALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLT NLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLR NNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLA YLD YYLDDIIDLFNCLTNVS SF SL VS VTIERVKDF SYNFGWQHLELVNCKFGQFPTLK LKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLS FNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGI FNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVL NMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFA- 45 - NAI-5007600029vlCTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVL SVLVVSVVAVLVYI< FYFHLMLLAGCII< YGRGENIYDAFVIYSSODEDWVRNELVI< N LEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQ TWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRK ALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 28)

[0145] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from 0X40. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g, CAR) comprises a transmembrane domain of 0X40 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g, CAR) is derived from mouse 0X40. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of mouse 0X40 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P47741 (SEQ ID NO: 29). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 212 to 236 of SEQ ID NO: 29. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 212 to 236 of SEQ ID NO: 29 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 212 to 236 of SEQ ID NO: 29. SEQ ID NO: 29 is provided below (amino acids 212 to 236 underlined).MYVWVQQPTALLLLALTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHT RDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQP RQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDR SLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLA PLTVLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI (SEQ ID NO: 29)

[0146] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from human 0X40. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of human 0X40 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the - 46 - NAI-5007600029vlamino acid sequence having a UniProt Reference No: P43489 (SEQ ID NO: 30). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 215 to 235 of SEQ ID NO: 30. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 amino acids, and / or up to about 35, or up to about 40 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 215 to 235 of SEQ ID NO: 30. SEQ ID NO: 30 is provided below (amino acids 215 to 235 underlined).MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSR CSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRA GTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICED RDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLG LLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 30)

[0147] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from a FcR. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of a FcR or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from a Fc-gamma receptor (FcyR). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of FcyR or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from FCERγ. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of FCERγ or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from mouse FCERγ. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of mouse FCERγ or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P20491 (SEQ ID NO: 35). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 24 to 44 of SEQ ID NO: 35. In certain embodiments, the - 47 - NAI-5007600029vlconsecutive portion has a length of at least about 5, at least about 10, at least about 15, or at least about 20 amino acids, and / or up to about 25, up to about 30, or up to about 35 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 24 to 44 of SEQ ID NO: 35 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 24 to 44 of SEQ ID NO: 35. SEQ ID NO: 35 is provided below (amino acids 24 to 44 underlined).MISAVILFLLLLVEQAAALGEPQLCYILDAVLFLYGIVLTLLYCRLKIQVRKAAIASRE KADAVYTGLNTRSQETYETLKHEKPPQ (SEQ ID NO: 35)

[0148] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from human FCERγ. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of human FCERγ or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P30273 (SEQ ID NO: 36). In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 24 to 44 of SEQ ID NO: 36. In certain embodiments, the consecutive portion has a length of at least about 5, at least about 10, at least about 15, or at least about 20 amino acids, and / or up to about 25, up to about 30, or up to about 35 amino acids. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises amino acids 24 to 44 of SEQ ID NO: 36 or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR)comprises amino acids 24 to 44 of SEQ ID NO: 36. SEQ ID NO: 36 is provided below (amino acids 24 to 44 underlined).MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYE KSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 36)

[0149] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises at least a portion of a synthetic, non-naturally occurring protein segment. In certain embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In certain embodiments, the protein segment has a length of at least about 10, at least about 15, at least about 20, at least about 25, at least about 30 amino acids, and / or up to about 35, or about 40 amino acids.5.2.3. Intracellular Domain

[0150] In certain embodiments, the chimeric receptor is a chimeric antigen receptor (CAR)- 48 - NAI-5007600029vlthat comprises an intracellular domain. In certain embodiments, the intracellular domain of the CAR is derived from a molecule that is capable of activating a dendritic cell (“dendritic cell-activating molecule” or “DC-activating molecule”). Upon binding of the extracellular antigen-binding domain of the CAR to the target antigen, the cell (e.g. a dendritic cell) comprising the CAR is activated. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of a DC-activating molecule. DC-activating molecules can activate DCs via various signaling pathways. In certain embodiments, the DC-activating molecule activates DCs through immunoreceptor tyrosine-based activation motif (IT M)-mediated signaling. ITAM is a conserved protein motif that is generally present in the tail portion of signaling molecules, such as a receptor or an adaptor protein that associates with the receptor, expressed in many immune cells. DC-activating molecules that are capable of activating DCs through IT AM-mediated signaling include, but are not limited to, CD3, CD3y, CD36, CD3s, a Fc receptor (including, but not limited to, a Fc-gamma receptor (FcyR), a Fc-epsilon receptor (FcsR), CD64, CD32A, or CD16A), CD79a, CD79b, TREM-1, and TREM-2.

[0151] In certain embodiments, the DC-activating molecule activates DCs through a Tolllike receptor (TLR)-mediated signaling pathway. The TLR-mediated signaling pathway involves a TLR and an adapter molecule. Individual TLRs interact with different combinations of adapter molecules and activate various transcription factors, e.g., nuclear factor (NF)-KB, activating protein- 1 and interferon regulatory factors, driving a specific immune response. In certain embodiments, the DC-activating molecule that is capable of activating DCs through a TLR-mediated signaling pathway is a TLR. TLRs include, but are not limited to TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11. In certain embodiments, the TLR is TLR4.

[0152] In certain embodiments, the DC-activating molecule is CD40.

[0153] In certain embodiments, the DC-activating molecule is selected from the group consisting of a TLR, CD40, CD3ζ, and a FcR. In certain embodiments, the intracellular domain of the chimeric receptor (e.g., CAR) comprises an intracellular domain of one DC-activating molecule or a fragment thereof. In certain embodiments, the DC-activating molecule is TLR4. In certain embodiments, the FcR is a Fc-gamma receptor (FcyR). In certain embodiments, the FcR is FCERγ (also known as “Fc receptor gamma-chain (FcRgamma)”, “Fc-epsilon Rl-gamma IgE”, “Fc receptor subunit gamma (FceRI gamma)”).

[0154] CD40

[0155] In certain embodiments, the intracellular domain of the CAR is derived from CD40.- 49 - NAI-5007600029vlIn certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of CD40 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse CD40. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse CD40 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P27512 (SEQ ID NO: 31). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, or at least about 70 amino acids, and / or up to about 60, up to about 75, or up to about 80 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. SEQ ID NO: 31 is provided below (amino acids 216 to 289 underlined).MVSLPRLCALWGCLLTAVHLGQCVTCSDKQYLHDGQCCDLCQPGSRLTSHCTALE KTQCHPCDSGEFSAQWNREIRCHQHRHCEPNQGLRVKKEGTAESDTVCTCKEGQHC TSKDCEACAQHTPCIPGFGVMEMATETTDTVCHPCPVGFFSNQSSLFEKCYPWTSCE DKNLEVLQKGTSQTNVICGLKSRMRALLVIPVVMGILITIFGVFLYIKKVVKKPKDNE ILPPAARRODPOEMEDYPGHNTAAPVOETLHGCOPVTQEDGKESRISVOEROVTDSI ALRPLV (SEQ ID NO: 31)

[0156] In certain embodiments, the intracellular domain of the CAR is derived from human CD40. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human CD40 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P25942 (SEQ ID NO: 32). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 216 to 277 of SEQ ID NO: 32. In certain embodiments, the consecutive portion has a length of at least about 50, at least about 60, or at least about 70 amino acids, and / or up to about 60, up to about 75, or up to about 80 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 277 of SEQ ID NO: 32 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 277 of SEQ ID NO: 32. SEQ ID NO: 32 is provided below (amino acids 216 to 277 underlined).- 50 - NAI-5007600029vlMVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTET ECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTS EACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKD LVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHP KOEPOEINFPDDLPGSNTAAPVOETLHGCOPVTQEDGKESRISVOERO (SEQ ID NO: 32)

[0157] In certain embodiments, the intracellular domain of the CAR is derived from CD40 and is not capable of activating an immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling. In certain embodiments, the intracellular domain of the CAR is derived from CD40 and lacks an intracellular domain of an activating receptor comprising an IT AM. In certain embodiments, the ITAM-containing activating receptor is selected from the group consisting of CD3ζ, CD3y, CD36, CD3s, a Fc receptor (including, but not limited to, a Fc-gamma receptor (FcyR), a Fc-epsilon receptor (FcsR), CD64, CD32A, or CD16A), CD79a, CD79b, TREM-1, and TREM-2. In certain embodiments, the ITAM-containing activating receptor is CD3.

[0158] CD3

[0159] In certain embodiments, the intracellular domain of the CAR is derived from CD3ζ. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of CD3ζ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse CD3ζ. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse CD3ζ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P24161 (SEQ ID NO: 33). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 52 to 164 of SEQ ID NO: 33. In certain embodiments, the consecutive portion has a length of at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or at least about 110 amino acids, and / or up to about 80, up to about 90, up to about 100, up to about 110, up to about 120, or up to about 130 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 33 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 33. SEQ ID NO: 33 is provided below (amino acids 52 to 164 underlined).- 51 - NAI-5007600029vlMKWKVSVLACILHVRFPGAEAOSFGLLDPKLCYLLDGILFIYGVIITALYLRAKFSRS AETAANLQDPNOLYNELNLGRREEYDVLEKKRARDPEMGGKOORRRNPOEGVYNA LOKDKMAEAYSEIGTKGERRRGKGHDGLYOGLSTATKDTYDALHMQTLAPR (SEP ID NO: 33)

[0160] In certain embodiments, the intracellular domain of the CAR is derived from human CD3ζ. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human CD3ζ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P20963 (SEQ ID NO: 34). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 52 to 164 of SEQ ID NO: 34. In certain embodiments, the consecutive portion has a length of at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or at least about 110 amino acids, and / or up to about 80, up to about 90, up to about 100, up to about 110, up to about 120, or up to about 130 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 34 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 34. SEQ ID NO: 34 is provided below (amino acids 52 to 164 underlined). MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSA DAPAYOOGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKPORRKNPOEGLYNEL OKDKMAEAYSEIGMKGERRRGKGHDGLYOGLSTATKDTYDALHMQALPPR (SEQ ID NO: 34)

[0161] Fc Receptor (FcR)

[0162] In certain embodiments, the intracellular domain of the CAR is derived from a FcR. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of FcR or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from a Fc-gamma receptor (FcyR). In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of FcyR or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from FCERγ. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of FCERγ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse FCERγ. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse FCERγ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is - 52 - NAI-5007600029vla consecutive portion of the amino acid sequence having a UniProt Reference No: P20491 (SEQ ID NO: 35). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 45 to 86 of SEQ ID NO: 35. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 20, at least about 30, or at least about 40 amino acids, and / or up to about 30, up to about 40, or up to about 50 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 35 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 35. SEQ ID NO: 35 is provided below (amino acids 45 to 86 underlined).MISAVILFLLLLVEQAAALGEPQLCYILDAVLFLYGIVLTLLYCRLKIQVRKAAIASRE KADAVYTGLNTRSOETYETLKHEKPPQ (SEQ ID NO: 35)

[0163] In certain embodiments, the intracellular domain of the CAR is derived from human FCERγ. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human FCERγ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P30273 (SEQ ID NO: 36). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 45 to 86 of SEQ ID NO: 36. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 20, at least about 30, or at least about 40 amino acids, and / or up to about 30, up to about 40, or up to about 50 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 36 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 36. SEQ ID NO: 36 is provided below (amino acids 45 to 86 underlined).MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIOVRKAAITSYE KSDGVYTGLSTRNOETYETLKHEKPPQ (SEQ ID NO: 36)

[0164] Toll-like Receptor (TLR)

[0165] In certain embodiments, the intracellular domain of the CAR is derived from a TLR. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of a TLR or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from TLR4. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of TLR4 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse TLR4. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of - 53 - NAI-5007600029vlmouse TLR4 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: Q9QUK6 (SEQ ID NO: 27). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the consecutive portion has a length of at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, or at least about 170 amino acids, and / or up to about 150, up to about 160, up to about 170, up to about 180, up to about 190, or up to about 200 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below (amino acids 660 to 835 underlined).MMPPWLLARTLIMALFFSCLTPGSLNPCIEVVPNITYQCMDQKLSKVPDDIPSSTKNI DLSFNPLKILKSYSFSNFSELQWLDLSRCEIETIEDKAWHGLHHLSNLILTGNPIQSFSP GSFSGLTSLENLVAVETKLASLESFPIGQLITLKKLNVAHNFIHSCKLPAYFSNLTNLV HVDLSYNYIQTITVNDLQFLRENPQVNLSLDMSLNPIDFIQDQAFQGIKLHELTLRGN FNSSNIMKTCLQNLAGLHVHRLILGEFKDERNLEIFEPSIMEGLCDVTIDEFRLTYTND FSDDIVKFHCLANVSAMSLAGVSIKYLEDVPKHFKWQSLSIIRCQLKQFPTLDLPFLK SLTLTMNKGSISFKKVALPSLSYLDLSRNALSFSGCCSYSDLGTNSLRHLDLSFNGAII MSANFMGLEELQHLDFQHSTLKRVTEFSAFLSLEKLLYLDISYTNTKIDFDGIFLGLTS LNTLKMAGNSFKDNTLSNVFANTTNLTFLDLSKCQLEQISWGVFDTLHRLQLLNMS HNNLLFLDSSHYNQLYSLSTLDCSFNRIETSKGILQHFPKSLAFFNLTNNSVACICEHQ KFLQWVKEQKQFLVNVEQMTCATPVEMNTSLVLDFNNSTCYMYKTIISVSVVSVIV VSTVAFLIYHFYFHLILIAGCKKYSRGESIYDAFVIYSSONEDWVRNELVKNLEEGVP RFHLCLHYRDFIPGVAIAANIIOEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAOTWOFL SSRSGIIFIVLEKVEKSLLRQOVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDG KASNPEQTAEEEQETATWT (SEQ ID NO: 27)

