Engineered dendritic cells and uses thereof

Abstract

Provided herein are nanobody- or monobody-based chimeric antigen receptors (CARs), and dendritic cells comprising such CARs. 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. 14857-005-228ENGINEERED DENDRITIC CELLS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Application No. 63 / 728,616, filed December 5, 2024, and U. S. Application No. 63 / 772,173, filed March 14, 2025, the disclosure of each of which is incorporated by reference herein in its entirety.GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under DP5OD02642705 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.SEQUENCE LISTING

[0003] 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 “14857-005-228_SL.xml”, was created on December 3, 2025, and is 62,466 bytes in size.1. FIELD

[0004] The present disclosure relates to chimeric antigen receptors (CARs) and immune cells (e.g., dendritic cells) comprising the CARs, and methods of making and using such cells.2. BACKGROUND

[0005] 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 for improved immunotherapy, e.g., improved curative systemic anti -tumor 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-5007599937vlis known as cross-presentation and is crucial for efficacious anti-tumor immune responses. (R. S. Laureano etal., Oncolmmunology (2022); 11: 1, 2096363).

[0006] 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

[0007] The present disclosure provides engineered dendritic cells (DCs) or precursors thereof. In certain embodiments, the DC or precursor thereof comprises: a chimeric antigen receptor (CAR), wherein the CAR comprises: a) an extracellular antigen-binding domain that binds to a target antigen, b) a transmembrane domain, and c) an intracellular domain derived from a molecule that is capable of activating a DC, wherein the DC-activating molecule is selected from the group consisting of CD3(^, a toll-like receptor (TLR), a Fc receptor (FcR), and CD40, and wherein the extracellular antigen-binding domain comprises i) a variable heavy domain of heavy chain (VHH domain) or ii) a monobody. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of the DC-activating molecule or a fragment thereof.

[0008] 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. In certain embodiments, the intracellular domain of the CAR comprises amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof.

[0009] In certain embodiments, the intracellular domain of the CAR is derived from CD3(^. 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: 34 or a fragment thereof.- 2 - NAI-5007599937vl

[0010] In certain embodiments, the intracellular domain of the CAR is derived from a Fc receptor (FcR). In certain embodiments, the FcR is Fc gamma receptor. In certain embodiments, the Fc gamma receptor is FCERG. In certain embodiments, the intracellular domain comprises amino acids 45 to 86 of SEQ ID NO: 35 or a fragment thereof. In certain embodiments, the intracellular domain comprises amino acids 45 to 86 of SEQ ID NO: 36 or a fragment thereof.

[0011] In certain embodiments, the intracellular domain is derived from CD40 and comprises amino acids 216 to 289 of SEQ ID NO: 31. In certain embodiments, the intracellular domain is derived from CD40 and comprises amino acids 216 to 277 of SEQ ID NO: 32.

[0012] In certain embodiments, the transmembrane domain is derived from CD8, a Tolllike receptor, CD40, CD3< a FcR, CD166, 0X40, CD28, 4-1BB, or ICOS.

[0013] In certain embodiments, the transmembrane domain of the CAR is derived from CD8. In certain embodiments, the CD8 is CD8a. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of CD8 or a fragment thereof. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of CD8a or a fragment thereof. In certain embodiments, the transmembrane domain of the CAR is derived from CD8a and comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the transmembrane domain of the CAR is derived from CD8a and comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

[0014] In certain embodiments, the transmembrane domain of the CAR is derived from a TLR. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of TLR or a fragment thereof. In certain embodiments, the TLR is TLR4. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of TLR4 or a fragment thereof. In certain embodiments, the transmembrane domain is derived from TLR4 and comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the transmembrane domain is derived from TLR4 and comprises amino acids 632-652 of SEQ ID NO: 28 or a fragment thereof.

[0015] In certain embodiments, the transmembrane domain of the CAR is derived from 0X40. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of 0X40 or a fragment thereof. In certain embodiments, the transmembrane domain is derived from 0X40 and comprises amino acids 212 to 236 of SEQ - 3 - NAI-5007599937vlID NO: 29 or a fragment thereof. In certain embodiments, the transmembrane domain is derived from 0X40 and comprises amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof.

[0016] 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, 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, CD3(^, a FcR, CD166, 0X40, IgGl, IgG4, CD28, 4-1BB, or ICOS.

[0017] In certain embodiments, the hinge region is derived from CD8. In certain embodiments, the hinge region comprises a native or modified extracellular domain of CD8 or a fragment thereof. In certain embodiments, the CD8 is CD8a. In certain embodiments, the hinge region comprises a native 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. In certain embodiments, the native extracellular domain of CD8a or fragment thereof comprises amino acids 159 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the native extracellular domain of CD8a or fragment thereof comprises amino acids 179 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region comprises a modified extracellular domain of CD8a 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.

[0018] 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 TLR is TLR4. 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 native extracellular domain of TLR4 or fragment thereof comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof.- 4 - NAI-5007599937vl

[0019] 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 hinge region comprises a modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, 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 modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0020] 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 hinge region comprises a native extracellular domain of TLR4 or a fragment thereof, and a modified extracellular domain of 0X40 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, 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 TLR4 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: 60. 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.

[0021] 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 - 5 - NAI-5007599937vlmaturation 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, CD 19, CD20, EGFR, Her2, EphA2, and beta amyloid. 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, p sori asis / p sori atic 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.

[0022] In certain embodiments, the extracellular antigen-binding domain comprises a VHH domain.

[0023] 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.

[0024] 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.- 6 - NAI-5007599937vl

[0025] 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.

[0026] 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 acid 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.

[0027] 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.

[0028] 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: 50. In certain embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 47, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 48, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0029] 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.

[0030] 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 - 7 - NAI-5007599937vlthe 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.

[0031] In certain embodiments, the dendritic cell or precursor thereof further comprises a signal peptide. In certain embodiments, the signal peptide is covalently connected to the N-terminus of the extracellular antigen-binding domain.

[0032] Furthermore, the present disclosure provides a chimeric antigen receptor (CAR) comprising: a) an extracellular antigen-binding domain that binds to a target antigen, b) a transmembrane domain, and c) an intracellular domain derived from CD40, a Toll-like receptor (TLR), or 0X40, wherein the extracellular antigen-binding domain comprises i) a variable heavy domain of heavy chain (VHH) domain or ii) a monobody, and wherein the intracellular domain is not capable of activating an immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling.

[0033] In certain embodiments, the intracellular domain is derived from a TLR. In certain embodiments, the TLR is TLR4. In certain embodiments, the intracellular domain is derived from TLR4 and comprises amino acids 660 to 835 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the intracellular domain is derived from TLR4 and comprises amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof.

[0034] In certain embodiments, the intracellular domain is derived from CD40. In certain embodiments, the intracellular domain is derived from CD40 and comprises amino acids 216 to 289 of SEQ ID NO: 31 or a fragment thereof. In certain embodiments, the intracellular domain is derived from CD40 and comprises amino acids 216 to 277 of SEQ ID NO: 32 or a fragment thereof.

[0035] In certain embodiments, the intracellular domain is derived from 0X40. In certain embodiments, the intracellular domain is derived from 0X40 and comprises amino acids 237 to 272 of SEQ ID NO: 29 or a fragment thereof. In certain embodiments, the intracellular domain is derived from 0X40 and comprises amino acids 236 to 277 of SEQ ID NO: 30 or a fragment thereof.

[0036] In certain embodiments, the transmembrane domain is derived from CD8, a Tolllike receptor, CD40, CD3< a FcR, CD166, 0X40, CD28, 4-1BB, or ICOS.

[0037] In certain embodiments, the transmembrane domain of the CAR is derived from CD8. In certain embodiments, the CD8 is CD8a. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of CD8a or a fragment thereof. In certain embodiments, the transmembrane domain is derived from CD8a and comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof. In certain - 8 - NAI-5007599937vlembodiments, the transmembrane domain of the CAR is derived from CD8a and comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

[0038] In certain embodiments, the transmembrane domain of the CAR is derived from TLR. In certain embodiments, the TLR is TLR4. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of TLR4 or a fragment thereof. In certain embodiments, the transmembrane domain of the CAR is derived from TLR4 and comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof. In certain embodiments, the transmembrane domain of the CAR is derived from TLR4 and comprises amino acids 632-652 of SEQ ID NO: 28 or a fragment thereof.

[0039] In certain embodiments, the transmembrane domain of the CAR is derived from 0X40. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of 0X40 or a fragment thereof. In certain embodiments, the transmembrane domain of the CAR is derived from 0X40 and comprises amino acids 212 to 236 of SEQ ID NO: 29 or a fragment thereof. In certain embodiments, the transmembrane domain is derived from 0X40 and comprises amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof.

[0040] In certain embodiments, the CAR further comprises d) a hinge region. In certain embodiments, the hinge region has a length of between about 5 and about 100 amino acids, or between about 5 and about 50 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, CD3(^, a FcR, CD166, 0X40, IgGl, IgG4, CD28, 4-1BB, or ICOS.

[0041] In certain embodiments, the hinge region is derived from CD8. In certain embodiments, the CD8 is CD8a. In certain embodiments, the hinge region comprises a native extracellular domain of CD8a or a fragment thereof. In certain embodiments, the hinge region is derived from CD8a and comprises amino acids 149 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region is derived from CD8a and comprises amino acids 159 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region is derived from CD8a and comprises amino acids 179 to 196 of SEQ ID NO: 25 or a fragment thereof. In certain embodiments, the hinge region comprises a modified extracellular domain of CD8a or a fragment thereof. In certain embodiments, the hinge region is derived from CD8a and comprises the amino acid sequence set forth in SEQ ID NO: 37 or a fragment thereof. In certain embodiments, the hinge region is derived from- 9 - NAI-5007599937vlCD8a and 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 TLR is TLR4. In certain embodiments, the hinge region comprises a native extracellular domain of TLR4 or a fragment thereof. In certain embodiments, the hinge region is derived from TLR4 and 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 extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the hinge region is derived from 0X40 and comprises amino acids 166 to 211 of SEQ ID NO: 29 or a fragment thereof. In certain embodiments, the hinge region is derived from 0X40 and comprises amino acids 167 to 214 of SEQ ID NO: 30 or a fragment thereof. In certain embodiments, the hinge region comprises a modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, 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 modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0044] In certain embodiments, the hinge region comprises a native or modified extracellular domain of TLR4 or a fragment thereof, and a native or modified extracellular domain of 0X40 or a fragment thereof. In certain embodiments, the hinge region comprises a native extracellular domain of TLR4 or a fragment thereof, and a modified extracellular domain of 0X40 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, 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 TLR4 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: 60. 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 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 antigen is a cancer - 10 - NAI-5007599937vlantigen. In certain embodiments, the extracellular antigen-binding domain comprises a VHH domain. In certain embodiments, the extracellular antigen-binding domain comprises a monobody.

[0046] 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.

[0047] Furthermore, the present disclosure provides nucleic acids encoding the CARs described herein, and vectors comprising the nucleic acids described herein. In certain embodiments, the nucleic acid is an mRNA.

[0048] The present disclosure further provides cells expressing the CARs, nucleic acids, or vectors described herein. In certain embodiments, the cell is an immune cell. In certain embodiments, the cell is a dendritic cell or precursor thereof.

[0049] 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. 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.

[0050] 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.

[0051] The present disclosure also provides various methods of using the presently disclosed cells and compositions. In one aspect, the present disclosure provides 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 tumor cells expressing the target antigen. In certain embodiments, the administration results in elimination of tumor cells that do not express the target antigen through epitope spreading. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cells are dendritic cells and the dendritic 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 - 11 - NAI-5007599937vlcertain 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, p sori asis / p sori atic 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 disorder is an infectious disease.

[0052] The present disclosure further provides methods of inducing an immune response in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells or 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. In certain embodiments, the cells or composition increase expression of one or more surface molecules expressed on DCs. In certain embodiments, the one or more surface molecules are selected from the group consisting of CD86, CD70, OX40L, CD40L, 4-1BBL, MHC-I, MHC-II, CCR7, and ICOSL. In certain embodiments, the one or more surface molecules are selected from the group consisting of CD86, CD70, MHC-I, and CCR7.

[0053] The present disclosure further provides methods of producing the dendritic cell or precursor thereof described herein. In certain embodiments, the method comprises introducing into a cell a messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid encoding the CAR. In certain embodiments, the mRNA further comprises a second nucleic acid encoding a self-cleaving peptide. In certain embodiments, the mRNA is introduced into the cell through electroporation.4. BRIEF DESCRIPTION OF THE FIGURES

[0054] FIGs. 1A-1E show the expression of scFv-based CARs comprising an IgHl signal peptide (SEQ ID NO: 40), an anti-EphA2 scFv (SEQ ID NO: 64) derived from a human antibody, an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) and either a short CD8P linker of 5 amino acids (SEQ ID NO: 41) (FIG. IB and FIG.1C), or a long CD8P amino acid linker of 35 amino acids (SEQ ID NO: 42) (FIG. ID, FIG.- 12 - NAI-5007599937vlIE), followed by a CD8P transmembrane domain (SEQ ID NO: 53), a Flt3 intracellular domain (SEQ ID NO: 54), a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) to allow for simultaneous assessment of CAR expression and transduction efficiency) followed by a stop codon. The CAR was retrovirally produced and transduced into primary mouse bone marrow cells on day 2 of differentiation in Flt3L differentiation media (transduction protocol described in Wu, Ohara etal., Nat Immunol(2022);23(11):1536-1550). The promoter used to drive CAR expression was changed to either a mouse CD11c promoter (FIG. IB, and FIG. ID), or a mouse CD11c promoter with additional BATF3 transcription factor binding sites (FIG. 1C, and FIG. IE). Both promoters effectively drove CAR transcription and the protein was effectively translated, as Thyl was expressed in >38% of cells in all conditions, however CAR surface expression (detected by a goat anti-human Fab2 (Jackson Labs)) was <1% in all conditions. FIG. 1 A shows FACS plots of primary eDCs generated using the SCF-Flt3L differentiation protocol from a C56B1 / 6 mouse gating scheme (Live, B220-MHCII+CD1 lc+) FACS plots of cDCls (Live, B220-MHCII+CD1 lc+XCRl+Sirpα-) looking at CAR (G4S (SEQ ID NO: 61)) surface expression and Thy 1.1 of CAR-DCs transduced with EphA2 -binding short Flt3 -signaling CAR using a CD11c promoter (FIG. IB), EphA2 -binding short Flt3 -signaling CAR using a BATF3+CD11c promoter (FIG. 1C), EphA2 -binding long Flt3 -signaling CAR using a CD11c promoter (FIG. ID), and EphA2 -binding long Flt3 -signaling CAR using a BATF3+ CD11c promoter (FIG. IE).

[0055] FIGs. 2A-2D show the expression of scFv-based CARs comprising various signal peptides including human CD11c (FIG. 2 A) and mouse CD11c (FIG. 2B), HLA-DRA (FIG.2C), and CD8a (FIG. 2D), followed by the anti-CD19 scFv FMC63 (Hyrenius-Wittsten, Su et al., Sci Transl Med (2021); 13(591)), an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon, was produced using in vitro transcription and delivered by electroporation to primary bone marrow derived DCs differentiated with Flt3L (Ou, Ferris et al., Eur J Immunol (2023);53(9): e2250201). No intracellular domain ((t) denotes lacking the intracellular domain) was used in the CARs to circumvent the added variable of tonic signaling or signaling induced CAR internalization. All CARs were effectively translated, as Thyl expression was achieved at high levels (>98%) in all cases, however, the CAR surface expression remained very low (<10% in all cases). The CAR expression was assessed using- 13 - NAI-5007599937vlan anti-G4S linker antibody (ACRO Biosystems), which recognizes the G4S (SEQ ID NO: 61) linker between the VH and VL domains of the scFv.

[0056] FIGs. 3A-3D show the expression of scFv-based CARs. For the HA(t) CAR ((t) denotes lacking the intracellular domain), the CAR comprises an IgH signal peptide (SEQ ID NO: 40), followed by an anti -HA scFv, an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 35 amino acids, followed by a CD8a transmembrane, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. The EphA2(t) CAR comprises an IgH signal peptide (SEQ ID NO: 40), followed by an anti-EphA2 scFv with a G4S (SEQ ID NO: 61) linker between the VH and VL, an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. The EphA2 FcR CAR comprises an IgH signal peptide (SEQ ID NO: 40), followed by an anti-EphA2 scFv with a G4S (SEQ ID NO: 61) linker between the VH and VL, an extracellular linker derived from the mouse FcR (common gamma chain) of 5 amino acids, followed by the FcR (common gamma chain) transmembrane domain, the FcR (common gamma chain) intracellular domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. The indicated CAR was retrovirally transduced into HoxB8-FL cells (Hacker 2023, Methods Mol Biol 2618: 93-107), then sorted twice, cultured for five passages, and differentiated into DCs using GM-CSF and IL-4. On Day 1 of differentiation, the cells were analyzed by flow cytometry for the surface CAR expression, and confirmed the CAR was still expressed at high levels in all conditions (HA CAR detected by the FLAG tag (SEQ ID NO: 52) in the linker domain, and EphA2 CARs detected by the anti-G4S antibody detecting the G4S linker (SEQ ID NO: 61) in the scFv) (FIG. 3B). Four days later (Day 5) in differentiation, when the cells uniformly began to express MHC-II and CD11c and were thus DCs, each DC population significantly downregulated CAR surface expression (FIG. 3B). FIG. 3 A shows scheme of the CAR design including the binding domain, extracellular hinge, transmembrane, and intracellular signaling domain. FIG. 3B shows flow cytometry for the surface CAR expression and MHC-II and CD11c expression. FIG. 3C shows percent of CAR surface expression of the different transduced CAR-Precursor DCs (Dayl Differentiated DCs) and the differentiated DCs (Day5 Differentiated DCs). FIG.3D shows combination of the percent of CAR surface expression of the 3 different CAR-precursor DCs (Dayl Differentiated DCs) and the CAR-differentiated DCs (Day5 Differentiated DCs). Representative data from one independent experiment is shown. Data - 14 - NAI-5007599937vlis shown as mean ± SD. Statistical significance was calculated with an unpaired two-tailed Welch’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

[0057] FIG. 4 shows the expression of VHH- or monobody -based CARs. Each CAR comprises an IgH signal peptide, followed by a camelid VHH domain targeting the indicated antigen (CD 19, CD20, B7H3 (also referred to as “aB7”), PSMA, or EGFR), and a FLAG tag (SEQ ID NO: 52) in the extracellular linker for easy detection. The structures of the CARs are described in Section 5.2.6. Each CAR is followed by a P2A cleavage sequence (SEQ ID NO: 55) and Thyl (SEQ ID NO: 56) followed by a stop codon. The surface CAR expression was examined by flow cytometry using anti-FLAG antibody, and construct expression was examined by anti-Thyl antibody.

[0058] FIGs. 5A-5E show the expression of the scFv-based CARs in different immune cells. FIGs. 5A-5D show expression of scFv based CARs lacking intracellular domain in different immune cells. Three non-signaling (t) CARs containing human CD19-specific scFv (FMC63) binding domains with different surface signaling peptides (SP) (hCDl 1c, mCDl 1c, and HLA-DRA) and one non-signaling EGFRvIII-specific scFv CAR were generated. FIG.5A shows the CAR construct design comprising the indicated signal peptide, followed by the indicated scFv binding domain, an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. No intracellular signaling domain was used in the CARs to avoid potential signaling-induced CAR internalization. CARs were produced using in vitro transcription and delivered by electroporation to primary cells - primary human T-cells isolated by Ficoll (DeSeim, Palomba et al. 2018, Mol Ther 26(11): 2542-2552) (FIG. 5B), primary mouse bone marrow derived macrophages differentiated in M-CSF (DeSeim, Miller et al. 2011, Dev Cell 21(5): 966-974) (FIG. 5C), or primary bone marrow derived DCs differentiated with Flt3L (Ou, Ferris et al. 2023, Eur J Immunol 53(9): e2250201) (FIG. 5D). The anti-EGFRvIII CAR (aEGFRvIII(t)) comprises an IgHl signal peptide (SEQ ID NO: 40), followed by an anti-EGFRvIII scFv, and an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. Representative flow plots are shown. The anti-CD19 CAR (aCD19(t)) comprises an IgHl signal peptide (SEQ ID NO: 40), followed by an anti-CD19 scFv FMC63 (Hyrenius-Wittsten, Su et al., Sci Transl Med (2021); 13(591)), and an- 15 - NAI-5007599937vlextracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. aCD19(t) VH CAR comprises an IgHl signal peptide (SEQ ID NO: 40), followed by the VH domain alone of the scFv, and an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. The VH domain alone of the scFv (aCD19(t) VH), which is similar in size to a camelid VHH, expressed well as a CAR on the surface of T-cells and macrophages, but not on DCs. FIG. 5E shows expression of scFv-based CARs in DCs, with various intracellular signaling domains. <zCD19 Flt3(s) is a CAR construct comprising a IgHl signal peptide (SEQ ID NO: 40), followed by the anti-CD19 scFv FMC63 (Hyrenius-Wittsten, Su et al. 2021), an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 5 amino acids, followed by a CD8a transmembrane domain, a Flt3 intracellular domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. <zCD19 Flt3(l) is a CAR construct comprising a IgHl signal peptide (SEQ ID NO: 40), followed by the anti-CD19 scFv FMC63 (Hyrenius-Wittsten, Su et al. 2021), an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a linker of 35 amino acids, followed by a CD8a transmembrane, a Flt3 intracellular domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. crEGFRvIII Flt3 is a CAR construct comprising a IgHl signal peptide (SEQ ID NO: 40), followed by the anti-EGFRvIII scFv, an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a Flt3 EC linker of 300 amino acids, followed by a Flt3 transmembrane domain, a Flt3 intracellular domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon. <zEphA2 CD40-Flt3 is a CAR construct comprising a IgHl signal peptide (SEQ ID NO: 40), followed by the anti-EphA2 scFv, an extracellular linker derived from a FLAG tag (8 amino acids; SEQ ID NO: 52) plus a CD8a EC linker of 35 amino acids, followed by a CD8a transmembrane domain, a CD40 intracellular domain, a Flt3 intracellular domain, a P2A cleavage sequence (SEQ ID NO: 55), and Thyl (SEQ ID NO: 56) followed by a stop codon.