[0166] In certain embodiments, the intracellular domain of the CAR is derived from human TLR4. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human TLR4 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: 000206-1 (SEQ ID NO: 28). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 653 to 839 of SEQ ID NO: 28. In - 54 - NAI-5007600029vlcertain embodiments, the consecutive portion has a length of at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, or at least about 180 amino acids, and / or up to about 150, up to about 160, up to about 170, up to about 180, up to about 190, or up to about 200 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. SEQ ID NO: 28 is provided below (amino acids 653 to 839 underlined).MMSASRLAGTLIPAMAFLSCVRPESWEPCVEVVPNITYQCMELNFYKIPDNLPFSTK NLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSL ALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLT NLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLR NNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLA YLD YYLDDIIDLFNCLTNVS SF SL VS VTIERVKDF SYNFGWQHLELVNCKFGQFPTLK LKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLS FNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGI FNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVL NMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFA CTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVL SVLVVSVVAVLVYI< FYFHLMLLAGCII< YGRGENIYDAFVIYSSODEDWVRNELVI< N LEEGVPPFOLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSOHFIOSRWCIFEYEIAQ TWOFLSSRAGIIFIVLOI< VEI< TLLROOVELYRLLSRNTYLEWEDSVLGRHIFWRRLRI< ALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 28)

[0167] In certain embodiments, the intracellular domain of the CAR is derived from a TLR (e.g., TLR4) and is not capable of activating an immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling. In certain embodiments, the intracellular domain of the CAR is derived from a TLR (e.g., TLR4) and lacks an intracellular domain of an activating receptor comprising an ITAM. In certain embodiments, the IT M-containing activating receptor is selected from the group consisting of CD3ζ, CD3y, CD36, CD3s, a Fc receptor (including, but not limited to, a Fc-gamma receptor (FcyR), a Fc-epsilon receptor (FcsR), CD64, CD32A, or CD16A), CD79a, CD79b, TREM-1, and TREM-2. In certain embodiments, the ITAM-containing activating receptor is CD3.

[0168] 0X40

[0169] In certain embodiments, the intracellular domain of the CAR comprises an- 55 - NAI-5007600029vlintracellular domain of 0X40 or a fragment thereof and is not capable of activating an immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling. In certain embodiments, the intracellular domain of the CAR is derived from 0X40 and lacks an intracellular domain of an activating receptor comprising an ITAM. In certain embodiments, the IT M-containing activating receptor is selected from the group consisting of CD3ζ, CD3y, CD36, CD3s, a Fc receptor (including, but not limited to, a Fc-gamma receptor (FcyR), a Fc-epsilon receptor (FcsR), CD64, CD32A, or CD16A), CD79a, CD79b, TREM-1, and TREM-2. In certain embodiments, the ITAM-containing activating receptor is CD3.

[0170] In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse 0X40 or a fragment thereof and lacks an intracellular domain of a DC-activating molecule. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P47741 (SEQ ID NO: 29). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 237 to 272 of SEQ ID NO: 29. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, or at least about 35 amino acids, and / or up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 237 to 272 of SEQ ID NO: 29 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 237 to 272 of SEQ ID NO: 29. SEQ ID NO: 29 is provided below (amino acids 237 to 272 underlined).MYVWVQQPTALLLLALTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHT RDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQP RQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDR SLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLA PLTVLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI (SEQ ID NO: 29)

[0171] In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human 0X40 or a fragment thereof and lacks an intracellular domain of a DC-activating molecule. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P43489 (SEQ ID NO: 30). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive - 56 - NAI-5007600029vlportion of amino acids 236 to 277 of SEQ ID NO: 30. In certain embodiments, the consecutive portion has a length of at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 amino acids, and / or up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 236 to 277 of SEQ ID NO: 30 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 236 to 277 of SEQ ID NO: 30. SEQ ID NO: 30 is provided below (amino acids 236 to 277 underlined).MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSR CSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRA GTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICED RDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLG LLGPLAILLALYLLRRDORLPPDAHKPPGGGSFRTPIOEEOADAHSTLAKI (SEQ ID NO: 30)

[0172] Flt3

[0173] In certain embodiments, the intracellular domain of the CAR is derived from FMS-like tyrosine kinase 3 (Flt3). In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of Flt3 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse Flt3. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse Flt3 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: Q00342 (SEQ ID NO: 45). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 565 to 992 of SEQ ID NO: 45. In certain embodiments, the consecutive portion has a length of at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, or at least about 400 amino acids, and / or up to about 300, up to about 350, up to about 400, or up to about 450 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 565 to 992 of SEQ ID NO: 45 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 565 to 992 of SEQ ID NO: 45. SEQ ID NO: 45 is provided below (amino acids 565 to 992 underlined).MRALAQRSDRRLLLLVVLSVMILETVTNQDLPVIKCVLISHENNGSSAGKPSSYRMV RGSPEDLQCTPRRQSEGTVYEAATVEVAESGSITLQVQLATPGDLSCLWVFKHSSLG- 57 - NAI-5007600029vlCQPHFDLQNRGIVSMAILNVTETQAGEYLLHIQSEAANYTVLFTVNVRDTQLYVLRR P YF RKMENQD A LLC I SEG VPEPT VEW VLC S SHRESCKEEGP AVVRKEEKVLHELFGT DIRCCARNALGRESTKLFTIDLNQAPQSTLPQLFLKVGEPLWIRCKAIHVNHGFGLTW ELEDKALEEGSYFEMSTYSTNRTMIRILLAFVSSVGRNDTGYYTCSSSKHPSQSALVT ILEKGFINATS SQEEYEIDPYEKFCF S VRFKAYPRIRCTWIF SQ ASFPCEQRGLEDGYSI SKFCDHKNKPGEYIFYAENDDAQFTKMFTLNIRKKPQVLANASASQASCSSDGYPLP SWTWKKCSDKSPNCTEEIPEGVWNKKANRKVFGQWVSSSTLNMSEAGKGLLVKCC AYNSMGTSCETIFLNSPGPFPFIQDNISFYATIGLCLPFIVVLIVLICHKYKKQFRYESOL OMIOVTGPLDNEYFYVDFRDYEYDLKWEFPRENLEFGKVLGSGAFGRVMNATAYGI SKTGVSIOVAVKMLKEKADSCEKEALMSELKMMTHLGHHDNIVNLLGACTLSGPV YLIFEYCC YGDLLNYLRSKREKFHRTWTEIFKEHNF SF YPTFQAHSNS SMPGSREVQL HPPLDQLSGFNGNSIHSEDEIEYENOKRLAEEEEEDLNVLTFEDLLCFAYOVAKGMEF LEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDILSDSSYVVRGNARLPVKWMAP ESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIOSGFKMEQPFYATE GIYFVMOSCWAFDSRKRPSFPNLTSFLGCOLAEAEEAMYONMGGNVPEHPSIYONR RPLSREAGSEPPSPQAQVKIHRERS (SEQ ID NO: 45)

[0174] In certain embodiments, the intracellular domain of the CAR is derived from human Flt3. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human Flt3 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P36888 (SEQ ID NO: 46). In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence that is a consecutive portion of amino acids 564 to 993 of SEQ ID NO: 46. In certain embodiments, the consecutive portion has a length of at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, or at least about 400 amino acids, and / or up to about 300, up to about 350, up to about 400, or up to about 450 amino acids. In certain embodiments, the intracellular domain of the CAR comprises amino acids 564 to 993 of SEQ ID NO: 46 or a fragment thereof. In certain embodiments, the intracellular domain of the CAR comprises amino acids 564 to 993 of SEQ ID NO: 46. SEQ ID NO: 46 is provided below (amino acids 564 to 993 underlined).MP AL ARDGGQLPLL VVF S AMIFGTITNQDLP V I KC VLINHKNND S S VGKS S S YPMVSE SPEDLGCALRPQSSGTVYEAAAVEVDVSASITLQVLVDAPGNISCLWVFKHSSLNCQ PHFDLQNRGVVSMVILKMTETQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRPYF RKMENQDALVCISESVPEPIVEWVLCDSQGESCKEESPAVVKKEEKVLHELFGTDIR- 58 - NAI-5007600029vlCCARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWEL ENKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIV EKGFINATNS SEDYEIDQYEEFCF S VRFKAYPQIRCTWTF SRKSFPCEQKGLDNGYSIS KFCNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPS WTWKKC SDK SPNCTEEITEGVWNRK ANRK VFGQW VS S STLNMSEAIKGFL VKCC A YNSLGTSCETILLNSPGPFPFIQDNISFYATIGVCLLFIVVLTLLICHKYKKQFRYESOL OMVOVTGSSDNEYFYVDFREYEYDL1< WEFPRENLEFGI< VLGSGAFGI< VMNATAYG ISKTGVSIOVAVKMLKEKADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIY LIFEYCCYGDLLNYLRSKREKFHRTWTEIFKEHNFSFYPTFOSHPNSSMPGSREVQIHP DSDOISGLHGNSFHSEDEIEYENOKRLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFK SCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDSNYVVRGNARLPVKWMAPESL FEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIONGFKMDOPFYATEEIY IIMOSCWAFDSRKRPSFPNLTSFLGCOLADAEEAMYONVDGRVSECPHTYONRRPFS REMDLGLLSPOAQVEDS (SEP ID NO: 46)

[0175] In certain embodiments, the presently disclosed chimeric receptor lacks an intracellular domain. Such chimeric receptors are not capable of activating an immune cell, e.g., a dendritic cell. Thus, they are also referred to as “non-signaling” CARs.5.2.4. Hinge Region

[0176] In certain embodiments, the presently disclosed chimeric receptor (e.g., CAR) further comprises a hinge region. A hinge region is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. In certain embodiments, the hinge region is located between the extracellular antigen-binding domain and the transmembrane domain. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the CAR can be used as the hinge region.

[0177] In certain embodiments, the hinge region has a length of about 5-100 amino acids, e.g., about 10-80 amino acids, about 20-80 amino acids, about 15-80 amino acids, about 15-75 amino acids, about 10-70 amino acids, about 20-70 amino acids, or about 20-50 amino acids. In certain embodiments, the hinge region has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, at least about 50 amino acids, at least about 55 amino acids, at least about 60 amino acids, at least about 65 amino acids, or at - 59 - NAI-5007600029vlleast about 70 amino acids, and / or up to about 30 amino acids, up to about 35 amino acids, up to about 40 amino acids, up to about 45 amino acids, up to about 50 amino acids, up to about 55 amino acids, up to about 60 amino acids, up to about 65 amino acids, up to about 70 amino acids, up to about 75 amino acids, or up to about 80 amino acids. In certain embodiments, the hinge region has a length of between about 10 amino acids and about 20 amino acids. In certain embodiments, the hinge region has a length of about 20 amino acids. In certain embodiments, the hinge region has a length of 18 amino acids. In certain embodiments, the hinge region has a length of between about 30 amino acids and about 40 amino acids. In certain embodiments, the hinge region has a length of about 40 amino acids. In certain embodiments, the hinge region has a length of 38 amino acids. In certain embodiments, the hinge region has a length of between about 40 amino acids and about 50 amino acids. In certain embodiments, the hinge region has a length of about 50 amino acids. In certain embodiments, the hinge region has a length of 48 amino acids. In certain embodiments, the hinge region has a length of between about 50 amino acids and about 80 amino acids. In certain embodiments, the hinge region has a length of about 70 amino acids.

[0178] In certain embodiments, the hinge region is derived from at least one naturally occurring protein. In certain embodiments, the hinge region comprises at least one extracellular domain of a naturally occurring protein or a fragment thereof. Non-limiting examples of the at least one naturally occurring proteins from which the hinge region can be derived from include CD8, a Toll-like receptor (including, but not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11), CD40, CD3ζ, a FcR, CD166, 0X40, CD28, 4-1BB, and ICOS. In certain embodiments, the hinge region and the transmembrane domain of the chimeric receptor (CAR) (e.g., any of those disclosed in Section 5.2.2 above) are derived from the same protein. In certain embodiments, the hinge region is derived from one naturally occurring protein. In certain embodiments, the hinge region comprises a native or modified extracellular domain of a naturally occurring protein or a fragment thereof. In certain embodiments, the hinge region and the transmembrane domain are derived from the same protein. In certain embodiments, the hinge region is derived from two or more naturally occurring proteins. In certain embodiments, the hinge region comprises native or modified extracellular domains of two or more naturally occurring proteins or fragments thereof. In certain embodiments, at least one of the two or more proteins from which the hinge region is derived is the same as the protein from which the transmembrane domain is derived.

[0179] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is - 60 - NAI-5007600029vlderived from CD8. The CD8 can be CD8a or CD8p. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of CD8 (CD8a or CD8P) or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from CD8a. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of CD8a or a fragment thereof.