[0059] FIGs. 6A-6G show the quantitative analysis of the CAR expressions of scFv-, nanobody-, and monobody-based CARs across immune cell types. FIG. 6A depicts the CAR comprising a signal peptide, followed by an antigen-binding domain (scFv, nanobody, or- 16 - NAI-5007599937vlmonobody), a linker region, transmembrane domain (TM), signaling domain, P2A sequence, and Thy 1.1 marker. Primary T-cells (FIG. 6B), macrophages (FIG. 6C) or DCs (FIG. 6D) were electroporated with the nanobody- and / or monobody-based CARs described in FIG. 4 (aPSMA CD40(l), aEGFR CD40(l), aCD20 CD40(l), and aCD19 CD40(l)), aB7H3 nanobody -based VHH without signaling domain (aB7H3(t)), aEphA2 monobody -based VHH without signaling domain (aEphA2 (t)) or the scFv-based CARs described in FIGs. 5 A-5D, and the percentage of cells expressing each CAR on the surface as well as the coexpression marker Thyl was quantified by flow cytometry. The scFv-based CAR surface expression was >50% on average in both T-cells and macrophages, but <10% on average in DCs, despite excellent Thyl expression of >90% (FIG. 6E). The nanobody- and monobodybased CARs, on the other hand, uniformly expressed on the surface to high levels in DCs (FIGs. 6F and 6G). To test whether it is simply a size limitation, the FMC63 scFv, which expresses well on T-cells and macrophages but not on DCs, was truncated to only contain a single VH domain, rather than a VH and VL domain separated by a G4S (SEQ ID NO: 61) linker. The VH domain alone of the scFv (aCD19(t) VH), which is similar in size to a camelid VHH, expressed well as a CAR on the surface of T-cells and macrophages, but not on DCs, indicating that size alone is not the determining factor for the ability of DCs to express CAR on the surface (FIGs 6B-6D).

[0060] FIGs. 7A-7C show the structures of the CARs comprising an scFv comprising a VH and a VL which are connected by a G4S (SEQ ID NO: 61) linker, a VHH or a monobody. FIG. 7A shows that each CAR comprises a signal peptide, followed by the target binding domain (either an scFv, a nanobody, or a monobody), an extracellular linker, followed by a transmembrane (TM) domain, a signaling domain, a P2A cleavage sequence, and Thyl followed by a stop codon (FIG. 7A). This generates either an scFv-based CAR (FIG. 7B) co-expressed with Thyl on the surface, or a nanobody / monobody -based CAR coexpressed with Thyl (FIG. 7C).

[0061] FIGs. 8A-8F show that the nanobody-based CAR DCs with various intracellular domains increased the proportion of DCs capturing tumor antigen in a target and signaling dependent manner. FIG. 8A shows the CAR construct design comprising the indicated signaling domain. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (mock), and cocultured in a 1:1 ratio with 1956 sarcoma tumor cells that had been loaded with the fluorescent AF488-ova protein, and either expressed B7H3 (by transduction and sorting using FACS) or did not. After two hours of- 17 - NAI-5007599937vlcoculture, the cells were analyzed by flow cytometry, and the percentage of AF488+ DCs was determined in each condition (FIGs. 8B-8F).

[0062] FIGs. 9A-9I show that the nanobody -based CAR DCs with various intracellular domains increased the amount of tumor captured by each DC in an antigen and signaling dependent manner. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (mock), and cocultured in a 1:1 ratio with 1956 sarcoma tumor cells that had been loaded with the fluorescent AF488-ova protein, and either expressed B7H3 (by transduction and sorting using FACS) or did not. After two hours of coculture, the cells were analyzed by flow cytometry, and the AF488 median fluorescent intensity (MFI) of the DC population, reflecting the amount of AF488 taken up from the tumor by the DC, was determined in each condition. “B7H3 on tumor” indicates whether the CAR DCs had been cocultured on tumor cells that express (+) or did not express (-) human B7H3. FIGs. 9A-9E, 9G and 91 show representative histogram of AF488-OVA for DC in each indicated condition. FIGs. 9F and 9H show mean fluorescent intensity (MFI) of AF488-ova in DCs in each indicated condition.

[0063] FIGs. 10A-10I show that the nanobody-based CAR DCs increased the crosspresentation of tumor-derived antigen on MHC-I in a CAR target antigen and CAR signaling dependent manner. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (mock), and cocultured with 1956 sarcoma tumor cells that had been loaded with the fluorescent AF488-ova protein, and either expressed B7H3 (by transduction and sorting using FACS) or did not. After 2 hours of coculture, the DCs were sorted using FACS, analyzed for AF488-ova MFI, and cultured alone for an additional 18 hours. The cells were then analyzed by flow cytometry using anti-SIINFEKL / MHC-I antibody to quantify the percentage of DCs cross-presenting SIINFEKL (SEQ ID NO: 62), a peptide derived from ovalbumin (originally present in the tumor) (FIGs. 10A-10G).Fluorophore quantification (Quantbrite) beads were used to calculate the number of actual molecules of captured AF488-ova after two hours of coculture of DCs with AF488-ova loaded tumor, as well as the actual number of molecules of cross-presented SIINFEKL (SEQ ID NO: 62) on MHC-I 18 hours later (FIG. 10H). Representative histograms from flow cytometry analysis shown as FIG. 101 (top to bottom: aB7(t); aB7 CD40(I); aB7 TLR; aB7 CD3Q.

[0064] FIGs. HA -11C show the results of antigen degradation rate of the nanobodybased CAR DCs. B7H3+ tumor cells were electroporated with AF488-ova, visualized for intracellular AF488 positivity, and cocultured with the indicated B7H3 targeting CAR DC or - 18 - NAI-5007599937vlmock DCs. After two hours of coculture, DCs were sorted by FACS, and analyzed at the indicated time points for AF488 MFI (FIG. 11 A). Quantum beads were used to determine the number of AF488 molecules / cell based on MFI. The rate of degradation from 2 to 22 hours, expressed as molecules per hour, was calculated (FIG. 1 IB). Antigen captured by FcR and CD3(^ CARs was degraded more rapidly than control CAR with no signaling, while antigen captured by TLR CARs was degraded more slowly (FIG. 1 IB). Representative histogram plots show as FIG. 11C (top to bottom: 48.5 hours; 22 hours; 2 hours; 0 hour).

[0065] FIGs. 12A-12P show the results of the ability of each CAR DC to cross-prime naive tumor reactive T-cells. The nanobody-based CARs with various intracellular domains improved the ability of DCs to cross-prime CD8 T-cells to tumor antigen, in a CAR target and CAR signaling dependent manner. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (mock), and cocultured with CellTrace Violet (CTV) labeled OTI T-cells and B2M ’’ 1956 sarcoma tumor cells that had been loaded with full length ovalbumin protein and either expressed B7H3 on the surface (by transduction and sorting using FACS) or did not. 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 (FIGs. 12B-12N). B7H3 targeting CAR were generated with a CD40 intracellular signaling domain and either a short, medium, or long extracellular linker from CD8a, and the long extracellular linker were found to function best, although all are better than the controls (FIG. 12F). FIG. 12A illustrates cross-priming of naive tumor-reactive T-cells by CAR-DCs. FIGs. 12B-12G, 12H (right panel), 121 (right panel), 12K, 12M, and 12N show representative histogram of proliferated OTI T-cells in each indicated condition. FIGs. 12H (left panel), 121 (left panel), 12 J, and 12L show representative FACS plots of CTV intensity and CD44 expression on OTI cells after 3 days of culture in each indicated condition. OTI proliferation is measured by CTV dilution, indicated by the gate. FIG. 120 shows proliferated OTI count in response to cross-priming by DC (mock), aB7(t) CAR DC, or aB7 TLR CAR DC with either IxlO3or 5xl03OVA-loaded [32M- B7H3+ tumor cells. FIG. 12P left panel shows proliferation count of OTI CD8 T-cells in response to ova-loaded tumor cells cross-presented by DCs electroporated with 12.5, 25, 50, or 100 |lg / ml of aB7(t) CAR mRNA. FIG. 12P right panel shows histogram plot of CAR (cameloid) intensity on DCs using various amounts of aB7(t) CAR mRNA.

[0066] FIGs. 13A-13C show the results of the tumor killing capacity of the presently disclosed CAR DC-primed T-cells. Primary bone marrow differentiated DCs were- 19 - NAI-5007599937vlelectroporated with the indicated CAR construct or were left untouched (Empty DCs), and cocultured with labeled OTI T-cells and B2M" ' 1956 sarcoma tumor cells that had been loaded with full length ovalbumin protein and expressed B7H3 on the surface (by transduction and sorting using FACS). 5,000 tumor cells, with 25,000 DCs and 25,000 T-cells were added to each well (as in all cross-presentation assays, unless otherwise indicated). After three days of coculture, new luciferase-ova-zsGreen expressing, B7H3 negative, 1956 tumor cells were added (5,000 tumor cells per well). 4 hours (FIGs. 13 A and 13C) or 24 hours (FIG. 13B) later, % tumor killing was determined using bioluminescence imaging; triton-x was used as the positive control for tumor killing.

[0067] FIGs. 14A-14F show that the nanobody-based CAR comprising a TLR4 signaling domain induced CD86 (FIG. 14 A), MHC-I (FIG. 14B), CD70 (FIG. 14C), CD40 (FIG. 14D), 4-1BBL (FIG. 14E), CCR7 (FIG. 14F) expression. Primary bone marrow differentiated DCs were electroporated with the B7 targeting TLR CAR construct (aB7 TLR), B7 targeting CAR with no intracellular signaling domain (aB7(t)), or nothing (mock), and cocultured with B7+ 1956 sarcoma tumor cells for 24 hours, followed by flow cytometry analysis. The median fluorescence intensity (MFI) of the indicated surface molecule on the DC population was quantified by flow cytometry. FIG. 14A shows MFI of CD86 in DCs. FIG. 14B shows MFI of MHC-I in DCs. FIG. 14C shows MFI of CD70 in DCs. FIG. 14D Left: MFI of CD40 in DCs. Middle: representative histogram of CD40 (top to bottom: DC; DC Thyl; aB7 (t); aB7 TLR). Right: Percent of CD40 positive DCs. FIG. 14E Left: MFI of 4-1BBL in DCs.Middle: representative histogram of 4-1BBL (top to bottom: DC; DC Thyl; aB7 (t); aB7 TLR). Right: Percent of 4-1BBL positive DCs. FIG.14 F Left: MFI of CCR7 in DCs.Middle: representative histogram of CCR7 (top to bottom: DC; DC Thyl; aB7 (t); aB7 TLR). Right: Percent of CCR7 positive DCs.

[0068] FIGs. 15A-15F show that the nanobody-based CAR comprising a CD40 intracellular signaling domain induced CD70 (FIG. 15A), OX40L (FIG. 15B) and 41BBL (FIG. 15C) costimulatory ligand expression, in a CAR target and CAR signaling dependent manner. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct (aB7 CD40(m)) or Thyl alone, and cocultured with B7+ or B7- 1956 sarcoma tumor cells for 24 hours, followed by flow cytometry analysis. The percentage of DCs expressing each costimulatory ligand was determined (FIGs. 15D-15F).

[0069] FIGs. 16A-16C show that the nanobody-based CARs comprising a CD40 intracellular signaling domain and various extracellular linker lengths induced CD70 (FIG.16B), CD86 (FIG. 16A) and 41BBL (FIG. 16C) costimulatory ligand expression, in a CAR - 20 - NAI-5007599937vltarget and CAR signaling dependent manner. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (Mock), and cocultured with B7+ 1956 sarcoma tumor cells for 24 hours, followed by flow cytometry analysis. The median fluorescence intensity (MFI) of each costimulatory ligand was determined.

[0070] FIGs. 17A and 17B show that the monobody -based CAR improved the ability of DCs to cross-prime CD8 T-cells to tumor antigen. Primary bone marrow differentiated DCs were electroporated with the indicated EphA2 targeting monobody with a CD40 intracellular signaling domain (EphA2 monobody CD40(s)), or Thyl alone, then cocultured with CellTrace Violet (CTV) labeled OTI T-cells and B2M- / - 1956 sarcoma tumor cells that had been loaded with full length ovalbumin protein (FIG. 17A). These tumor cells naturally expressed mouse EphA2 on the surface, and the EphA2 monobody used recognizes both human and mouse EphA2 (though human with higher affinity). 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. 17B).

[0071] FIGs. 18A and 18B show the antigen-specific T cell activity induced by the presently disclosed CAR DCs. FIG. 18A Primary bone marrow differentiated DCs were electroporated with the anti-B7H3 nanobody-based CAR comprising a CD40 intracellular signaling domain (aB7 CD40(m)), or Thyl alone (25k DCs in each well), and cocultured with CellTrace Violet (CTV) labeled CD45.1 OTI CD8 T-cells plus CD45.2 WT CD8 T-cells (12.5k OTI and 12.5k WT T-cells per well), and B2M- / - 1956 sarcoma tumor cells (5k per well) that had been loaded with full length ovalbumin protein and expressed B7H3 on the surface (by transduction and sorting using FACS). After three days of coculture, the cells were analyzed by flow cytometry (FIG. 18A middle panel), and proliferated OTI and WT T-cell numbers were quantified as the number of CD44+ CTV diluted CD45.1 or CD45.2 T-cells in each well (T-cell back gates include CD8+, TCRvbeta+, live / dead, forward / side gates) (FIG. 18A right panel). The CAR DCs robustly activated tumor reactive OTI T-cells, but not nearby WT T-cells. FIG.18B left panel shows proliferation count of either OVA-specific OTI CD8 T-cells (OTI) or WT CD8 T-cells (WT) in response to OVA-loaded 1956 cells cross-presented by Thyl DCs, aB7(t) CAR DCs and aB7 TLR CAR DCs. FIG.18B right panel shows representative FACS plots of CTV intensity and CD44 expression on (left) OTI cells or (right) WT T-cells after 3 days of culture. OTI / WT proliferation is measured by CTV dilution, indicated by the gate.

[0072] FIG. 19 depicts the in vivo activity of the presently disclosed CAR DCs. 1656 sarcoma was injected orthotopically into the flank of B6 mice, and allowed to establish for - 21 - NAI-5007599937vlfour days. Primary bone marrow differentiated DCs were electroporated with the anti-B7H3 nanobody-based CAR comprising a CD40 intracellular signaling domain (aB7 CD40(m)), or control human CD 19 targeting nanobody CAR DCs with an identical extracellular, transmembrane and intracellular domain (aCD19 CD40 (m)). Six days later, tumor draining nodes removed and stained for mutant PSMD6 tetramer (recognizing 1956 tumor neoantigen specific T-cells). The number of tetramer positive (tumor neoantigen specific) T-cells per node was quantified.

[0073] FIG. 20 depicts the in vivo activity of the presently disclosed CAR DCs. A 50:50 mixture of B7H3 expressing (B7+) and non-expressing (B7-) 1956 sarcoma was injected orthotopically into the flank (1.5 million cells) of WT B6 mice. On day 3 and 6 after tumor implantation, mice were treated with either 0.5 or 2 million aB7H3 CD40 CAR DCs (aB7 CD40(m)), 2 million control Thyl DCs, or PBS, intratum orally. Tumor size was monitored over time.

[0074] FIGs. 21A-21C depict the in vivo activity of the presently disclosed CAR DCs. A 75:25 mixture of B7H3 expressing (B7+) and non-expressing (B7-) 1956 sarcoma was injected orthotopically into the flank of WT B6 mice. On day 8 and 11 after tumor implantation, mice were treated with either 2 million aB7 TLR CAR DCs (FIG. 21 A), aB7(t) CAR DCs (FIG. 2 IB), or control Thyl DCs (FIG. 21C), intratum orally. Tumor size was monitored over time.

[0075] FIGs. 22A-22C show that TLR CAR and FcRy CAR induced CD70 expression. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (Mock), and cocultured with B7H3 expressing 1956 sarcoma tumor cells for 24 hours, followed by flow cytometry analysis. The percentage of DCs expressing CD70 (FIG. 22B), as well as the median fluorescent intensity (MFI) of the CD70 expressed on the surface of the CAR DCs (FIG. 22A), was determined. Representative flow plot shown as FIG. 22C.

[0076] FIGs. 23A-23C show that TLR CAR and FcRy CAR induced CD86 expression. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (Mock), and cocultured with B7H3 expressing 1956 sarcoma tumor cells for 24 hours, followed by flow cytometry analysis. The percentage of DCs expressing CD86 (FIG. 23B), as well as the median fluorescent intensity (MFI) of the CD86 expressed on the surface of the CAR DCs (FIG. 23 A), was determined. Representative flow plot shown as FIG. 23C with dotted line at the MFI for Mock DCs.- 22 - NAI-5007599937vl

[0077] FIGs. 24A-24C show that TLR CAR and FcRy CAR induced CCR7 expression. Primary bone marrow differentiated DCs were electroporated with the indicated CAR construct or nothing (Mock), and cocultured with B7H3 expressing 1956 sarcoma tumor cells for 24 hours, followed by flow cytometry analysis. The percentage of DCs expressing CCR7 (FIG. 24B), as well as the median fluorescent intensity (MFI) of the CCR7 expressed on the surface of the CAR DCs (FIG. 24A), was determined. Representative flow plot shown as FIG. 24C.

[0078] FIGs. 25A and 25B show the complete rejection of re-injected tumors in previously cured mice, regardless of B7H3 expression, demonstrating the generation of antitumor memory by treating with aB7 TLR CAR DCs. A 75:25 mixture of B7H3 expressing (B7+) (FIG. 25 A) and non-expressing (B7-) (FIG. 25B) 1956 sarcoma was injected orthotopically into the flank of WT B6 mice on day 0 (1 million tumor cells per injection). On day 8 and 11 after tumor implantation, mice that were treated with 2 million aB7 TLR CAR DCs or aB7(t) CAR DCs, intratum orally. Mice that exhibited a complete response were rechallenged at day 102 with 1 million 1956 sarcoma cells in the left flank that expressed no B7H3 CAR target antigen (B7H3(-)), as well as 1 million 1956 sarcoma cells in the right flank that expressed B7H3 on 100% of the cells (B7H3(+)). Tumor regrowth was monitored on both sides. Regardless of B7H3 expression, re-injected tumor was completely rejected in previously cured mice, demonstrating generation of anti -tumor memory.

[0079] FIGs. 26A-26D show that murine TLR4 CAR-DC with an 0X40 EC domain improved T-cell proliferation. FIG. 26A shows FACS plots of DCs electroporated with different murine TLR4 signaling CARs with various EC and TM domains. FIG. 26B shows Gating scheme of cross-priming assay for OVA-specific OTI CD8 T-cell in response to OVA-loaded β2M⁻ tumor cells by DCs. FIG. 26C shows proliferation count of OTI T-cells in response to cross-priming aB7(t) CAR (“A”) DCs, aB7 TLR CAR (“OX4050T420”) DCs, and aB7 TLR (20) CAR (“T420”) DCs. FIG. 26D shows proliferated OTI counts for aB7(t) CAR DCs and multiple different CAR-DCs. Representative data from one of two independent experiments are shown. Data is shown as mean ± SD. Statistical significance was calculated with an ordinary one-way ANOVA with multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

[0080] FIGs. 27A and 27B show human TLR4 CAR-DC with an 0X40 EC domain improved T-cell proliferation. FIG. 27A shows FACS plots of primary DCs of CAR (FLAG or cameloid) and Thyl of DCs electroporated with different human TLR4 signaling CARs- 23 - NAI-5007599937vlwith various EC and TM domains. FIG. 27B shows proliferated OTI counts in response to being cross-primed by various CAR-DC as indicated. Representative data from one experiment is shown. Data is shown as mean ± SD. Statistical significance was calculated with an ordinary one-way ANOVA with multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

[0081] FIGs. 28A-28E show aB7 TLR CAR DC treatment leads to complete tumor rejection. FIG. 28A shows schematic of tumor injection in C57B1 / 6 mice injected with 1061956 cells with 75% B7H3+and 25% B7H3' and not treated (n=l 1) (FIG. 28B) or day 8 and 11 intratumorally injected with Thyl DC (n=19) (FIG. 28C), aB7(t) CAR DC (n=43) (FIG.28D), or aB7 TLR CAR DC (n=30) (FIG. 28E) and followed for tumor growth. Data represent pooled biologically independent samples from four independent experiments.