[0180] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from mouse CD8a. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of mouse CD8a or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P01731 (SEQ ID NO: 25). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 28 to 196 of SEQ ID NO: 25. In certain embodiments, the consecutive portion has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, and / or up to about 30 amino acids, up to about 35 amino acids, up to about 40 amino acids, up to about 45 amino acids, or up to about 50 amino acids. In certain embodiments, the consecutive portion has a length of about 20 amino acids. In certain embodiments, the consecutive portion has a length of 18 amino acids. In certain embodiments, the consecutive portion has a length of about 40 amino acids. In certain embodiments, the consecutive portion has a length of 38 amino acids. In certain embodiments, the consecutive portion has a length of about 50 amino acids in length. In certain embodiments, the consecutive portion has a length of 48 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 28 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 149 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 159 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 145 to 196 of SEQ ID NO: 25. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 179 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain - 61 - NAI-5007600029vlembodiments, the hinge region of the chimeric receptor e.g., CAR) comprises amino acids 179 to 196 of SEQ ID NO: 25. SEQ ID NO: 25 is provided below (amino acids 149 to 196 italicized; amino acids 159 to 196 bolded; amino acids 179 to 196 underlined).MASPLTRFLSLNLLLLGESIILGSGEAKPQAPELRIFPKKMDAELGQKVDLVCEVLGS VSQGC S WLFQNS S SKLPQPTF VVYM AS SHNKITWDEKLNS SKLF S AMRDTNNK YVL TLNKFSKENEGYYFCSVISNSVMYFSSVVPVLQKFA ZZZ PFVVTPAPEHPTGTNSPVGPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSRKRVCKCPR PLVRQEGKPRPSEKIV (SEQ ID NO: 25)

[0181] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from human CD8a. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of human CD8a or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P01732 (SEQ ID NO: 26). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 22 to 182 of SEQ ID NO: 26. In certain embodiments, the consecutive portion has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, and / or up to about 30 amino acids, up to about 35 amino acids, up to about 40 amino acids, up to about 45 amino acids, or up to about 50 amino acids. In certain embodiments, the consecutive portion has a length of about 20 amino acids. In certain embodiments, the consecutive portion has a length of 18 amino acids. In certain embodiments, the consecutive portion has a length of about 40 amino acids. In certain embodiments, the consecutive portion has a length of 38 amino acids. In certain embodiments, the consecutive portion has a length of about 50 amino acids in length. In certain embodiments, the consecutive portion has a length of 48 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 22 to 182 of SEQ ID NO: 26 or a fragment thereof.

[0182] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from CD8p. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of CD8P or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises the amino acid sequence set forth in SEQ ID NO: 41, which is provided below. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises the amino acid sequence set - 62 - NAI-5007600029vlforth in SEQ ID NO: 42, which is provided below.CSLTT (SEQ ID NO: 41)TTAPTKKTTLKMKKKKQCPFPHPETQKGLTCSLTT (SEQ ID NO: 42)

[0183] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from a TLR. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of a TLR or a fragment thereof. Non-limiting examples of TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11. In certain embodiments, the TLR is TLR4. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from mouse TLR4. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of mouse TLR4 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: Q9QUK6 (SEQ ID NO: 27). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 26 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 100 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 150 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 200 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 250 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 300 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 350 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 400 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 450 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 500 to 638 of SEQ ID - 63 - NAI-5007600029vlNO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 550 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 600 to 638 of SEQ ID NO: 27. In certain embodiments, the consecutive portion has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids. In certain embodiments, the consecutive portion has a length up to about 100 amino acids, up to about 150 amino acids, up to about 200 amino acids, or up to about 250 amino acids.

[0184] In certain embodiments, the hinge region comprises a less structured portion of the extracellular domain of TLR4. In some embodiments, the less structured portion comprises a linear region of the TLR4 extracellular domain, such as a region of the TLR extracellular domain that does not fold into a non-linear three-dimensional structure (e.g., a loop or horseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the TLR4 extracellular domain does not comprise a leucine-rich repeat (LRR) motif. The LRRs generally contain a conserved 11 -residue segment with the highly conserved segment, LxxLxLxxNxL, in which " L" is Leu, He, Vai, or Phe and " N" is Asn, Thr, Ser, or Cys and "x" is any amino acid (see, e.g., Matsushima N, et al. BMC Genomics. 2007 May 21;8: 124; see also Martin EC, et al. Genes (Basel). 2020 Mar 8;11(3):286). In some embodiments, the less structured portion of the TLR4 extracellular domain is located between the C-terminal end of the last LRR motif and the N-terminal end of the transmembrane domain of TLR4. For example, in some embodiments, the less structured portion of the TLR4 extracellular domain is located between amino acids 582 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion of the TLR4 extracellular domain is located between the C-terminal end of the last LRR motif and the N-terminal end of the transmembrane domain of TLR4 and has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, or at least about 50 amino acids. In certain embodiments, the less structured portion is a portion of the extracellular domain of TLR4 that is proximate to the transmembrane domain of TLR4.- 64 - NAI-5007600029vl

[0185] In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the less structured portion comprises at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, or at least about 20 amino acids. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 54 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 50 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 40 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 30 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 20 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 54 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 50 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 40 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 30 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 20 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 54 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 50 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion - 65 - NAI-5007600029vlcomprises at least about 15 amino acids and up to about 40 amino acids of the extracellular domain of mouse TLR4. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 30 amino acids of the extracellular domain of mouse TLR4.

[0186] In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 600 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 610 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 619 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 621 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 626 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 628 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g, CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 600 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g, CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 610 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 619 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 621 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 626 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 628 to 638 of SEQ ID NO: 27. In certain embodiments, the consecutive portion has a length of at least about 4 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, or at least about 30 amino acids, and / or up to about 20 amino acids, up to about 25 amino acids, up to about 30 amino acids, or up to about 40 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 600 to 638 of SEQ ID NO: 27, or a portion thereof comprising at least about 4 amino acids, at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, or - 66 - NAI-5007600029vlat least about 35 consecutive amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 619 to 638 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 621 to 638 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 626 to 638 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 628 to 638 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 619 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 621 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 626 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 628 to 638 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below (amino acids 619 to 638 underlined).MMPPWLLARTLIMALFFSCLTPGSLNPCIEVVPNITYQCMDQKLSKVPDDIPSSTKNI DLSFNPLKILKSYSFSNFSELQWLDLSRCEIETIEDKAWHGLHHLSNLILTGNPIQSFSP GSFSGLTSLENLVAVETKLASLESFPIGQLITLKKLNVAHNFIHSCKLPAYFSNLTNLV HVDLSYNYIQTITVNDLQFLRENPQVNLSLDMSLNPIDFIQDQAFQGIKLHELTLRGN FNSSNIMKTCLQNLAGLHVHRLILGEFKDERNLEIFEPSIMEGLCDVTIDEFRLTYTND FSDDIVKFHCLANVSAMSLAGVSIKYLEDVPKHFKWQSLSIIRCQLKQFPTLDLPFLK SLTLTMNKGSISFKKVALPSLSYLDLSRNALSFSGCCSYSDLGTNSLRHLDLSFNGAII MSANFMGLEELQHLDFQHSTLKRVTEFSAFLSLEKLLYLDISYTNTKIDFDGIFLGLTS LNTLKMAGNSFKDNTLSNVFANTTNLTFLDLSKCQLEQISWGVFDTLHRLQLLNMS HNNLLFLDSSHYNQLYSLSTLDCSFNRIETSKGILQHFPKSLAFFNLTNNSVACICEHQ KFLQWVKEQKQFLVNVEQMTCATPVEMNTSLVLDFNNSTCYMYKTIISVSVVSVIV VSTVAFLIYHFYFHLILIAGCKKYSRGESIYDAFVIYSSQNEDWVRNELVKNLEEGVP RFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAQTWQFL SSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDG KASNPEQTAEEEQETATWT (SEQ ID NO: 27)

[0187] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from human TLR4. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of human TLR4 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR)- 67 - NAI-5007600029vlcomprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: 000206 (SEQ ID NO: 28). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acid 24 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 100 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 150 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 200 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 250 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 300 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 350 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 400 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 450 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 500 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 550 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 600 to 631 of SEQ ID NO: 28. In certain embodiments, the consecutive portion has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids. In certain embodiments, the consecutive portion has a length up to about 100 amino acids, up to about 150 amino acids, up to about 200 amino acids, or up to about 250 amino acids.- 68 - NAI-5007600029vl

[0188] In certain embodiments, the hinge region comprises a less structured portion of the extracellular domain of TLR4. In some embodiments, the less structured portion comprises a linear region of the TLR4 extracellular domain, such as a region of the TLR extracellular domain that does not fold into a non-linear three-dimensional structure (e.g., a loop or horseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the TLR4 extracellular domain does not comprise a leucine-rich repeat (LRR) motif. In some embodiments, the less structured portion of the TLR4 extracellular domain is located between the C-terminal end of the last LRR and the N-terminal end of the transmembrane domain of TLR4. For example, in some embodiments the less structured portion of the TLR4 extracellular domain is located between amino acids 585 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion of the TLR4 extracellular domain is located between the C-terminal end of the last LRR motif and the N-terminal end of the transmembrane domain of TLR4 and has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, or at least about 50 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 581 to 631 of SEQ ID NO: 28, or a portion thereof comprising at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, at least about 35 consecutive amino acids, or at least about 40 consecutive amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acids 592 to 631 of SEQ ID NO: 28, or a portion thereof at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, at least about 35 consecutive amino acids, or at least about 40 consecutive amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 566 to 631 of SEQ ID NO: 28, or a portion thereof comprising at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, at least about 35 consecutive amino acids, or at least about 40 consecutive amino acids. In certain embodiments, the less structured portion is a - 69 - NAI-5007600029vlportion of the extracellular domain of TLR4 that is proximate to the transmembrane domain ofTLR4.

[0189] In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the less structured portion comprises at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, or at least about 20 amino acids. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 54 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 50 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 40 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 30 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 20 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 54 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 50 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 40 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 30 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 20 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 54 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured - 70 - NAI-5007600029vlportion comprises at least about 15 amino acids and up to about 50 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 40 amino acids of the extracellular domain of human TLR4. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 30 amino acids of the extracellular domain of human TLR4.

[0190] In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 576 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 578 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 600 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 610 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 612 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 622 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 628 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 576 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 578 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 600 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 610 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 622 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 628 to 631 of SEQ ID NO: 28. In certain embodiments, the consecutive portion has a length of at least about 4 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, or at least about 30 amino acids, and / or up to about 20 amino acids, up to about 25 amino acids, up to about 30 amino acids, or up to about 40 amino acids. In certain embodiments, the consecutive portion has a length that is up to about 20 amino acids. In - 71 - NAI-5007600029vlcertain embodiments, the consecutive portion has a length of about 20 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 576 to 631 of SEQ ID NO: 28, or a portion thereof comprising at least about 4 consecutive amino acids, at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, at least about 35 consecutive amino acids, or at least about 40 consecutive amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 472 to 631 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 520 to 631 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 578 to 631 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 612 to 631 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 628 to 631 of SEQ ID NO: 28 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 472 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 520 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 578 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 612 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 622 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 628 to 631 of SEQ ID NO: 28. SEQ ID NO: 28 is provided below (amino acids 612 to 631 underlined).MMSASRLAGTLIPAMAFLSCVRPESWEPCVEVVPNITYQCMELNFYKIPDNLPFSTK NLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSL ALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLT NLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLR NNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLA YLD YYLDDIIDLFNCLTNVS SF SL VS VTIERVKDF SYNFGWQHLELVNCKFGQFPTLK- 72 - NAI-5007600029vlLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLS FNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGI FNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVL NMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFA CTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCOMNKTIIGVSVL SVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKN LEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQ TWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRK ALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 28)

[0191] Identification of the less structured portion of the TLR4 extracellular domain is with the skillset of a skilled artisan. Exemplary, but non-limiting examples for identifying a less structured portion include using structural prediction tools (e.g., AlphaFold (see Jumper, J et al. Highly accurate protein structure prediction with AlphaFold. Nature (2021); Fleming J. et al. AlphaFold Protein Structure Database and 3D-Beacons: New Data and Capabilities.Journal of Molecular Biology, (2025); alphafold.ebi.ac.uk) or other computational structureprediction algorithms), crystal structures - analysis tool, and sequence-analysis tools. In some embodiments, the less structured portion of the TLR4 extracellular domain comprises a disordered region predicted by one or more computational structure-prediction tools. In some embodiments, the less structured portion of the TLR4 extracellular domain corresponds to the residues that are unresolved or poorly resolved in crystal structures of the TLR4 extracellular domain. In some embodiments, the less structured portion of the TLR4 extracellular domain is delineated by the absence of LRR motifs and the absence of hydrophobicity characteristic of a transmembrane domain. In certain embodiments, the less structured domain acts as a dynamic or flexible connection, allowing extracellular domain to undergo relative movement with respect to the membrane-anchored transmembrane domain, facilitating signal transduction upon ligand binding.