[0082] FIGs. 29A-29E show aB7 TLR (also called “B7(T4)”) CAR DC migrates to tdLN greater than Thyl DC and peaks at 72hrs. FIG. 29A shows schematic of CD45.2+C57BL / 6 mice injected with 1061956 B7H3+cells and after 5 days 2xl06CD45.1+CTFR+Thyl DCs were coinjected intratumorally with 2xl06CD45.1+CTV+aB7 TLR CAR DC. tdLNs were isolated 1, 2, 3, or 6 days after DC injection. FIG. 29B shows gating scheme of injected DCs from tdLN (live, CD45.1+CTV+or CTFR+). FIG. 29C shows Thyl DC counts per tdLN were determined using CTFR while aB7 TLR CAR DC counts per tdLN were determined using CTV (n=10 each) (FIG. 29D). FIG. 29E shows DC count per tdLN of Thyl DC compared to aB7 TLR CAR DC (n=24, two independent experiments). Data is shown as mean ± SD. Statistical significance was calculated with an ordinary one-way ANOVA with multiple comparisons. Statistical significance was calculated with a paired two-tailed t test. *P < 0.05, **P < 0.01, ***p < 0.001, **** P < 0.0001.

[0083] FIGs. 30A-30D show aB7 TLR CAR DC transports more tumor antigen to tdLN. FIG. 30A shows schematic of CD45.2+C57B1 / 6 mice injected with 1.5xl061956 B7H3+zsgreen+cells and after 5 days 2xl06CD45.1+CTFR+Thyl DC or CD45.1+CTFR+aB7(t) CAR DCs were coinjected intratumorally with 2xl06CD45.1+CTV+aB7 TLR CAR DC and tdLNs were isolated 3 days later. FIG. 30B shows DC count per tdLN of aB7(t) CAR DC compared to aB7 TLR CAR DC (n=15). FIG. 30C shows zsGreen percent of Thyl DC compared to zsGreen percent of aB7 TLR CAR DC (n=24, two independent experiments). FIG. 30D shows zsGreen percent of aB7(t) CAR DC compared to zsGreen percent of B7 TLR CAR DC (n=15, one experiment). Data is shown as mean ± SD.- 24 - NAI-5007599937vlStatistical significance was calculated with a paired two-tailed t test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

[0084] FIGs. 31A-31C show CAR-DCs produce antitumor memory response. FIG. 31 A shows schematic of C57B1 / 6 mice injected with 1061956 cells with 75% B7H3+ and 25% B7H3- and then on day 8 and 11 post tumor injection were injected with either aB7(t) CAR DC (n=l) or aB7 TLR CAR DC (n=5) and the mice that had complete rejections were reinjected subcutaneously with 106B7H3+ 1956 into one flank (FIG. 3 IB) and 106B7H3-1956 cells into the other flank (FIG. 31C). Tumor growth was followed.

[0085] FIGs. 32A and 32B show aB7 TLR CAR DC increases number and scope of cross presented tumor peptides. FIG. 32A shows the number of unique cross-presented tumor peptides by Thyl DC, aB7(t) CAR DC, and aB7 TLR CAR DC. FIG. 32B shows the number of shared cross-presented tumor peptides by all three DCs, Thyl DC and aB7(t) CAR DC, Thyl DC and aB7 TLR CAR DC, and aB7(t) CAR DC and aB7 TLR CAR DC.

[0086] FIGs. 33A-33C show aB7 TLR CAR DC increases mPsmd6 percent of CD8 T-cells. FIG. 33A shows schematic of C57B1 / 6 mice injected with 1061956 cells with 75% B7H3+and 25% B7H3' and then on day 8 and 11 post tumor injection were injected with aB7(t) CAR DC or aB7 TLR CAR DC and then day 14 spleens and tumors were isolated. FIG. 33B shows percent of CD8 T-cells from the spleen that were tetramer+ (mPsmd6-specific) (live, CD45+TCRβ+CD8α+CD4⁻ Tet+) in aB7(t) CAR DC or aB7 TLR CAR DC (n=10). FIG. 33C shows percent of CD8 T-cells from the tumor that were tetramer+ (mPsmd6-specific) (live, CD45+TCRβ+CD8α+CD4⁻ Tet+) in aB7(t) CAR DC (n=9) or aB7 TLR CAR DC (n=l 1) (from two independent experiments). Data is shown as mean ± SD. Statistical significance was calculated with an unpaired two-tailed Welch’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

[0087] FIG. 34 shows schematic for CD8 T-cell isolations for further transcriptional assessment. Schematic of C57B1 / 6 mice injected with 1061956 cells (75% B7H3+and 25% B7H3') and on day 8 and 11 post tumor injection were injected with aB7(t) CAR DC or aB7 TLR CAR DC and then day 14 tdLNs, spleen, and tumors were isolated. CD8 T-cells from tdLNs were assessed using scRNAseq and TCR sequencing. CD8 T-cells from tumors were sorted for Tet+ and Tet- and assessed using scRNAseq and TCR sequencing. CD8 T-cells from spleen and tdLNs were adoptively transferred to new mice.

[0088] FIG. 35 shows T-cells in the tdLN show differentially expressed genes after intratumoral treatment with aB7 TLR CAR DC compared with aB7(t) CAR DC. Significant - 25 - NAI-5007599937vl(p<0.05) DEGs (rows) with functional description from CD8 T-cells from tdLNs of mice treated with aB7(t) CAR DC (column left) or aB7 TLR CAR DC (column right) in a heat map of average log2 Fold change.

[0089] FIGs. 36A and 36B show CAR-DCs lead to oligoclonally expanded CD8 T-cells in both Tet+ and Tet- TILs. CD8 T-cell clones that were highly prevalent in the tumor (>100 clones) and present in the tdLN were plotted by frequency. Tet+ and Tet- clones, identified by sorting the tetramer+ and tetramer- TILs and independently sequencing their TCRs, are labeled at the right of each plot. Clone frequency is shown for T-cells from aB7 TLR CAR (FIG. 36A) and aB7(t) CAR DC (FIG. 36B) treated mice.

[0090] FIGs. 37A and 37B show tumor-infiltrating T-cells differentially expressed genes (DEGs) after intratumoral treatment with aB7 TLR CAR DC compared with aB7(t) CAR DC. FIG. 37A shows significant (p<0.05) DEGs (rows) with functional description from Tet+ CD8 TILs of mice treated with aB7(t) CAR DC (column left) or aB7 TLR CAR DC (column right) in a heat map of average log2 Fold change. FIG. 37B shows significant (p<0.05) DEGs (rows) with functional description from Tet- CD8 TILs of mice treated with aB7(t) CAR DC (column left) or aB7 TLR CAR DC (column right) in a heat map of average log2 Fold change.

[0091] FIG. 38 shows aB7 TLR CAR DC CD8 TILs show significantly elevated IFN responsive genes. DEGs (rows) with functional description from Tet+ CD8 TILs (left two columns) or Tet- CD8 TILs (right two columns) of mice treated with aB7(t) CAR DC or aB7 TLR CAR DC in a heat map of average log2 Fold change.

[0092] FIGs. 39A-39H show adoptively transferred CD8 T-cells from aB7 TLR CAR DC treated mice maintain a greater portion of Tet+ CD8 T-cells. FIG. 39A shows schematic of Rag mice injected with 0.1 x 1051956 cells (50% β2M⁻) and next day adoptively transferred CD8 T-cells from previously treated mice with aB7(t) CAR DC or aB7 TLR CAR DC and then spleens and tumors were isolated day 11. FIG. 39B shows Tet+ percent of CD8 T-cells (live, CD45.1+TCRβ+CD8α+CD4⁻Tet+) from the spleen and tumor of Rag mice that were adoptively transferred with CD8 T-cells (CD45.1+) originally from aB7(t) CAR DC or aB7 TLR CAR DC treated mice (n=5). Data is shown as mean ± SD. Statistical significance was calculated with an unpaired two-tailed Welch’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, **** p < 0.0001. The top 50 clones from Rag1- / - ' mouse tumors were matched to initial tumor TILs from either aB7(t) CAR DC (FIG. 39C) or aB7 TLR CAR DC (FIG. 39D) treated mice to identify which clones persisted and re-infiltrated tumor. FIGs. 39E-39H - 26 - NAI-5007599937vlshow that CAR-DCs enhance persistence and recall of tumor-specific CD8+T-cell clones in secondary Rag2⁻ / ⁻ hosts. To functionally test whether CAR-DCs endow tumor-reactive T cells with superior persistence and recall capacity, an adoptive transfer experiment was performed into secondary tumor-bearing Rag2⁻ / ⁻ mice. CD8+T cells were isolated from the tumor-draining lymph nodes and spleens of wild-type donor mice that had been treated 8 days earlier with either CAR-DCs (aB7 TLR CAR DC) or binding-only control DCs (aB7(t) CAR DC), labelled with CellTrace Violet (CTV), and transferred intravenously into Rag2⁻ / ⁻ recipients bearing mixed B7H3⁺ / B7H3⁻ tumors in which 50% of tumor cells were β2M⁻. This tumor model ensured antigen re-encounter while preventing complete tumor clearance, allowing recovery of tumor-infiltrating lymphocytes (TILs). Two weeks after transfer, tumors from recipient mice were harvested for TCR sequencing. Tetramer CD8+T cells specific for the mPSMD6 neoantigen were TCR sequenced in the original wild-type donor tumors, and total CD8+T cells were TCR sequenced from the secondary Rag2⁻ / ⁻ tumors, allowing for clonal mapping. FIG 39E illustrates Bar graph showing, for each treatment group, the total number of unique tetramer⁺ CD8+T-cell clones identified in the secondary tumor that were also detected in the corresponding primary tumor (x-axis) and the frequency of each of these shared tetramer4clones within the secondary tumor (y-axis). This represents a pool of tumor-reactive T-cell clones that had: (1) been primed and expanded in the original tumor-bearing host, (2) survived transfer into a lymphopenic environment, (3) undergone homeostatic proliferation, (4) trafficked back into a second tumor and (5) re-expanded upon antigen re-encounter. CAR-DC (aB7 TLR CAR DC) treatment increased both the number and frequency of recalled tetramer+T-cell clones compared to binding-only DCs (aB7(t) CAR DC). FIG. 39F shows mean frequency of the top 25 most abundant shared tetramer+CD8+T-cell clones depicted in FIG. 39E, demonstrating superior expansion of dominant mPSMD6-specific clones in CAR-DC (aB7 TLR CAR DC) -treated mice. FIG. 39G illustrates Bar graph showing, for each treatment group, the total number of unique CD8+T-cell clones (tetramer+and tetramer⁻) identified in the secondary tumour that were also present in the primary tumour (x-axis) and the frequency of each of these shared clones in the secondary tumour (y-axis). These T-cell clones recovered from tumors initially, and again on rechallenge, have high probability of representing tumor-specific T-cells. FIG. 39H shows mean frequency of the top 50 most abundant shared CD8+T-cell clones depicted in FIG. 39G, illustrating broader expansion of recalled CD8+T-cell clonotypes following CAR-DC treatment.- 27 - NAI-5007599937vl

[0093] FIG. 40 shows adoptively transferred CD8 T-cells from B7 TLR CAR DC treated mice repopulate tumor and have significant elevation in activation and memory related genes. Significant (p<0.05) DEGs (rows) with functional description from CD8 TILs isolated from Rag mice that were adoptively transferred IV with CD8 T-cells from mice treated with aB7(t) CAR DC (column left) or B7 TLR CAR DC (column right) in a heat map of average log2 Fold change.

[0094] FIG. 41 shows iCD103 culture produces greater cDC1s. FACS plots of primary CD103+ cDCls generated using the iCD103 differentiation protocol from a CD45.1+ C56B1 / 6 mouse gating scheme (MHCII+CD1 lc+XCRl+Sirpα-).5. DETAILED DESCRIPTION

[0095] The present disclosure provides nanobody- or monobody-based chimeric antigen receptors (CARs). A nanobody- or monobody-based CAR is a CAR comprising an extracellular antigen-binding domain that comprises a variable heavy domain of heavy chain (VHH) or a monobody. The present disclosure also provides immune cells e.g., dendritic cells and precursors thereof) comprising a nanobody or monobody-based chimeric antigen receptor (CAR). 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).

[0096] The present disclosure is based, at least in part, on the discovery that the presently disclosed nanobody / monobody -based CAR DCs surprisingly exhibit enhanced CAR expression on the cell surface of the CAR-DCs as compared to conventional scFv-based CAR-DCs (the extracellular antigen-binding domain of the CAR comprises a single chain variable fragment (scFv)). Additional surprising effects of the nanobody / monobody CAR engineered dendritic cells include, but are not limited to, improved antigen uptake, enhanced cross-presentation capability, increased ability to cross-prime tumor-reactive T-cells, enhanced promotion of T-cell proliferation, increased expression of DC surface proteins, and improved cytotoxicity (e.g., anti -turn or activity) compared to conventional scFv-based CAR-DCs. Overall, these engineered dendritic cells offer advantages over non-engineered dendritic cells and dendritic cells comprising a conventional scFv-based CARs, which make the presently disclosed CAR-DCs suitable candidates for immunotherapy.

[0097] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:5.1. Definitions;- 28 - NAI-5007599937vl5.2. 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

[0098] 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). Unless 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.

[0099] 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);- 29 - NAI-5007599937vland 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 or 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.

[0100] 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 - 30 - NAI-5007599937vlcomprise 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 et al., 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.

[0101] As used herein, the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably. A “CDR” refers to one of three hypervariable 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.

[0102] 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 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35 A 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- 31 - NAI-5007599937vlnumbering 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 numbering 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 et al., supra).

[0103] 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.

[0104] 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%,- 32 - NAI-5007599937vlat 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.

[0105] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences ( / .<?., % 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.

[0106] 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.

[0107] The percent identity between two amino acid sequences can be determined using the algorithm ofE. 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 acid 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.- 33 - NAI-5007599937vlBiol. 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.

[0108] The term “isolated cell” refers to a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0109] 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 relationships 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 ( / .<?., 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.

[0110] 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 - 34 - NAI-5007599937vlseparate 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.

[0111] 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.

[0112] “Allogeneic” refers to a graft derived from a different individual of the same species.

[0113] 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.

[0114] “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.

[0115] 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 - 35 - NAI-5007599937vlconsequences 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.

[0116] As used herein, in certain embodiments, a subject is a mammal, such as a nonprimate 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 embodiment, the subject is a mammal (e.g., a human) who is at risk of developing a disease or disorder.

[0117] 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, alleviation 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.

[0118] 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).

[0119] 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.

[0120] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0121] 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.- 36 - NAI-5007599937vl

[0122] 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.

[0123] 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

[0124] 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, an intracellular domain, and a transmembrane 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 (e.g., a 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 antigenbinding 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.

[0125] 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.

[0126] 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.- 37 - NAI-5007599937vl5.2.1. Extracellular Antigen-Binding Domain

[0127] 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. In 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.

[0128] 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.

[0129] In some embodiments, the extracellular antigen-binding domain is monovalent. In some embodiments, the extracellular antigen-binding domain comprises a single VHH - 38 - NAI-5007599937vldomain. In some embodiments, the extracellular antigen-binding domain comprises a single monobody domain. In some embodiments, the extracellular antigen-binding domain is bivalent. In some embodiments, the extracellular antigen-binding domain comprises two VHH domains. In some embodiments, the extracellular antigen-binding domain comprises two monobody domains. In some embodiments, the extracellular antigen-binding 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.

[0130] In some embodiments, a CAR comprising a VHH extracellular antigen-binding 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 antigenbinding 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 - 39 - NAI-5007599937vlVHH 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.

[0131] In some embodiments, a CAR comprising a VHH extracellular antigen-binding 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- 40 - NAI-5007599937vlextracellular antigen-binding domain exhibits about a 100-fold greater expression on the cell surface of cDCls compared to a traditional scFv-based CARs.

[0132] 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 comprising 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 antigenbinding 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 antigenbinding 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 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 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.- 41 - NAI-5007599937vl

[0133] 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 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 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.

[0134] 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 - 42 - NAI-5007599937vltarget 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.

[0135] 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, the 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, ROR1 (Receptor Tyrosine Kinase-Like Orphan Receptor 1), GPC3 (Glypican-3), MAGE-A4 (Melanoma-Associated Antigen A4), B7-H4, and Axl.

[0136] 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.

[0137] In certain embodiments, the target antigen is selected from the group consisting of B7H3, CD19, CD20, PSMA, EGFR, Her2, and EphA2.- 43 - NAI-5007599937vl

[0138] 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 framework. 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.

[0139] 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)

[0140] 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 - 44 - NAI-5007599937vlhuman 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.

[0141] 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, 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-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)

[0142] 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.

[0143] 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 - 45 - NAI-5007599937vlsystem, 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 comprises 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)

[0144] 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.

[0145] 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- 46 - NAI-5007599937vlcomprises 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) QVQLQESGGGSVQAGGSLRLRCIISRYGVTLPYMAWFRQGPGEEREGVAAITLRGST LYADNVKGRFTLSQDPPKRALFLQMNNLQPEDSGMYYCAAGTSARSLSPSDYGYRG RGTQVTVSS (SEQ ID NO: 16)

[0146] 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.

[0147] 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)- 47 - NAI-5007599937vlQVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGG YTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQ RPLDYDYWGQGTQVTVSS (SEQ ID NO: 20)

[0148] In certain embodiments, the extracellular antigen-binding domain of the presently disclosed chimeric receptor (e.g., CAR) comprises a monobody. In certain embodiments, the target 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 an 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)

[0149] 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.

[0150] In certain embodiments, the target antigen is an antigen associated with a virus. Non-limiting examples of viruses include Coronoviridae (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).- 48 - NAI-5007599937vl

[0151] 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 the 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.

[0152] 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 coll), 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).

[0153] 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.

[0154] 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 - 49 - NAI-5007599937vlcertain embodiments, the extracellular antigen-binding domain of the chimeric receptor (e.g., CAR) comprises a VHH domain that binds to beta amyloid.

[0155] 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: 50. 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-beta amyloid VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the anti-beta amyloid VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 50. SEQ ID NOs: 47-50 are provided below.GSTFRNYN (SEQ ID NO: 47)VSRTGIST (SEQ ID NO: 48)AAGRPGVGAVNRAMDYDY (SEQ ID NO: 49) QVQLQASGGGLVQTGDSLRLSCADSGSTFRNYNIGWFRQTPGQAREFVAAVSRTGIS THVADSLQGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAGRPGVGAVNRAMDY DYWGQGTQVTVSS (SEQ ID NO: 50)5.2.2. Transmembrane Domain

[0156] 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 aFcR, 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.

[0157] 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.- 50 - NAI-5007599937vl

[0158] 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 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 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)

[0159] 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 - 51 - NAI-5007599937vlof 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 TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSL SARYV (SEQ ID NO: 26)

[0160] 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- 52 - NAI-5007599937vlSLTLTMNKGSISFKKVALPSLSYLDLSRNALSFSGCCSYSDLGTNSLRHLDLSFNGAII MSANFMGLEELQHLDFQHSTLKRVTEFSAFLSLEKLLYLDISYTNTKIDFDGIFLGLTS LNTLKMAGNSFKDNTLSNVFANTTNLTFLDLSKCQLEQISWGVFDTLHRLQLLNMS HNNLLFLDSSHYNQLYSLSTLDCSFNRIETSKGILQHFPKSLAFFNLTNNSVACICEHQ KFLQWVKEQKQFLVNVEQMTCATPVEMNTSLVLDFNNSTCYMYKTIISVSVVSVIV VSTVAFLIYHFYFHLILIAGCKKYSRGESIYDAFVIYSSONEDWVRNELVKNLEEGVP RFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSRWCIFEYEIAQTWQFL SSRSGIIFIVLEKVEKSLLRQQVELYRLLSRNTYLEWEDNPLGRHIFWRRLKNALLDG KASNPEQTAEEEQETATWT (SEQ ID NO: 27)

[0161] 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- 53 - NAI-5007599937vlCTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVL SVLVVSVVAVLVYI< FYFHLMLLAGCII< YGRGENIYDAFVIYSSODEDWVRNELVI< N LEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQ TWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRK ALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 28)

[0162] 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)

[0163] 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 - 54 - NAI-5007599937vlamino 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)

[0164] 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 FCERG. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of FCERG or a fragment thereof. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from mouse FCERG. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of mouse FCERG 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 - 55 - NAI-5007599937vlconsecutive 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)

[0165] In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) is derived from human FCERG. In certain embodiments, the transmembrane domain of the chimeric receptor (e.g., CAR) comprises a transmembrane domain of human FCERG 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)

[0166] 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.- 56 - NAI-5007599937vl5.2.3 Intracellular Domain

[0167] In certain embodiments, the chimeric receptor is a chimeric antigen receptor (CAR) that 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 tyrosinebased 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.

[0168] 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 embodiment, the TLR is TLR4.

[0169] In certain embodiments, the DC-activating molecule is CD40.