[0192] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from 0X40. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from mouse 0X40. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of mouse 0X40 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt- 73 - NAI-5007600029vlReference No: P47741 (SEQ ID NO: 29). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 20 to 211 of SEQ ID NO: 29. In certain embodiments, the consecutive portion has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, and / or up to about 30 amino acids, up to about 40 amino acids, up to about 50 amino acids, up to about 60 amino acids, up to about 70 amino acids, up to about 80 amino acids, up to about 90 amino acids, or up to about 100 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 50 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 45 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 40 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 35 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 30 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 25 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 20 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 15 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 10 amino acids. In certain embodiments, the consecutive portion has a length of between about 10 amino acids and about 60 amino acids. In certain embodiments, the consecutive portion has a length of between about 10 amino acids and about 50 amino acids. In certain embodiments, the consecutive portion has a length of between about 10 amino acids and about 40 amino acids. In certain embodiments, the consecutive portion has a length of between about 20 amino acids and about 40 amino acids. In certain embodiments, the consecutive portion has a length of about 40 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 20 to 211 of SEQ ID NO: 29, or a portion thereof comprising at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, at least about 35 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, and / or up to about 30 consecutive amino acids, up to about 40 consecutive amino acids, up to about 50 consecutive amino acids, up to about 60 consecutive amino acids, up to about 70 consecutive amino acids,- 74 - NAI-5007600029vlup to about 80 consecutive amino acids, up to about 90 consecutive amino acids, or up to about 100 consecutive amino acids.

[0193] In certain embodiments, the hinge region comprises a less structured portion of the extracellular domain of 0X40. In some embodiments, the less structured portion comprises a linear region of the 0X40 extracellular domain, such as a region of the 0X40 extracellular domain that does not fold into a non-linear three-dimensional structure (e.g., a loop or horseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the 0X40 extracellular domain does not comprise a leucine-rich repeat (LRR) motif. In some embodiments, the less structured portion of the 0X40 extracellular domain is located between the C-terminal end of the last LRR and the N-terminal end of the transmembrane domain of 0X40. In certain embodiments, the less structured portion is a portion of the extracellular domain of 0X40 that is proximate to the transmembrane domain of 0X40.

[0194] In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 70 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 60 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 50 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 40 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 30 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 20 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 60 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 50 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 40 amino - 75 - NAI-5007600029vlacids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 30 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 20 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 60 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 50 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 40 amino acids of the extracellular domain of mouse 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 30 amino acids of the extracellular domain of mouse 0X40.

[0195] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 165 to 211 of SEQ ID NO: 29. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 166 to 211 of SEQ ID NO: 29. SEQ ID NO: 29 is provided below (amino acids 165 to 211 underlined, and amino acids 166 to 211 italicized).MYVWVQQPTALLLLALTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHT RDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQP RQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCED7? SLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLA VLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI (SEQ ID NO: 29)

[0196] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from human 0X40. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of human 0X40 or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P43489 (SEQ ID NO: 30). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 29 to 214 of SEQ ID NO: 30. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 157 to 214 of SEQ ID NO: 30. In certain embodiments, the consecutive portion has a length of at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino - 76 - NAI-5007600029vlacids, at least about 50 amino acids, or at least about 60 amino acids, and / or up to about 30 amino acids, up to about 40 amino acids, up to about 50 amino acids, up to about 60 amino acids, or up to about 70 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 50 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 45 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 40 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 35 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 30 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 25 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 20 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 15 amino acids. In certain embodiments, the consecutive portion has a length of between about 5 and about 10 amino acids. In certain embodiments, the consecutive portion has a length of between about 10 amino acids and about 60 amino acids. In certain embodiments, the consecutive portion has a length of between about 10 amino acids and about 50 amino acids. In certain embodiments, the consecutive portion has a length of between about 10 amino acids and about 40 amino acids. In certain embodiments, the consecutive portion has a length of between about 20 amino acids and about 40 amino acids. In certain embodiments, the consecutive portion has a length of about 40 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 29 to 214 of SEQ ID NO: 30, or a portion thereof comprising at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, at least about 35 consecutive amino acids, at least about 40 consecutive amino acids, at least about 50 consecutive amino acids, at least about 60 consecutive amino acids, and / or up to about 30 consecutive amino acids, up to about 40 consecutive amino acids, up to about 50 consecutive amino acids, up to about 60 consecutive amino acids, up to about 70 consecutive amino acids, up to about 80 consecutive amino acids, up to about 90 consecutive amino acids, or up to about 100 consecutive amino acids.

[0197] In certain embodiments, the hinge region comprises a less structured portion of the extracellular domain of 0X40. In some embodiments, the less structured portion comprises a linear region of the 0X40 extracellular domain, such as a region of the 0X40 extracellular domain that does not fold into a non-linear three-dimensional structure (e.g., a loop or - 77 - NAI-5007600029vlhorseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the 0X40 extracellular domain does not comprise a leucine-rich repeat (LRR) motif. In some embodiments, the less structured portion of the 0X40 extracellular domain is located between the C-terminal end of the last LRR and the N-terminal end of the transmembrane domain of 0X40. In certain embodiments, the less structured portion is a portion of the extracellular domain of 0X40 that is proximate to the transmembrane domain of 0X40.

[0198] In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 70 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 60 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 50 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 40 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 30 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 5 amino acids and up to about 20 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 60 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 50 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 40 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 30 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 10 amino acids and up to about 20 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 60 amino acids of the extracellular domain of - 78 - NAI-5007600029vlhuman 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 50 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 40 amino acids of the extracellular domain of human 0X40. In certain embodiments, the less structured portion comprises at least about 15 amino acids and up to about 30 amino acids of the extracellular domain of human 0X40.

[0199] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 167 to 214 of SEQ ID NO: 30. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 168 to 214 of SEQ ID NO: 30. SEQ ID NO: 30 is provided below (amino acids 167 to 214 underlined, and amino acids 168 to 214 italicized).MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSR CSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRA GTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICED RDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLG LGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 30)

[0200] Identification of the less structured portion of the 0X40 extracellular domain is with the skillset of a skilled artisan. Exemplary, but nonlimiting examples for identifying a less structured portion includes using structural prediction tools (e.g., AlphaFold (see Jumper, J et al. Highly accurate protein structure prediction with AlphaFold. Nature (2021); Fleming J. et al. AlphaFold Protein Structure Database and 3D-Beacons: New Data and Capabilities.Journal of Molecular Biology, (2025); alphafold.ebi.ac.uk) or other computational structureprediction algorithms), crystal structures - analysis tool, and sequence-analysis tools. In some embodiments, the less structured portion of the 0X40 extracellular domain comprises a disordered region predicted by one or more computational structure-prediction tools. In some embodiments, the less structured portion of the 0X40 extracellular domain corresponds to the residues that are unresolved or poorly resolved in crystal structures of the 0X40 extracellular domain. In some embodiments, the less structured portion of the 0X40 extracellular domain is delineated by the absence of LRR motifs and the absence of hydrophobicity characteristic of a transmembrane domain. In certain embodiments, the less structured domain acts as a dynamic or flexible connection, allowing extracellular domain to undergo relative movement with respect to the membrane-anchored transmembrane domain, facilitating signal transduction upon ligand binding.- 79 - NAI-5007600029vl

[0201] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises a modified extracellular domain of a naturally occurring protein. Non-limiting examples of modifications include insertions, deletions, and substitutions. In certain embodiments, the modification enriches the N-linked glycosylation content of the original amino acid sequence. In certain embodiments, the modification results in a hinge region comprising at least one N-linked glycosylation consensus sequence. A “N-linked glycosylation consensus sequence” is a sequence that relates to N-linked glycosylation. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the X is Gly. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Gly-Thr (NGT). In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Ser, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Ser (NFS). In certain embodiments, the X is Ala. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Ala-Ser (NAS). In certain embodiments, the X is Vai. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Val-Ser (NVS). In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Cys, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Cys (NFC). In certain embodiments, the modification results in a hinge region comprising at least one (1) and no more than five (5) N-linked glycosylation consensus sequences. In certain embodiments, the modification comprises an insertion of one or more N-linked glycosylation consensus sequences.

[0202] In certain embodiments, the modification comprises a substitution of one or more native (or wild-type) residues to one or more N-linked glycosylation consensus sequences. The embodiment encompasses that an extracellular domain, such as a hinge region, with an enriched N-linked glycosylation content can improve the chimeric receptor expression, target binding, and function on the cell surface of a dendritic cell.

[0203] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises a modified extracellular domain of CD8a or a fragment thereof. In certain embodiments, the modified extracellular domain of CD8a comprises amino acids 159 to 196 of SEQ ID NO: 25 and one or more (e.g., between 1 and 5) N-linked glycosylation consensus sequences. In certain embodiments, the modified extracellular domain of CD8a comprises - 80 - NAI-5007600029vlamino acids 159 to 196 of SEQ ID NO: 25 and one N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the modified extracellular domain of CD8a comprises a mutant variant of amino acids 159 to 196 of SEQ ID NO: 25, wherein at least one native amino acid residue is substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the modified extracellular domain of CD8a comprises a mutant variant of amino acids 159 to 196 of SEQ ID NO: 25, wherein three native amino acid residues are substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the modified extracellular domain of CD8a comprises the amino acid sequence set forth in SEQ ID NO: 37. SEQ ID NO: 37 is provided below (NFT glycosylation site underlined).RTPSPVHPTGTSQPQNFTDCRPRGSVKGTGLDFACDIY (SEQ ID NO:37)

[0204] In certain embodiments, the modified extracellular domain of CD8a comprises amino acids 149 to 196 of SEQ ID NO: 25 and one or more (e.g., between 1 and 5) N-linked glycosylation consensus sequences. In certain embodiments, the modified extracellular domain of CD8a comprises amino acids 149 to 196 of SEQ ID NO: 25 and one N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the modified extracellular domain of CD8a comprises a mutant variant of amino acids 149 to 196 of SEQ ID NO: 25, wherein at least one native amino acid residue is substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the modified extracellular domain of CD8a comprises a mutant variant of amino acids 149 to 196 of SEQ ID NO: 25, wherein three native amino acid residues are substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the modified extracellular domain of CD8a comprises the amino acid sequence set forth in SEQ ID NO: 38. SEQ ID NO: 38 is provided below (NFT glycosylation site - 81 - NAI-5007600029vlunderlined).VNSTTTKPVLRTPSPVHPTGTSQPQNFTDCRPRGSVKGTGLDFACDIY (SEQ ID NO: 38)

[0205] In certain embodiments, the modified extracellular domain of CD8a comprises amino acids 179 to 196 of SEQ ID NO: 25 and one or more (e.g., between 1 and 5) N-linked glycosylation consensus sequences. In certain embodiments, the modified extracellular domain of CD8a comprises amino acids 179 to 196 of SEQ ID NO: 25 and one N-linked glycosylation consensus sequence. In certain embodiments, the modified extracellular domain of CD8a comprises a mutant variant of amino acids 179 to 196 of SEQ ID NO: 25, wherein at least one native amino acid residue is substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the modified extracellular domain of CD8a comprises a mutant variant of amino acids 179 to 196 of SEQ ID NO: 25, wherein three amino acid residues are substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT).

[0206] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises a modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the modified extracellular domain of 0X40 comprises amino acids 166 to 211 of SEQ ID NO: 29 and one or more (e.g., between 1 and 5) N-linked glycosylation consensus sequences. In certain embodiments, the modified extracellular domain of 0X40 comprises amino acids 166 to 211 of SEQ ID NO: 29 and one N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the modified extracellular domain of 0X40 comprises the amino acid sequence set forth in SEQ ID NO: 39. SEQ ID NO: 39 is provided below (NFT glycosylation site underlined).EDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPNFTSPPTLVTPEGP (SEQ ID NO: 39)

[0207] In certain embodiments, the modified extracellular domain of 0X40 comprises a mutant variant of amino acids 166 to 211 of SEQ ID NO: 29, wherein at least one native amino acid residue is substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the modified extracellular domain of 0X40 comprises a mutant variant - 82 - NAI-5007600029vlof amino acids 166 to 211 of SEQ ID NO: 29, wherein three native amino acid residues are substituted with aN-linked glycosylation consensus sequence.

[0208] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises a modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the modified extracellular domain of 0X40 comprises amino acids 167 to 214 of SEQ ID NO: 30 and one or more (e.g., between 1 and 5) N-linked glycosylation consensus sequences. In certain embodiments, the modified extracellular domain of 0X40 comprises amino acids 167 to 214 of SEQ ID NO: 30 and one N-linked glycosylation consensus sequence. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-X-Thr, wherein X is any amino acid except Pro. In certain embodiments, the X is Phe. In certain embodiments, the N-linked glycosylation consensus sequence is Asn-Phe-Thr (NFT). In certain embodiments, the modified extracellular domain of 0X40 comprises the amino acid sequence set forth in SEQ ID NO: 65. SEQ ID NO: 65 is provided below (NFT glycosylation site underlined).EDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTNFTRPVEVPGGRA (SEQ ID NO: 65)

[0209] In certain embodiments, the modified extracellular domain of 0X40 comprises the amino acid sequence set forth in SEQ ID NO: 84. SEQ ID NO: 84 is provided below (NFS glycosylation site underlined).EDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPNFSTRPVEVPGGRA (SEQ ID NO: 84)

[0210] In certain embodiments, the modified extracellular domain of 0X40 comprises a mutant variant of amino acids 167 to 214 of SEQ ID NO: 30, wherein at least one native amino acid residue is substituted with a N-linked glycosylation consensus sequence. In certain embodiments, the modified extracellular domain of 0X40 comprises a mutant variant of amino acids 167 to 214 of SEQ ID NO: 30, wherein three native amino acid residues are substituted with aN-linked glycosylation consensus sequence. The embodiment encompasses that for a CAR whose intracellular domain is derived from a TLR (e.g., TLR4) (e.g., the intracellular domain of the CAR comprises an intracellular domain of a TLR (e.g., TLR4) or a fragment thereof), the structure of the hinge region can contribute to the CAR expression on the cell surface and the binding of the CAR to the target antigen. This is likely due to the complex structure of the TLR.

[0211] In certain embodiments, the hinge region comprises a less structured portion of the extracellular domain of a TLR (c.g, TLR4). In some embodiments, the less structured portion - 83 - NAI-5007600029vlcomprises a linear region of the TLR4 extracellular domain, such as a region of the TLR extracellular domain that does not fold into a non-linear three-dimensional structure (e.g., a loop or horseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the TLR4 extracellular domain does not comprise a leucine-rich repeat (LRR) motif. In some embodiments, the less structured portion of the TLR4 extracellular domain is located between the C-terminal end of the last LRR and the N-terminal end of the transmembrane domain of TLR4. In certain embodiments, the less structured portion is a portion of the extracellular domain of the TLR that is proximate to the transmembrane domain of the TLR e.g., TLR4). In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 600 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 610 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 619 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 621 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 626 to 638 of SEQ ID NO: 27. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 628 to 638 of SEQ ID NO: 27. In certain embodiments, the hinge region further comprises a less structured portion of an extracellular domain of a second protein that comprises a relatively unstructured extracellular domain. Non-limiting examples of the second protein include 0X40, CD8, CD40, CD28, and IgG. In some embodiments, the second protein is selected from the group consisting of 0X40, CD8, CD40, CD28, and IgG. In some embodiments, the less structured portion of the second protein comprises a linear region of the extracellular domain of the second protein, such as a portion of an extracellular domain of a second protein that does not fold into a nonlinear three-dimensional structure (e.g., a loop or horseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the second protein does not comprise a leucine-rich repeat (LRR) motif. In some embodiments, the less structured portion of the second protein extracellular domain - 84 - NAI-5007600029vlis located between the C-terminal end of the last LRR and the N-terminal end of the transmembrane domain of the second protein. In certain embodiments, the less structured portion is a portion of the extracellular domain of the second protein that is proximate to the transmembrane domain of the second protein. In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the second protein is 0X40. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises an extracellular domain of TLR4 or a fragment thereof (e.g., any of the extracellular domain of TLR4 or fragment thereof disclosed herein), and an extracellular domain of 0X40 or a fragment thereof disclosed herein (e.g., any of the extracellular domain of 0X40 or fragment thereof disclosed herein). In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 619 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 621 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 626 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 628 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39.

[0212] In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 576 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 578 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 600 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and is within amino acids 610 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 612 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 622 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and within amino acids 628 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is - 85 - NAI-5007600029vlderived from TLR4 and comprises or consists of amino acids 578 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 612 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 622 to 631 of SEQ ID NO: 28. In certain embodiments, the less structured portion is derived from TLR4 and comprises or consists of amino acids 628 to 631 of SEQ ID NO: 28. In certain embodiments, the hinge region further comprises a less structured portion of an extracellular domain of a second protein that comprises a relatively unstructured extracellular domain. Non-limiting examples of the second protein include 0X40, CD8, CD40, CD28, and IgG. In some embodiments, the second protein is selected from the group consisting of 0X40, CD8, CD40, CD28, and IgG. In some embodiments, the less structured portion of the second protein comprises a linear region of the extracellular domain of the second protein, such as a portion of an extracellular domain of a second protein that does not fold into a non-linear three-dimensional structure (e.g., a loop or horseshoe-shaped structure) in its native, inactive / resting state under physiological conditions. In some embodiments, the less structured portion of the second protein does not comprise a leucine-rich repeat (LRR) motif. In some embodiments, the less structured portion of the second protein extracellular domain is located between the C-terminal end of the last LRR and the N-terminal end of the transmembrane domain of the second protein. In certain embodiments, the less structured portion is a portion of the extracellular domain of the second protein that is proximate to the transmembrane domain of the second protein. In some embodiments, the less structured portion comprises a sequence that includes at least one cysteine residue capable of forming a disulfide bond that contributes to stability and function of the CAR. In some embodiments, the less structured portion does not comprise residues predicted to form a-helical secondary structure. In some embodiments, the less structured portion excludes structured regions that contain a-helices. In certain embodiments, the second protein is 0X40. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises an extracellular domain of TLR4 or a fragment thereof (e.g., any of the extracellular domain of TLR4 or fragment thereof disclosed herein), and an extracellular domain of 0X40 or a fragment thereof disclosed herein (e.g., any of the extracellular domain of 0X40 or fragment thereof disclosed herein). In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 578 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises - 86 - NAI-5007600029vlamino acids 612 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 622 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 628 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 578 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 612 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 622 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the hinge region of the CAR whose intracellular domain is derived from TLR4 comprises amino acids 628 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84.

[0213] Identification of the less structured portion of the second protein extracellular domain is with the skillset of a skilled artisan. Exemplary, but non-limiting examples for identifying a less structured portion include using structural prediction tools (e.g., AlphaFold (see Jumper, J et al. Highly accurate protein structure prediction with AlphaFold. Nature (2021); Fleming J. et al. AlphaFold Protein Structure Database and 3D-Beacons: New Data and Capabilities. Journal of Molecular Biology, (2025); alphafold.ebi.ac.uk) or other computational structure-prediction algorithms), crystal structures - analysis tool, and sequence-analysis tools. In some embodiments, the less structured portion of the second protein extracellular domain comprises a disordered region predicted by one or more computational structure-prediction tools. In some embodiments, the less structured portion of the second protein extracellular domain corresponds to the residues that are unresolved or poorly resolved in crystal structures of the second protein extracellular domain. In some embodiments, the less structured portion of the second protein extracellular domain is delineated by the absence of LRR motifs and the absence of hydrophobicity characteristic of a transmembrane domain. In certain embodiments, the less structured domain acts as a dynamic or flexible connection, allowing extracellular domain to undergo relative movement with respect to the membrane-anchored transmembrane domain, facilitating signal transduction upon ligand binding.

[0214] In certain embodiments, the hinge region of the CAR is derived from a FcR. In - 87 - NAI-5007600029vlcertain embodiments, the hinge region of the CAR comprises an extracellular domain of FcR or a fragment thereof. In certain embodiments, the hinge region of the CAR is derived from a Fc-gamma receptor (FcyR). In certain embodiments, the hinge region of the CAR comprises an extracellular domain of FcyR or a fragment thereof. In certain embodiments, the hinge region of the CAR is derived from FCERγ. In certain embodiments, the hinge region of the CAR comprises an extracellular domain of FCERγ or a fragment thereof. In certain embodiments, the hinge region of the CAR is derived from mouse FCERγ. In certain embodiments, the hinge region of the CAR comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P20491 (SEQ ID NO: 35). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 19 to 23 of SEQ ID NO: 35. In certain embodiments, the consecutive portion has a length of at least about 2 amino acids, and / or up to about 5 amino acids. In certain embodiments, the consecutive portion has a length of about 5 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 19 to 23 of SEQ ID NO: 35. SEQ ID NO: 35 is provided below (amino acids 19 to 23 underlined).MISAVILFLLLLVEQAAALGEPQLCYILDAVLFLYGIVLTLLYCRLKIQVRKAAIASRE KADAVYTGLNTRSQETYETLKHEKPPQ (SEQ ID NO: 35)

[0215] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from human FCERγ. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an extracellular domain of human FCERγ or a fragment thereof. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of the amino acid sequence having a UniProt Reference No: P30273 (SEQ ID NO: 36). In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises an amino acid sequence that is a consecutive portion of amino acids 19 to 23 of SEQ ID NO: 36. In certain embodiments, the consecutive portion has a length of at least about 2 amino acids, and / or up to about 5 amino acids. In certain embodiments, the consecutive portion has a length of about 5 amino acids. In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) comprises amino acids 19 to 23 of SEQ ID NO: 36. SEQ ID NO: 36 is provided below (amino acids 19 to 23 underlined).MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYE KSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 36)- 88 - NAI-5007600029vl5.2.5. Signal Peptide

[0216] The presently disclosed chimeric receptor (e.g., CAR) can further comprise a signal peptide (also known as a signal sequence or leader). In certain embodiments, the signal peptide is covalently connected to the N-terminus of the extracellular antigen-binding domain. In general, signal peptides are peptide sequences that target a polypeptide to the desired site in a cell, or the plasma membrane. A signal peptide can be a signal sequence of a naturally occurring protein (e.g., CD8) or a synthetic, non-naturally occurring signal sequence.

[0217] In certain embodiments, the signal peptide is derived from IgH. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. SEQ ID NO: 40 is provided below.MEFGLSWVFLVALFRGVQC (SEQ ID NO: 40)5.2.6. Exemplary CARs5,2.6.1 _ Exemplary nanobody-based CARs (VHH CAR)

[0218] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises a VHH domain (any of the VHH domains disclosed in Section 5.2.1.1 above), a transmembrane domain (any of the transmembrane domains disclosed in Section 5.2.2 above), and an intracellular domain (any of the intracellular domains disclosed in Section 5.2.3 above). In certain embodiments, the VHH CAR further comprises a hinge region (any of the hinge regions disclosed in Section 5.2.4 above).

[0219] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 179 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR- 89 - NAI-5007600029vlcomprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0220] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 159 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0221] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and - 90 - NAI-5007600029vla CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “aB7H3 CD40 CAR” in Section 6. Examples.

[0222] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “aB7H3 CD40(m)” in Section 6. Example.

[0223] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-PSMA VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain - 91 - NAI-5007600029vlderived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-PSMA VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the anti-PSMA VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the anti-PSMA VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0224] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-PSMA VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-PSMA VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the anti-PSMA VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the anti-PSMA VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises - 92 - NAI-5007600029vlthe amino acid sequence set forth in SEQ ID NO: 40.

[0225] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “aCD19 CD40(m)” in Section 6. Example.

[0226] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR- 93 - NAI-5007600029vlcomprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “CD 19 CD40 CAR” in Section 6 Examples.

[0227] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD20 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-CD20 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the anti-CD20 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the anti-CD20 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0228] In certain embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD20 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-CD20 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the anti-CD20 VHH domain comprises a CDR1 comprising the amino acid sequence set forth - 94 - NAI-5007600029vlin SEQ ID NO: 13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the anti-CD20 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0229] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-EGFR VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-EGFR VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the anti-EGFR VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the anti-EGFR VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0230] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-EGFR VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular - 95 - NAI-5007600029vldomain derived from CD40 (e.g., an intracellular domain of CD40). In certain embodiments, the anti-EGFR VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the anti-EGFR VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the anti-EGFR VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0231] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD3ζ (e.g., an intracellular domain of CD3Q. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 33. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.- 96 - NAI-5007600029vl

[0232] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from CD3ζ (e.g., an intracellular domain of CD3Q. In certain embodiments, the antiCD 19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 33. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0233] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from FcsRy (e.g., an intracellular domain of FcsRy). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 35. In - 97 - NAI-5007600029vlcertain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0234] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from CD8a (e.g., a modified extracellular domain of CD8a), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and an intracellular domain derived from FcsRy (e.g., an intracellular domain of FcsRy). In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 35. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0235] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from 0X40 (e.g., a modified extracellular domain of 0X40), a transmembrane domain derived from 0X40 (e.g., a transmembrane domain of 0X40), and an intracellular domain derived from 0X40 (e.g., an intracellular domain of 0X40). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in - 98 - NAI-5007600029vlSEQ ID NO: 4. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 212 to 236 of SEQ ID NO: 29. In certain embodiments, the intracellular domain of the CAR comprises amino acids 237 to 272 of SEQ ID NO: 29. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0236] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from 0X40 (e.g., a modified extracellular domain of 0X40), a transmembrane domain derived from 0X40 (e.g, a transmembrane domain of 0X40), and an intracellular domain derived from 0X40 (e.g, an intracellular domain of 0X40). In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 212 to 236 of SEQ ID NO: 29. In certain embodiments, the intracellular domain of the CAR comprises amino acids 237 to 272 of SEQ ID NO: 29. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0237] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from TLR4 (e.g., an extracellular domain of TLR4 or a fragment thereof), a transmembrane domain derived from TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the - 99 - NAI-5007600029vlanti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 619 to 638 of SEQ ID NO: 27. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0238] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from human TLR4 (e.g., an extracellular domain of TLR4 or a fragment thereof), a transmembrane domain derived from human TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from human TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 578 to 631 of SEQ ID NO: 28. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0239] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from human TLR4 (e.g., an extracellular domain of TLR4 or a fragment - 100 - NAI-5007600029vlthereof), a transmembrane domain derived from human TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from human TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 520 to 631 of SEQ ID NO: 28. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0240] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from human TLR4 (e.g., an extracellular domain of TLR4 or a fragment thereof), a transmembrane domain derived from human TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from human TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 472 to 631 of SEQ ID NO: 28. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the - 101 - NAI-5007600029vlsignal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0241] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from TLR4 and 0X40 e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 619 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “aB7 TLR” in Section 6. Example.