[0170] In certain embodiments, the DC-activating molecule is selected from the group consisting of a TLR, CD40, CD3(^, 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 FCERG (also known as “Fc receptor gamma-chain (FcRgamma)”, “Fc-epsilon Rl-gamma IgE”, “Fc receptor subunit gamma (FceRI gamma)”).- 57 - NAI-5007599937vlIn certain embodiments, the DC-activating molecule is FMS-like tyrosine kinase 3 receptor (Flt3). In specific embodiments, the intracellular domain of the CAR does not comprise FMS-like tyrosine kinase 3 (FLT3) or a fragment thereof.

[0171] CD40

[0172] In certain embodiments, the intracellular domain of the CAR is derived from CD40. In 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)

[0173] 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 - 58 - NAI-5007599937vl60, 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).MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTET ECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTS EACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKD LVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHP KOEPOEINFPDDLPGSNTAAPVOETLHGCOPVTQEDGKESRISVOERO (SEQ ID NO: 32)

[0174] 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 (IT M)-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 ITAM. In certain embodiments, the ITAM-containing activating receptor is selected from the group consisting of 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. In certain embodiments, the ITAM-containing activating receptor is CD3.

[0175] CD3

[0176] In certain embodiments, the intracellular domain of the CAR is derived from CD3(^. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of CD3(^ or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse CD3(^. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse CD3(^ 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- 59 - NAI-5007599937vlembodiments, 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).MKWKVSVLACILHVRFPGAEAQSFGLLDPKLCYLLDGILFIYGVIITALYLRAKFSRS AETAANLODPNOLYNELNLGRREEYDVLEKKRARDPEMGGKOQRRRNPOEGVYNA LOKDKMAEAYSEIGTKGERRRGKGHDGLYOGLSTATKDTYDALHMQTLAPR (SEQ ID NO: 33)

[0177] In certain embodiments, the intracellular domain of the CAR is derived from human CD3(^. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human CD3(^ 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 DAPAYOOGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPOEGLYNEL OKDKMAEAYSEIGMKGERRRGKGHDGLYOGLSTATKDTYDALHMQALPPR (SEQ ID NO: 34)

[0178] Fc Receptor (FcR)

[0179] 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 FCERG. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain - 60 - NAI-5007599937vlof FCERG or a fragment thereof. In certain embodiments, the intracellular domain of the CAR is derived from mouse FCERG. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of mouse FCERG 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: 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)

[0180] In certain embodiments, the intracellular domain of the CAR is derived from human FCERG. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain of human FCERG 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)

[0181] Toll-like Receptor (TLR)

[0182] 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 - 61 - NAI-5007599937vlthe 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 mouse 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)

[0183] 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 - 62 - NAI-5007599937vlintracellular 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 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, 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)

[0184] 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 CD3(^, CD3y, CD36, CD3s, a Fc receptor (including, but not limited to, a Fc-gamma receptor (FcyR), a Fc-epsilon receptor - 63 - NAI-5007599937vl(FcsR), CD64, CD32A, or CD16A), CD79a, CD79b, TREM-1, and TREM-2. In certain embodiments, the ITAM-containing activating receptor is CD3.

[0185] 0X40

[0186] In certain embodiments, the intracellular domain of the CAR comprises an intracellular 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 ITAM-containing activating receptor is selected from the group consisting of 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. In certain embodiments, the ITAM-containing activating receptor is CD3.

[0187] 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)

[0188] 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 - 64 - NAI-5007599937vlof 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 portion 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)

[0189] 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

[0190] 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.

[0191] 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,- 65 - NAI-5007599937vlat 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 least 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.

[0192] 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, CD3(^, 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 - 66 - NAI-5007599937vlproteins 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.

[0193] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived 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.

[0194] 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 - 67 - NAI-5007599937vl159 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 embodiments, 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 TLNKFSKENEGYYFCSVISNSVMYFSSVVPVLQKF WZZZAPFZ / JTPAPEHPTG'TNgP / gPEDC / gP / gG'AWrG'TGZD ACD / FIWAPLAGICVALLLSLIITLICYHRSRKRVCKCPR PLVRQEGKPRPSEKIV (SEQ ID NO: 25)

[0195] 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.

[0196] 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.,- 68 - NAI-5007599937vlCAR) 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 forth in SEQ ID NO: 42, which is provided below.CSLTT (SEQ ID NO: 41)TTAPTKKTTLKMKKKKQCPFPHPETQKGLTCSLTT (SEQ ID NO: 42)

[0197] 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 - 69 - NAI-5007599937vlcertain 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 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 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.

[0198] 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 - 70 - NAI-5007599937vlleast 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.

[0199] 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,- 71 - NAI-5007599937vlthe 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 comprises 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.

[0200] 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)- 72 - NAI-5007599937vlcomprises 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 at 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)- 73 - NAI-5007599937vl

[0201] 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) comprises 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 - 74 - NAI-5007599937vlacids, 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.

[0202] 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 - 75 - NAI-5007599937vlamino 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 portion of the extracellular domain of TLR4 that is proximate to the transmembrane domain ofTLR4.

[0203] 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- 76 - NAI-5007599937vlembodiments, 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 portion 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.

[0204] 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 - 77 - NAI-5007599937vlacids, 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 certain 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- 78 - NAI-5007599937vlALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLT NLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLR NNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLA YLD YYLDDIIDLFNCLTNVS SF SL VS VTIERVKDF SYNFGWQHLELVNCKFGQFPTLK LKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLS FNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGI FNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVL NMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFA CTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCOMNKTIIGVSVL SVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKN LEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQ TWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRK ALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 28)

[0205] Identification of the less structured portion of the TLR4 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 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.

[0206] 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 - 79 - NAI-5007599937vl0X40. 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 Reference 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 - 80 - NAI-5007599937vlabout 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.

[0207] 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.

[0208] 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 - 81 - NAI-5007599937vlup 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 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 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.

[0209] 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? SLLATLLWETQRPTFRPTTVOSTTVWPRTSELPSPPTLVTPEGP^ VEEGEGEGEE^PE VLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI (SEQ ID NO: 29)

[0210] 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,- 82 - NAI-5007599937vlthe 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 acids, 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 - 83 - NAI-5007599937vlabout 100 consecutive amino acids.

[0211] 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.

[0212] 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- 84 - NAI-5007599937vlstructured 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 human 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

[0213] 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 RDPPATOPOETOGPPARPITVQPTEAWPRTSOGPSTRPVEVPGGRAN MLGEGEVEGE LGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 30)

[0214] 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 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 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 - 85 - NAI-5007599937vlis 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.

[0215] 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. 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.- 86 - NAI-5007599937vl

[0216] 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 amino 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)

[0217] 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- 87 - NAI-5007599937vlembodiments, 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 underlined).VNSTTTKPVLRTPSPVHPTGTSQPQNFTDCRPRGSVKGTGLDFACDIY (SEQ ID NO: 38)

[0218] 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).

[0219] 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).- 88 - NAI-5007599937vlEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPNFTSPPTLVTPEGP (SEQ ID NO: 39)

[0220] 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 of amino acids 166 to 211 of SEQ ID NO: 29, wherein three native amino acid residues are substituted with a N-linked glycosylation consensus sequence.

[0221] 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: 51. SEQ ID NO: 51 is provided below (NFT glycosylation site underlined).EDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTNFTRPVEVPGGRA (SEQ ID NO: 51)

[0222] In certain embodiments, the modified extracellular domain of 0X40 comprises the amino acid sequence set forth in SEQ ID NO: 60. SEQ ID NO: 60 is provided below (NFS glycosylation site underlined).EDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPNFSTRPVEVPGGRA (SEQ ID NO: 60)

[0223] 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 a N-linked glycosylation consensus sequence.

[0224] The embodiment encompasses that for a CAR whose intracellular domain is - 89 - NAI-5007599937vlderived 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. In certain embodiments, the hinge region comprises a less structured portion of the extracellular domain of a TLR (e.g., 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 horseshoeshaped 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 - 90 - NAI-5007599937vlregion 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 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.

[0225] 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- 91 - NAI-5007599937vlstructured 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 derived 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- 92 - NAI-5007599937vlextracellular 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: 51. 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: 51. 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: 51. 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: 51. 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: 60. 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: 60. 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: 60. 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: 60.

[0226] Identification of the less structured portion of the second protein 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 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 - 93 - NAI-5007599937vldelineated 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.

[0227] In certain embodiments, the hinge region of the CAR is derived from a FcR. In certain 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 FCERG. In certain embodiments, the hinge region of the CAR comprises an extracellular domain of FCERG or a fragment thereof. In certain embodiments, the hinge region of the CAR is derived from mouse FCERG. 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)

[0228] In certain embodiments, the hinge region of the chimeric receptor (e.g., CAR) is derived from human FCERG. In certain embodiments, the hinge region of the chimeric receptor e.g., CAR) comprises an extracellular domain of human FCERG 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.- 94 - NAI-5007599937vlIn 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)5.2.5. Signal Peptide

[0229] 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.

[0230] 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)

[0231] Exemplary VHH CARs each comprising an extracellular antigen-binding domain that comprises a VHH domain are generated as shown in Section 6 below.

[0232] 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).

[0233] 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 - 95 - NAI-5007599937vlembodiments, 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 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 “aB7 CD40(s)” or “aB7H3 CD40(s)” in Section 6. Examples.

[0234] 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.- 96 - NAI-5007599937vl

[0235] 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 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 “aB7 CD40(l)” or “aB7H3 CD40(l)” in Section 6. Examples.

[0236] 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- 97 - NAI-5007599937vlintracellular 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 “aB7 CD40(m)” or “aB7H3 CD40(m)” in Section 6. Examples.

[0237] 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 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 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.

[0238] 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 - 98 - NAI-5007599937vlSEQ 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 the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “aPSMA CD40(l)” in Section 6. Examples.

[0239] 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)” or “al 9 CD40(m)” in Section 6. Examples.

[0240] 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 - 99 - NAI-5007599937vltransmembrane 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 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(l)” or “al 9 CD40(l)” in Section 6. Examples.

[0241] 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 - 100 - NAI-5007599937vlsignal peptide is derived from IgHl. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0242] 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 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 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 “aCD20 CD40(l)” in Section 6. Examples.

[0243] 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 - 101 - NAI-5007599937vlsequence 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.

[0244] 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 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 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 “aEGFR CD40(l)” in Section 6. Examples.

[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 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 CD3(^ (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 - 102 - NAI-5007599937vlanti-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. This VHH CAR is also referred as “aB7 CD3< ’ or “aB7H3 CD3 ’ in Section 6. Examples.

[0246] 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 CD3(^ (e.g, an intracellular domain of CD3Q. 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 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. This VHH CAR is also referred as “aCD19 CD3< ’ or “al9 CD3 ’ in Section 6. Examples.