[0242] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from TLR4 and 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the - 102 - NAI-5007600029vlhinge region comprises amino acids 621 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0243] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from TLR4 and 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 626 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0244] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from TLR4 and 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1,- 103 - NAI-5007600029vla CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 628 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0245] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from human TLR4 and human 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from human TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from human TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 578 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.- 104 - NAI-5007600029vl

[0246] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from human TLR4 and human 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from human TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from human TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 622 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.In some embodiments, provided herein is a VHH CAR comprising an extracellular antigenbinding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from human TLR4 and human 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from human TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from human TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 628 to 631 of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the transmembrane - 105 - NAI-5007600029vldomain of the CAR comprises amino acids 632 to 652 of SEQ ID NO: 28. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0247] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from TLR4 and 0X40 (e.g., an extracellular domain of TLR4 and a modified extracellular domain of 0X40), a transmembrane domain derived from TLR4 (e.g., a transmembrane domain of TLR4), and an intracellular domain derived from TLR4 (e.g., an intracellular domain of TLR4). In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises amino acids 619 to 638 of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27. In certain embodiments, the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0248] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-beta amyloid VHH domain, a hinge region derived from FcsRy (e.g., an extracellular domain of FcsRy or a fragment thereof), a transmembrane domain derived from FcsRy (e.g., a transmembrane domain of FcsRy), and an intracellular domain derived from FcsRy (e.g., an intracellular domain of FcsRy). In certain embodiments, the anti -beta amyloid VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, the anti-beta amyloid VHH domain comprises a CDR1 comprising the - 106 - NAI-5007600029vlamino acid sequence set forth in SEQ ID NO: 61, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the anti-beta amyloid VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, the hinge region comprises amino acids 19 to 23 of SEQ ID NO: 35. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 24 to 44 of SEQ ID NO: 35. In certain embodiments, the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 35. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0249] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-CD19 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and does not comprise an intracellular domain. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-CD19 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the anti-CD19 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0250] In some embodiments, provided herein is a VHH CAR comprising an extracellular antigen-binding domain that comprises an anti-B7H3 VHH domain, a hinge region derived from CD8a (e.g., an extracellular domain of CD8a or a fragment thereof), a transmembrane domain derived from CD8a (e.g., a transmembrane domain of CD8a), and does not comprise an intracellular domain. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1, a CDR2, and a CDR3 of a VHH domain comprising the amino acid sequence set forth - 107 - NAI-5007600029vlin SEQ ID NO: 4. In certain embodiments, the anti-B7H3 VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-B7H3 VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the hinge region comprises amino acids 149 to 196 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25. In certain embodiments, the VHH CAR further comprises a signal peptide covalently connected to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.[0...

Claims

WHAT IS CLAIMED:

1. A dendritic cell or a precursor thereof comprising:(I) a) a chimeric antigen receptor (CAR) that binds to a target antigen, and b) a membranebound chimeric interferon (IFN) molecule, or(II) a) a CAR that binds to a target antigen, and b) a membrane-bound chimeric interleukin-2 (IL-2) family cytokine molecule.

2. The dendritic cell or precursor thereof of claim 1, wherein the dendritic cell or a precursor thereof comprises: a) a CAR that binds to a target antigen, and b) a membranebound chimeric IFN molecule.

3. The dendritic cell or precursor thereof of claim 1 or 2, wherein the IFN is a Type I IFN.

4. The dendritic cell or precursor thereof of any one of claims 1-3, wherein the IFN is IFN-a or IFN-p.

5. The dendritic cell or precursor thereof of claim 1 or 2, wherein the IFN is IFN-y.

6. The dendritic cell or precursor thereof of any one of claims 1-5, wherein the membrane-bound chimeric IFN molecule comprises i) an IFN domain.

7. The dendritic cell or precursor thereof of claim 6, wherein the IFN domain comprises an active form of the IFN.

8. The dendritic cell or precursor thereof of claim 7, wherein the active form of the IFN comprises a native active form of the IFN or a variant thereof.

9. The dendritic cell or precursor thereof of claim 7 or claim 8, wherein the active form of the IFN comprises a native active form of the IFN.

10. The dendritic cell or precursor thereof of claim 8 or claim 9, wherein the IFN is IFN-y, and the native active form of IFN-y comprises the amino acid sequence set forth in SEQ ID NO: 47 or a fragment thereof.

11. The dendritic cell or precursor thereof of claim 8 or claim 9, wherein the IFN is IFN-- 191 - NAI-5007600029vly, and the native active form of IFN-y comprises the amino acid sequence set forth in SEQ ID NO: 48 or a fragment thereof.

12. The dendritic cell or precursor thereof of claim 8 or claim 9, wherein the IFN is IFN-a.

13. The dendritic cell or precursor thereof of claim 12, wherein the IFN is IFN-al.

14. The dendritic cell or precursor thereof of any one of claims 8, 9, 12 and 13, wherein the IFN is IFN-al, and the native active form of IFN-al comprises the amino acid sequence set forth in SEQ ID NO: 49 or a fragment thereof.

15. The dendritic cell or precursor thereof of any one of claims 8, 9, 12 and 13, wherein the IFN is IFN-al, and the native active form of IFN-al comprises the amino acid sequence set forth in SEQ ID NO: 50 or a fragment thereof.

16. The dendritic cell or precursor thereof of claim 8 or claim 9, wherein the IFN is IFN-P, and the native active form of IFN-P comprises the amino acid sequence set forth in SEQ ID NO: 51 or a fragment thereof.

17. The dendritic cell or precursor thereof of claim 8 or claim 9, wherein the IFN is IFN-P, and the native active form of IFN-P comprises the amino acid sequence set forth in SEQ ID NO: 52 or a fragment thereof.

18. The dendritic cell or precursor thereof of any one of claims 6-17, wherein the membrane-bound IFN molecule further comprises ii) an anchor domain that anchors the IFN domain to the membrane of the dendritic cell or precursor thereof.

19. The dendritic cell or precursor thereof of claim 18, wherein the anchor domain comprises a GPI-anchored polypeptide or a transmembrane domain derived from CD8, a Toll-like receptor (TLR), a FcR, CD166, or 0X40.

20. The dendritic cell or precursor thereof of claim 19, wherein the GPI-anchored polypeptide is derived from CD59, CD55, CD48, CD24, CD14, Thy-1, or Ly-6.

21. The dendritic cell or precursor thereof of claim 19 or claim 20, wherein the GPI-anchored polypeptide is derived from CD59.- 192 - NAI-5007600029vl22. The dendritic cell or precursor thereof of claim 21, wherein the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 53 or a fragment thereof.

23. The dendritic cell or precursor thereof of claim 19 or claim 20, wherein the GPI-anchored polypeptide is derived from CD55.

24. The dendritic cell or precursor thereof of claim 23, wherein the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 54 or a fragment thereof.

25. The dendritic cell or precursor thereof of any one of claims 6-24, wherein the membrane-bound chimeric IFN molecule further comprises iii) a hinge domain.

26. The dendritic cell or precursor thereof of claim 25, wherein the hinge domain is positioned between i) the IFN domain and ii) the anchor domain.

27. The dendritic cell or precursor thereof of claim 25 or claim 26, wherein the hinge domain has a length of at least about 5 amino acids and / or up to about 30 amino acids.

28. The dendritic cell or precursor thereof of any one of claims 25-27, wherein the hinge domain has a length of between about 5 and about 30 amino acids.

29. The dendritic cell or precursor thereof of any one of claims 25-28, wherein the hinge domain has a length of about 30 amino acids.

30. The dendritic cell or precursor thereof of any one of claims 25-29, wherein the hinge domain is derived from CD8, CD55, a TLR, CD40, CD3ζ, a FcR, CD166, IgGl or IgG4.

31. The dendritic cell or precursor thereof of any one of claims 25-30, wherein the hinge domain is derived from CD8.

32. The dendritic cell or precursor thereof of any one of claims 25-31, wherein the hinge domain comprises a native or modified extracellular domain of CD8a or a fragment thereof.

33. The dendritic cell or precursor thereof of claim 32, wherein the hinge domain comprises amino acids 149 to 176 of SEQ ID NO: 26 or a fragment thereof.

34. The dendritic cell or precursor thereof of any one of claims 25-30, wherein the hinge - 193 - NAI-5007600029vldomain is derived from CD55.

35. The dendritic cell or precursor thereof of any one of claims 25-30, and 34, wherein the hinge domain comprises a native or modified extracellular domain of CD55 or a fragment thereof.

36. The dendritic cell or precursor thereof of claim 35, wherein the hinge domain comprises amino acids 323 to 340 of SEQ ID NO: 55 or a fragment thereof.

37. The dendritic cell or precursor thereof of any one of claims 6-36, wherein the membrane-bound chimeric IFN molecule further comprises: iv) a signal peptide.

38. The dendritic cell or precursor thereof of claim 37, wherein the signal peptide is covalently connected to the N-terminus of the IFN domain.

39. The dendritic cell or precursor thereof of claim 1, wherein the dendritic cell or a precursor comprises: a) a CAR that binds to a target antigen, and b) a membrane-bound chimeric IL-2 family cytokine molecule.

40. The dendritic cell or precursor thereof of claim 39, wherein the IL-2 family cytokine is selected from the group consisting of IL-2, interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin- 15 (IL-15), and interleukin-21 (IL-21).

41. The dendritic cell or precursor thereof of claim 39, wherein the IL-2 family cytokine is IL-2.

42. The dendritic cell or precursor thereof of any one of claims 39-41, wherein the membrane-bound chimeric IL-2 family cytokine molecule comprises i) a cytokine domain.

43. The dendritic cell or precursor thereof of claim 42, wherein the cytokine domain comprises an active form of the IL-2 family cytokine.

44. The dendritic cell or precursor thereof of claim 43, wherein the active form of the IL-2 family cytokine comprises a native active form of the IL-2 family cytokine or a variant thereof.

45. The dendritic cell or precursor thereof of claim 43 or claim 44, wherein the active form of the IL-2 family cytokine comprises a native active form of the IL-2 family cytokine.- 194 - NAI-5007600029vl46. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-2, and the native active form of IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 67 or a fragment thereof.

47. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-2, and the native active form of IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 68 or a fragment thereof.

48. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-15, and the native active form of IL-15 comprises the amino acid sequence set forth in SEQ ID NO: 70 or a fragment thereof.

49. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-15, and the native active form of IL-15 comprises the amino acid sequence set forth in SEQ ID NO: 71 or a fragment thereof.

50. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-21, and the native active form of IL-21 comprises the amino acid sequence set forth in SEQ ID NO: 72 or a fragment thereof.

51. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-21, and the native active form of IL-21 comprises the amino acid sequence set forth in SEQ ID NO: 73 or a fragment thereof.

52. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-7, and the native active form of IL-7 comprises the amino acid sequence set forth in SEQ ID NO: 74 or a fragment thereof.

53. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-7, and the native active form of IL-7 comprises the amino acid sequence set forth in SEQ ID NO: 75 or a fragment thereof.

54. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-9, and the native active form of IL-9 comprises the amino acid sequence set forth in SEQ ID NO: 76 or a fragment thereof.

55. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2- 195 - NAI-5007600029vlfamily cytokine is IL-9, and the native active form of IL-9 comprises the amino acid sequence set forth in SEQ ID NO: 77 or a fragment thereof.

56. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-4, and the native active form of IL-4 comprises the amino acid sequence set forth in SEQ ID NO: 78 or a fragment thereof.

57. The dendritic cell or precursor thereof of claim 44 or claim 45, wherein the IL-2 family cytokine is IL-4, and the native active form of IL-4 comprises the amino acid sequence set forth in SEQ ID NO: 79 or a fragment thereof.

58. The dendritic cell or precursor thereof of any one of claims 39-44, wherein the cytokine domain comprises a variant of a native active form of the IL-2 family cytokine.

59. The dendritic cell or precursor thereof of claim 58, wherein the variant comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the native active form of the IL-2 family cytokine.

60. The dendritic cell or precursor thereof of claim 58 or claim 59, wherein the variant comprises an amino acid sequence that is at least about 95% identical to the native active form of the IL-2 family cytokine.

61. The dendritic cell or precursor thereof of any one of claims 58-60, wherein the variant comprises at least one modification that increases the activation of cytotoxic T cells or increases the activation of regulatory T cells.

62. The dendritic cell or precursor thereof of claim 61, wherein the at least one modification is selected from deletion, insertion, substitutions, and combination thereof.

63. The dendritic cell or precursor thereof of claim 61 or claim 62, wherein the at least one modification comprises one or more substitutions.

64. The dendritic cell or precursor thereof of any one of claims 58-63, wherein the IL-2 family cytokine is IL-2, and the variant comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 68.

65. The dendritic cell or precursor thereof of any one of claims 58-64, wherein the IL-2 - 196 - NAI-5007600029vlfamily cytokine is IL-2, and the variant comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO: 67 or a fragment thereof, or SEQ ID NO: 68 or a fragment thereof.

66. The dendritic cell or precursor thereof of any one of claims 58-65, wherein the IL-2 family cytokine is IL-2, and the variant comprises at least one amino acid substitution of the amino acid sequence set forth in SEQ ID NO: 67 or SEQ ID NO: 68.

67. The dendritic cell or precursor thereof of claim 66, wherein the at least one amino acid substitution is selected from the group consisting of F42, L80, R81, L85, 186, 192, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 67.

68. The dendritic cell or precursor thereof of claim 67, wherein the at least one amino acid substitution is selected from the group consisting of F42A, L80F, R81D, L85V, I86V, I92F, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 67.

69. The dendritic cell or precursor thereof of any one of claims 58-68, wherein the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 69 or a fragment thereof.

70. The dendritic cell or precursor thereof of claim 66, wherein the at least one amino acid substitution comprises N88 of the amino acid sequence set forth in SEQ ID NO: 67.

71. The dendritic cell or precursor thereof of claim 70, wherein the at least one amino acid substitution comprises N88R of the amino acid sequence set forth in SEQ ID NO: 67.

72. The dendritic cell or precursor thereof of any one of claims 58-66, 70, and 71, wherein the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 80 or a fragment thereof.

73. The dendritic cell or precursor thereof of claim 70, wherein the at least one amino acid substitution comprises N88D of the amino acid sequence set forth in SEQ ID NO: 67.

74. The dendritic cell or precursor thereof of any one of claims 58-66, 70, and 73, wherein the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 81 or a fragment thereof.- 197 - NAI-5007600029vl75. The dendritic cell or precursor thereof of claim 66, wherein the at least one amino acid substitution is selected from the group consisting of N103, V106, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 68.

76. The dendritic cell or precursor thereof of claim 75, wherein the at least one amino acid substitution is selected from the group consisting of N103R, V106D, and any combinations of the foregoing of the amino acid sequence set forth in SEQ ID NO: 68.

77. The dendritic cell or precursor thereof of any one of claims 58-66, 75, and 76, wherein the IL-2 family cytokine is IL-2, and the variant comprises the amino acid sequence set forth in SEQ ID NO: 82 or a fragment thereof.

78. The dendritic cell or precursor thereof of any one of claims 42-77, wherein the membrane-bound chimeric IL-2 family cytokine molecule further comprises ii) an anchor domain that anchors the cytokine domain to the membrane of the dendritic cell or precursor thereof.

79. The dendritic cell or precursor thereof of claim 78, wherein the anchor domain comprises a GPI-anchored polypeptide, or a transmembrane domain derived from CD8, a TLR, a FcR, CD166, or OX40.

80. The dendritic cell or precursor thereof of claim 79, wherein the GPI-anchored polypeptide is derived from CD59, CD55, CD48, CD24, CD14, Thy-1, or Ly-6.

81. The dendritic cell or precursor thereof of claim 79 or claim 80, wherein the GPI-anchored polypeptide is derived from CD59.

82. The dendritic cell or precursor thereof of claim 81, wherein the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 53 or a fragment thereof.

83. The dendritic cell or precursor thereof of claim 79 or claim 80, wherein the GPI-anchored polypeptide is derived from CD55.

84. The dendritic cell or precursor thereof of claim 83, wherein the GPI-anchored polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 54 or a fragment thereof.- 198 - NAI-5007600029vl85. The dendritic cell or precursor thereof of any one of claims 42-84, wherein the membrane-bound chimeric IL-2 family cytokine molecule further comprises iii) a hinge domain.

86. The dendritic cell or precursor thereof of claim 85, wherein the hinge domain is positioned between i) the cytokine domain and ii) the anchor domain.

87. The dendritic cell or precursor thereof of claim 85 or claim 86, wherein the hinge domain has a length of at least about 5 amino acids and / or up to about 30 amino acids.

88. The dendritic cell or precursor thereof of any one of claims 85-87, wherein the hinge domain has a length of between about 5 and about 30 amino acids.

89. The dendritic cell or precursor thereof of any one of claims 85-88, wherein the hinge domain has a length of about 30 amino acids.

90. The dendritic cell or precursor thereof of any one of claims 85-89, wherein the hinge domain is derived from CD8, CD55, a TLR, CD40, CD3ζ, a FcR, CD166, IgGl, or IgG4.

91. The dendritic cell or precursor thereof of any one of claims 85-90, wherein the hinge domain is derived from CD8.

92. The dendritic cell or precursor thereof of any one of claims 85-91, wherein the hinge domain comprises a native or modified extracellular domain of CD8a or a fragment thereof.

93. The dendritic cell or precursor thereof of claim 92, wherein the hinge domain comprises amino acids 149 to 176 of SEQ ID NO: 26 or a fragment thereof.

94. The dendritic cell or precursor thereof of any one of claims 85-90, wherein the hinge domain is derived from CD55.

95. The dendritic cell or precursor thereof of any one of claims 85-90 and 94, wherein the hinge domain comprises a native or modified extracellular domain of CD55 or a fragment thereof.

96. The dendritic cell or precursor thereof of claim 95, wherein the hinge domain comprises amino acids 323 to 340 of SEQ ID NO: 55 or a fragment thereof.

97. The dendritic cell or precursor thereof of any one of claims 42-96, wherein the- 199 - NAI-5007600029vlmembrane-bound chimeric IL-2 family cytokine molecule further comprises: iv) a signal peptide.

98. The dendritic cell or precursor thereof of claim 97, wherein the signal peptide is covalently connected to the N-terminus of the cytokine domain.

99. The dendritic cell or precursor thereof of any one of claims 1-98, wherein the CAR comprises: a) an extracellular antigen-binding domain that binds to the target antigen, b) a transmembrane domain, and c) an intracellular domain.

100. The dendritic cell or precursor thereof of claim 99, wherein the intracellular domain is derived from a molecule that is capable of activating a dendritic cell (DC), wherein the Deactivating molecule is selected from the group consisting of CD3ζ, a toll-like receptor (TLR), a Fc receptor (FcR), and CD40.

101. The dendritic cell or precursor thereof of claim 100, wherein the intracellular domain of the CAR comprises an intracellular domain of the DC-activating molecule or a fragment thereof.

102. The dendritic cell or precursor thereof of claim 100, wherein the TLR is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11.

103. The dendritic cell or precursor thereof of claim 102, wherein the TLR is TLR4.

104. The dendritic cell or precursor thereof of any one of claims 100-103, wherein the intracellular domain of the CAR is derived from TLR4.

105. The dendritic cell or precursor thereof of claim 104, wherein the intracellular domain of the CAR comprises amino acids 660 to 835 of SEQ ID NO: 27 or a fragment thereof or amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof.

106. The dendritic cell or precursor thereof of claim 100 or 101, wherein the intracellular domain of the CAR is derived from CD3ζ.

107. The dendritic cell or precursor thereof of claim 106, wherein the intracellular domain of the CAR comprises amino acids 52 to 164 of SEQ ID NO: 33 or a fragment thereof or amino acids 52 to 164 of SEQ ID NO: 34 or a fragment thereof.- 200 - NAI-5007600029vl108. The dendritic cell or precursor thereof of claim 100, wherein the FcR is Fc gamma receptor.

109. The dendritic cell or precursor thereof of claim 108, wherein the Fc gamma receptor is FCERγ.

110. The dendritic cell or precursor thereof of any one of claims 100, 101, 108, and 109, wherein the intracellular domain of the CAR is derived from FCERγ.

111. The dendritic cell or precursor thereof of claim 110, wherein the intracellular domain of the CAR comprises amino acids 45 to 86 of SEQ ID NO: 35 or a fragment thereof or amino acids 45 to 86 of SEQ ID NO: 36 or a fragment thereof.

112. The dendritic cell or precursor thereof of claim 100 or claim 101, wherein the intracellular domain of the CAR is derived from CD40.

113. The dendritic cell or precursor thereof of claim 112, wherein the intracellular domain of the CAR comprises amino acids 216 to 289 of SEQ ID NO: 31 or a fragment thereof or amino acids 216 to 277 of SEQ ID NO: 32 or a fragment thereof.

114. The dendritic cell or precursor thereof of claim 99, wherein the intracellular domain is derived from Flt3.

115. The dendritic cell or precursor thereof of claim 114, wherein the intracellular domain of the CAR comprises amino acids 565 to 992 of SEQ ID NO: 45 or a fragment thereof or amino acids 564 to 993 of SEQ ID NO: 46 or a fragment thereof.

116. The dendritic cell or precursor thereof of claim 99, wherein the intracellular domain of the CAR is derived from 0X40.

117. The dendritic cell or precursor thereof of claim 116, wherein the intracellular domain of the CAR comprises amino acids 237 to 272 of SEQ ID NO: 29 or a fragment thereof or amino acids 236 to 277 of SEQ ID NO: 30 or a fragment thereof.

118. The dendritic cell or precursor thereof of any one of claims 99-117, wherein the transmembrane domain of the CAR is derived from CD8, a Toll-like receptor, CD40, CD3ζ, a FcR, CD166, 0X40, CD28, 4-1BB, or ICOS.- 201 - NAI-5007600029vl119. The dendritic cell or precursor thereof of claim 118, wherein the transmembrane domain of the CAR is derived from CD8a.

120. The dendritic cell or precursor thereof of claim 119, wherein the transmembrane domain of the CAR comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof or amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

121. The dendritic cell or precursor thereof of claim 118, wherein the transmembrane domain of the CAR is derived from CD8p.

122. The dendritic cell or precursor thereof of claim 121, wherein the transmembrane domain of the CAR comprises amino acids 176 to 196 of SEQ ID NO: 43 or a fragment thereof or amino acids 171 to 191 of SEQ ID NO: 44 or a fragment thereof.

123. The dendritic cell or precursor thereof of claim 118, wherein the transmembrane domain of the CAR is derived from TLR4.

124. The dendritic cell or precursor thereof of claim 123, wherein the transmembrane domain of the CAR comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof or amino acids 632 to 652 of SEQ ID NO: 28 or a fragment thereof.

125. The dendritic cell or precursor thereof of claim 118, wherein the transmembrane domain of the CAR is derived from 0X40.

126. The dendritic cell or precursor thereof of claim 125, wherein the transmembrane domain of the CAR comprises amino acids 212 to 236 of SEQ ID NO: 29 or a fragment thereof or amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof.

127. The dendritic cell or precursor thereof of claim 118, wherein the transmembrane domain of the CAR is derived from FcεRγ.

128. The dendritic cell or precursor thereof of claim 127, wherein the transmembrane domain of the CAR comprises amino acids 24 to 44 of SEQ ID NO: 35 or a fragment thereof or amino acids 24 to 44 of SEQ ID NO: 36 or a fragment thereof.

129. The dendritic cell or precursor thereof of any one of claims 99-128, wherein the CAR further comprises a hinge region.- 202 - NAI-5007600029vl130. The dendritic cell or precursor thereof of claim 129, wherein the hinge region has a length of between about 5 and about 100 amino acids, between about 10 and about 50 amino acids, or between about 50 and about 100 amino acids.

131. The dendritic cell or precursor thereof of claim 129 or claim 130, wherein the hinge region has a length of about 20, about 40, or about 50, or about 70 amino acids.

132. The dendritic cell or precursor thereof of any one of claims 129-131, wherein the hinge region is positioned between the extracellular antigen-binding domain and the transmembrane domain.

133. The dendritic cell or precursor thereof of any one of claims 129-132, wherein the hinge region is derived from CD8, a TLR, CD40, CD3ζ, a FcR, CD166, OX40, CD28, 4-1BB, or ICOS.

134. The dendritic cell or precursor thereof of any one of claims 129-133, wherein the hinge region is derived from CD8.

135. The dendritic cell or precursor thereof of any one of claims 129-134, wherein the hinge region comprises a native or modified extracellular domain of CD8a or a fragment thereof.

136. The dendritic cell or precursor thereof of claim 135, wherein the native extracellular domain of CD8a or fragment thereof comprises amino acids 149 to 196 of SEQ ID NO: 25 or a fragment thereof, amino acids 159 to 196 of SEQ ID NO: 25 or a fragment thereof, or amino acids 179 to 196 of SEQ ID NO: 25 or a fragment thereof.

137. The dendritic cell or precursor thereof of claim 135, wherein the modified extracellular domain of CD8a or fragment thereof comprises i) the amino acid sequence set forth in SEQ ID NO: 37 or a fragment thereof or ii) the amino acid sequence set forth in SEQ ID NO: 38 or a fragment thereof.

138. The dendritic cell or precursor thereof of any one of claims 129-131, wherein the hinge region is derived from a TLR.

139. The dendritic cell or precursor thereof of any one of claims 129-131, and 138, wherein the hinge region comprises a native or modified extracellular domain of a TLR.- 203 - NAI-5007600029vl140. The dendritic cell or precursor thereof of any one of claims 129-131, 138, and 139, wherein the hinge region comprises a native or modified extracellular domain of TLR4 or a fragment thereof.

141. The dendritic cell or precursor thereof of claim 140, wherein the native extracellular domain of TLR4 or fragment thereof comprises amino acids 619 to 638 of SEQ ID NO: 27 or a fragment thereof.

142. The dendritic cell or precursor thereof of claim 140, wherein the native extracellular domain of TLR4 or fragment thereof comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof.

143. The dendritic cell or precursor thereof of any one of claims 129-131, wherein the hinge region is derived from 0X40.

144. The dendritic cell or precursor thereof of any one of claims 129-131, and 142, wherein the hinge region comprises a native or modified extracellular domain of 0X40 or a fragment thereof.

145. The dendritic cell or precursor thereof of claim 144, wherein the native extracellular domain of 0X40 or fragment thereof comprises amino acids 166 to 211 of SEQ ID NO: 29 or a fragment thereof.

146. The dendritic cell or precursor thereof of claim 144, wherein the native extracellular domain of 0X40 or fragment thereof comprises amino acids 167 to 214 of SEQ ID NO: 30 or a fragment thereof.

147. The dendritic cell or precursor thereof of claim 144, wherein the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 39.

148. The dendritic cell or precursor thereof of claim 144, wherein the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 84.

149. The dendritic cell or precursor thereof of any one of claims 129-131, wherein the hinge region comprises a native or modified extracellular domain of TLR or a fragment- 204 - NAI-5007600029vlthereof, and a native or modified extracellular domain of 0X40 or a fragment thereof.

150. The dendritic cell or precursor thereof of claim 148, wherein the native extracellular domain of TLR or fragment thereof comprises amino acids 619 to 638 of SEQ ID NO: 27 or a fragment thereof, and the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 39.

151. The dendritic cell or precursor thereof of claim 148, wherein the native extracellular domain of TLR or fragment thereof comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof, and the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 84.

152. The dendritic cell or precursor thereof of any one of claims 149 to 151, wherein the intracellular domain of the CAR is derived from a TLR.

153. The dendritic cell or precursor thereof of any one of claims 149 to 152, wherein the intracellular domain of the CAR is derived from TLR4.

154. The dendritic cell or precursor thereof of any one of claim 149-153, wherein the transmembrane domain of the CAR is derived from a TLR.

155. The dendritic cell or precursor thereof of any one of claims 149-154, wherein the transmembrane domain of the CAR is derived from TLR4.

156. The dendritic cell or precursor thereof of any one of claims 1-155, wherein the target antigen is a tumor antigen.

157. The dendritic cell or precursor thereof of claim 156, wherein the tumor is a solid tumor.

158. The dendritic cell or precursor thereof of claim 156 or claim 157, wherein the target antigen is selected from the group consisting of epidermal growth factor receptor (EGFR), Prostate-specific membrane antigen (PSMA), IL13Ra2 (Interleukin- 13 Receptor Alpha 2), MUC1 (Mucin 1), Claudin 18.2, Mesothelin, GD2, CEA, FAP, R0R1 (Receptor Tyrosine Kinase-Like Orphan Receptor 1), GPC3 (Glypican-3), MAGE-A4 (Mel anoma- Associated Antigen A4), B7-H4, and Axl.

159. The dendritic cell or precursor thereof of claim 156, wherein the tumor is a liquid - 205 - NAI-5007600029vltumor.

160. The dendritic cell or precursor thereof of claim 156 or claim 159, wherein the target antigen is selected from the group consisting of CD 19, CD20, B-cell maturation antigen (BCMA), G-protein-coupled receptor class 5 member D (GPRC5D), Fc receptor-like 5 (FCRL5), CD22, CD33, CD123, and CD30.

161. The dendritic cell or precursor thereof of claim 156, wherein the target antigen is overexpressed in one or more types of tumor or cancer tissues but has no or a limited expression in essential normal tissues.

162. The dendritic cell or precursor thereof of claim 156 or claim 161, wherein the target antigen is selected from the group consisting of B7H3, EphA2, and Her2.

163. The dendritic cell or precursor thereof of any one of claims 1-162, wherein the target antigen is a cancer antigen.

164. The dendritic cell or precursor thereof of claim 163, wherein the target antigen is selected from the group consisting of B7H3, PSMA, CD19, CD20, EGFR, Her2, EphA2, and beta amyloid.

165. The dendritic cell or precursor thereof of any one of claims 1-155, wherein the target antigen is associated with an autoimmune disease, optionally wherein the autoimmune disease is a T-cell mediated autoimmune disease, optionally wherein the autoimmune disease is selected from the group consisting of type 1 diabetes (T1D), multiple sclerosis (MS), primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune myositis, psoriasis / psoriatic arthritis, and autoimmune vasculitis.

166. The dendritic cell or precursor thereof of claim 165, wherein the target antigen is selected from the group consisting of CD19, CD20, Sulfonylurea Receptor 1 (SURI), Myelin Basic Protein (MBP), Proteolipid Protein (PLP), Myelin Oligodendrocyte Glycoprotein (MOG), Myelin-Associated Glycoprotein (MAG), Oligodendrocyte Myelin Glycoprotein (OMgp), Galactocerebroside (GalC), cytokeratins, Aquaporin-1 (AQP1), CD55, VCAM-1 (Vascular Cell Adhesion Molecule 1), Desmin, Dystrophin, CD56, myeloperoxidase (MPO), proteinase 3 (PR3), CD177, and keratin.

167. The dendritic cell or precursor thereof of any one of claims 99-166, wherein the - 206 - NAI-5007600029vlextracellular antigen-binding domain of the CAR comprises a variable heavy domain of heavy chain (VHH) domain.

168. The dendritic cell or precursor thereof of claim 167, wherein:the target antigen is B7H3 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 4; the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3; and / or the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4;the target antigen is PSMA and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 8; the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; and / or the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8;the target antigen is CD 19 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 12; the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11; and / or the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12;the target antigen is CD20 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 16; the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; and / or the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 16;the target antigen is EGFR and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 20; the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; and / or the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 20; orthe target antigen is beta amyloid and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 64; the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 61, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 62, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 63; and / or the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 64.- 207 - NAI-5007600029vl169. The dendritic cell or precursor thereof of any one of claims 99-166, wherein the extracellular antigen-binding domain comprises a monobody.

170. The dendritic cell or precursor thereof of claim 169, wherein the monobody is based on a fibronectin type III (FN3) domain.

171. The dendritic cell or precursor thereof of claim 169 or claim 170, wherein the monobody comprises a BC loop, a DE loop, and an FG loop.

172. The dendritic cell or precursor thereof of any one of claims 169-171, wherein the target antigen is EphA2, and the monobody comprises a BC loop comprising the amino acid sequence set forth in SEQ ID NO: 21, a DE loop comprising the amino acid sequence set forth in SEQ ID NO: 22, and an FG loop comprising the amino acid sequence set forth in SEQ ID NO: 23; and / or the monobody comprises the amino acid sequence set forth in SEQ ID NO: 24.

173. The dendritic cell or precursor thereof of any one of claims 99-166, wherein the extracellular antigen-binding domain of the CAR comprises an antigen-binding fragment.

174. The dendritic cell or precursor thereof of any one of claims 99-166 and 173, wherein the extracellular antigen-binding domain of the CAR comprises a single chain variable fragment (scFv).

175. The dendritic cell or precursor thereof of any one of claims 99-174, wherein the CAR further comprises a signal peptide.

176. The dendritic cell or precursor thereof of claim 175, wherein the signal peptide is covalently connected to the N-terminus of the extracellular antigen-binding domain.

177. The dendritic cell or precursor thereof of any one of claims 1-176, wherein the CAR and / or the membrane-bound IFN molecule is recombinantly expressed.

178. The dendritic cell or precursor thereof of any one of claims 1-177, wherein the CAR is expressed from a vector or a mRNA.

179. The dendritic cell or precursor thereof of any one of claims 1-178, wherein the membrane-bound chimeric IFN molecule or the membrane-bound chimeric IL-2 family cytokine molecule is expressed from a vector or a mRNA.- 208 - NAI-5007600029vl180. The dendritic cell or precursor thereof of any one of claims 1-179, wherein the cell is a conventional dendritic cell (cDC).

181. The dendritic cell or precursor thereof of any one of claims 1-180, wherein the cell is conventional type 1 dendritic cells (cDCls).

182. The dendritic cell or precursor thereof of any one of claims 1-181, wherein the cell is murine cDCl or human cDCl.

183. The dendritic cell or precursor thereof of any one of claims 1-182, wherein the cell is a monocyte-derived dendritic cell (MoDC).

184. A composition comprising an effective amount of the cells of any one of claims 1-183.

185. The composition of claim 184, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

186. A method of treating a disease or a disorder in a subject, comprising administering to the subject an effective amount of the cells of any one of claims 1-183, or the composition of claim 184 or claim 185.

187. The method of claim 186, wherein the disease or disorder is tumor.

188. The method of claim 186 or claim 187, wherein the administration results in elimination of a tumor cell expressing the target antigen.

189. The method of any one of claims 186-188, wherein the administration results in elimination of a tumor cell that does not express the target antigen through epitope spreading.

190. The method of any one of claims 186-189, wherein the disease or disorder is cancer.

191. The method of any one of claims 186-190, wherein the cells are capable of cross priming a T cell.

192. The method of claim 191, wherein the T cell is reactive to a target cell expressing the target antigen to which the CAR binds.

193. The method of claim 191 or claim 192, wherein the T cell is a CD8+T cell.- 209 - NAI-5007600029vl194. The method of claim 186, wherein the disease or disorder is an autoimmune disease.

195. The method of claim 194, wherein the autoimmune disease is a T-cell mediated autoimmune disease.

196. The method of claim 194 or claim 195, wherein the autoimmune disease is selected from the group consisting of type 1 diabetes (T1D), multiple sclerosis (MS), primary biliary cirrhosis (PBC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune myositis, psoriasis / psoriatic arthritis, and autoimmune vasculitis.

197. The method of any one of claims 194-196, wherein the cells are capable of cross priming a T cell.

198. The method of claim 197, wherein the T cell is a regulatory T cell (Treg).

199. The method of claim 186, wherein the disease or disorder is a neurodegenerative disease.

200. The method of claim 186, wherein the disease or disorder is associated with beta amyloid accumulation.

201. The method of claim 200, wherein the disease or disorder associated with beta amyloid accumulation is selected from the group consisting of Alzheimer’s disease, Down syndrome, Cerebral Amyloid Angiopathy (CAA), Dementia with Lewy Bodies (DLB), Parkinson’s Disease Dementia (PDD), and Traumatic Brain Injury (TBI).

202. The method of claim 186, wherein the disease or disorder is an infectious disease.

203. A method of inducing an immune response in a subject, comprising administering to the subject an effective amount of the dendritic cells or precursors thereof of any one of claims 1-183, or the composition of claim 184 or claim 185.

204. The method of claim 203, wherein the immune response is a T cell-mediated immune response.

205. The method of claim 204, wherein the T cell is reactive to a target cell expressing the target antigen to which the CAR binds.

206. The method of claim 204 or claim 205, wherein the T cell is reactive to a tumor cell - 210 - NAI-5007600029vlthat does not express the target antigen through epitope spreading.

207. The method of any one of claims 204-206, wherein the T cell is a CD8+T cell.

208. The method of any one of claims 204-206, wherein the T cell is a regulatory T cell (Treg).

209. The method of any one of claims 186-208, wherein the method further comprises administering to the subject a second therapeutic agent.

210. The method of claim 209, wherein the second therapeutic agent is a soluble programmed-death 1 (PD-1) inhibitor.

211. The method of claim 210, wherein the soluble PD-1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof.

212. The method of claim 209, wherein the second therapeutic agent is soluble programmed death ligand 1 (PD-L1) inhibitor.

213. The method of claim 212, wherein the soluble PD-L1 inhibitor is an anti-PD-Ll antibody or an antigen-binding fragment thereof.

214. A nucleic acid composition comprising (a) a first nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a target antigen, and (b) a second nucleic acid encoding a membrane-bound chimeric interferon (IFN) molecule or a membrane-bound chimeric IL-2 family cytokine molecule.

215. The nucleic acid composition of claim 214, wherein the first nucleic acid is operably linked to a promoter, and / or the second nucleic acid is operably linked to a promoter, or the first and second nucleic acids are joined by a self-cleavage sequence.

216. The nucleic acid composition of claim 214 or claim 215, wherein the first nucleic acid is a mRNA, and / or the second nucleic acid is a mRNA.

217. The nucleic acid composition of claim 214 or claim 215, wherein the first nucleic acid is comprised in a vector and / or the second nucleic acid is comprised in a vector.

218. A vector comprising the nucleic acid composition of any one of claims 214-217.- 211 - NAI-5007600029vl219. A method of producing an antigen-specific dendritic cell or a precursor thereof, the method comprising: introducing into a dendritic cell or a precursor thereof (a) a first nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a target antigen, and (b) a second nucleic acid encoding a membrane-bound interferon (IFN) molecule or a membranebound chimeric IL-2 family cytokine molecule.

220. The method of claim 219, wherein the first nucleic acid is operably linked to a promoter, and / or the second nucleic acid is operably linked to a promoter.

221. The method of claim 219 or claim 220, wherein the first nucleic acid is comprised in a mRNA and / or the second nucleic acid is comprised in a mRNA.

222. The method of claim 219 or claim 220, wherein the first nucleic acid is comprised in a vector and / or the second nucleic acid is comprised in a vector.

223. The method of any one of claims 219-222, the method comprising introducing into the dendritic cell or precursor thereof the nucleic acid composition of any one of claims 214-217.- 212 - NAI-5007600029vl

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