[0247] In some embodiments, provided herein is a VHH CAR comprising an- 103 - NAI-5007599937vlextracellular 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 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 FcR” or “aB7H3 FcR” in Section 6. Examples.

[0248] 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 - 104 - NAI-5007599937vlID 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. This VHH CAR is also referred as “aCD19 FcR” or “al9 FcR” in Section 6. Examples.

[0249] 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 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 SEQ 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. This VHH CAR is also referred as “aB70X40” or “aB7H3 0X40” in Section 6. Examples.

[0250] 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,- 105 - NAI-5007599937vland 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. This VHH CAR is also referred as “aCD190X40” or “al 90X40” in Section 6. Examples.

[0251] 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 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. 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(20) ” or “aB7H3 TLR(20) ” in Section 6. Examples.

[0252] 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 - 106 - NAI-5007599937vlthereof), 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. This VHH CAR is also referred as “T455” or “aB7H3 T455” in Section 6. Examples.

[0253] 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 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.- 107 - NAI-5007599937vlIn 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 “T4112” or “aB7H3 T4112” in Section 6. Examples.

[0254] 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 signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 40. This VHH CAR is also referred as “T4160” or “aB7H3 T4160” in Section 6. Examples.

[0255] 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 sequen...

Claims

1. WHAT IS CLAIMED:

1. A dendritic cell or a precursor thereof comprising: a chimeric antigen receptor (CAR), wherein the CAR comprises: a) an extracellular antigen-binding domain that binds to a target antigen, b) a transmembrane domain, and c) an intracellular domain 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 CD3(^, a toll-like receptor (TLR), a Fc receptor (FcR), and CD40, and wherein the extracellular antigen-binding domain comprises i) a variable heavy domain of heavy chain (VHH) domain or ii) a monobody.

2. The dendritic cell or precursor thereof of claim 1, wherein the intracellular domain comprises an intracellular domain of the DC-activating molecule or a fragment thereof.

3. The dendritic cell or precursor thereof of claim 1 or 2, wherein the TLR is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11.

4. The dendritic cell or precursor thereof of claim 3, wherein the TLR is TLR4.

5. The dendritic cell or precursor thereof of any one of claim 1-4, wherein the intracellular domain of the CAR is derived from TLR4 and comprises amino acids 660 to 835 of SEQ ID NO: 27 or a fragment thereof.

6. The dendritic cell or precursor thereof of any one of claims 1-4, wherein the intracellular domain of the CAR is derived from TLR4 and comprises amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof.

7. The dendritic cell or precursor thereof of claim 1 or 2, wherein the intracellular domain of the CAR is derived from CD3(^ and comprises amino acids 52 to 164 of SEQ ID NO: 33 or a fragment thereof.

8. The dendritic cell or precursor thereof of claim 1 or 2, wherein the intracellular domain of the CAR is derived from CD3(^ and comprises amino acids 52 to 164 of SEQ ID NO: 34 or a fragment thereof.

9. The dendritic cell or precursor thereof of claim 1, wherein the FcR is Fc gamma receptor.11.- 167 - NAI-5007599937vl 10. The dendritic cell or precursor thereof of claim 9, wherein the Fc gamma receptor is FCERG.

11. The dendritic cell or precursor thereof of any one of claims 1, 2, 9, and 10, wherein the intracellular domain is derived from FCERG and comprises amino acids 45 to 86 of SEQ ID NO: 35 or a fragment thereof.

12. The dendritic cell or precursor thereof of any one of claims 1, 2, 9, and 10, wherein the intracellular domain is derived from FCERG and comprises amino acids 45 to 86 of SEQ ID NO: 36 or a fragment thereof.

13. The dendritic cell or precursor thereof of claim 1 or 2, wherein the intracellular domain is derived from CD40 and comprises amino acids 216 to 289 of SEQ ID NO: 31.

14. The dendritic cell or precursor thereof of claim 1 or 2, wherein the intracellular domain is derived from CD40 and comprises amino acids 216 to 277 of SEQ ID NO: 32.

15. The dendritic cell or precursor thereof of any one of claims 1-14, wherein the transmembrane domain is derived from CD8, a Toll-like receptor, CD40, CD3(^, a FcR, CD166, 0X40, CD28, 4-1BB, or ICOS.

16. The dendritic cell or precursor thereof of any one of claims 1-15, wherein the transmembrane domain of the CAR is derived from CD8.

17. The dendritic cell or precursor thereof of claim 16, wherein the CD8 is CD8a.

18. The dendritic cell or precursor thereof of any one of claims 1-17, wherein the transmembrane domain of the CAR comprises a transmembrane domain of CD8 or a fragment thereof.

19. The dendritic cell or precursor thereof of any one of claims 1-18, wherein the transmembrane domain of the CAR comprises a transmembrane domain of CD8a or a fragment thereof.

20. The dendritic cell or precursor thereof of any one of claims 1-19, wherein the transmembrane domain of the CAR is derived from CD8a and comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof.22.- 168 - NAI-5007599937vl 21. The dendritic cell or precursor thereof of any one of claims 1-19, wherein the transmembrane domain of the CAR is derived from CD8a and comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

22. The dendritic cell or precursor thereof of any one of claims 1-15, wherein the transmembrane domain of the CAR is derived from a TLR.

23. The dendritic cell or precursor thereof of any one of claims 1-15, and 22, wherein the transmembrane domain of the CAR comprises a transmembrane domain of TLR or a fragment thereof.

24. The dendritic cell or precursor thereof of any one of claims 1-15, 22, and 23, wherein the TLR is TLR4.

25. The dendritic cell or precursor thereof of any one of claims 1-15 and 22-24, wherein the transmembrane domain of the CAR comprises a transmembrane domain of TLR4 or a fragment thereof.

26. The dendritic cell or precursor thereof of any one of claims 1-15 and 22-25, wherein the transmembrane domain is derived from TLR4 and comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof.

27. The dendritic cell or precursor thereof of any one of claims 1-15 and 22-25, wherein the transmembrane domain is derived from TLR4 and comprises amino acids 632-652 of SEQ ID NO: 28 or a fragment thereof.

28. The dendritic cell or precursor thereof of any one of claims 1-15, wherein the transmembrane domain of the CAR is derived from 0X40.

29. The dendritic cell or precursor thereof of any one of claims 1-15, and 28, wherein the transmembrane domain of the CAR comprises a transmembrane domain of 0X40 or a fragment thereof.

30. The dendritic cell or precursor thereof of any one of claims 1-15, 28, and 29, wherein the transmembrane domain is derived from 0X40 and comprises amino acids 212 to 236 of SEQ ID NO: 29 or a fragment thereof.

31. The dendritic cell or precursor thereof of any one of claims 1-15, and 28-30, wherein - 169 - NAI-5007599937vlthe transmembrane domain is derived from 0X40 and comprises amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof.

32. The dendritic cell or precursor thereof of any one of claims 1-31, wherein the CAR further comprises d) a hinge region.

33. The dendritic cell or precursor thereof of claim 32, wherein the hinge region has a length of between about 5 and about 100 amino acids.

34. The dendritic cell or precursor thereof of claim 32 or claim 33, wherein the hinge region has a length of between about 10 and about 50 amino acids.

35. The dendritic cell or precursor thereof of any one of claims 32-34, wherein the hinge region has a length of about 20, about 40, or about 50 amino acids.

36. The dendritic cell or precursor thereof of claim 32 or claim 33, wherein the hinge region has a length of between about 50 and about 100 amino acids.

37. The dendritic cell or precursor thereof of any one of claims 32, 33, and 36, wherein the hinge region has a length of about 70 amino acids.

38. The dendritic cell or precursor thereof of any one of claims 32-37, wherein the hinge region is positioned between the extracellular antigen-binding domain and the transmembrane domain.

39. The dendritic cell or precursor thereof of any one of claims 32-38, wherein the hinge region is derived from CD8, a TLR, CD40, CD3< a FcR, CD166, 0X40, IgGl, IgG4, CD28, 4-1BB, or ICOS.

40. The dendritic cell or precursor thereof of any one of claims 32-39, wherein the hinge region is derived from CD 8.

41. The dendritic cell or precursor thereof of any one of claims 32-40, wherein the hinge region comprises a native or modified extracellular domain of CD8 or a fragment thereof.

42. The dendritic cell or precursor thereof of claim 40 or claim 41, wherein the CD8 is CD8a.

43. The dendritic cell or precursor thereof of any one of claims 40-42, wherein the hinge - 170 - NAI-5007599937vlregion comprises a native extracellular domain of CD8a or a fragment thereof.

44. The dendritic cell or precursor thereof of claim 43, 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.

45. The dendritic cell or precursor thereof of claim 43, wherein the native extracellular domain of CD8a or fragment thereof comprises amino acids 159 to 196 of SEQ ID NO: 25 or a fragment thereof.

46. The dendritic cell or precursor thereof of claim 43, wherein the native extracellular domain of CD8a or fragment thereof comprises amino acids 179 to 196 of SEQ ID NO: 25 or a fragment thereof.

47. The dendritic cell or precursor thereof of any one of claims 40-42, wherein the hinge region comprises a modified extracellular domain of CD8a or a fragment thereof.

48. The dendritic cell or precursor thereof of claim 47, 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.

49. The dendritic cell or precursor thereof of any one of claims 32-39, wherein the hinge region is derived from a TLR.

50. The dendritic cell or precursor thereof of any one of claims 32-39, and 49, wherein the hinge region comprises a native or modified extracellular domain of a TLR.

51. The dendritic cell or precursor thereof of claim 49 or claim 50, wherein the TLR is TLR4.

52. The dendritic cell or precursor thereof of any one of claims 32-39, 50, and 51, wherein the hinge region comprises a native extracellular domain of TLR4 or a fragment thereof.

53. The dendritic cell or precursor thereof of claim 52, 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.55.- 171 - NAI-5007599937vl 54. The dendritic cell or precursor thereof of claim 52, 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.

55. The dendritic cell or precursor thereof of any one of claims 32-39, wherein the hinge region is derived from 0X40.

56. The dendritic cell or precursor thereof of any one of claims 32-39, and 55, wherein the hinge region comprises a native extracellular domain of 0X40 or a fragment thereof.

57. The dendritic cell or precursor thereof of claim 56, 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.

58. The dendritic cell or precursor thereof of claim 56, 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.

59. The dendritic cell or precursor thereof of any one of claims 32-39, and 55, wherein the hinge region comprises a modified extracellular domain of 0X40 or a fragment thereof.

60. The dendritic cell or precursor thereof of claim 59, wherein the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 39.

61. The dendritic cell or precursor thereof of claim 59, wherein the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 60.

62. The dendritic cell or precursor thereof of any one of claims 32-39, wherein 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.

63. The dendritic cell or precursor thereof of any one of claims 32-39 and 62, wherein the hinge region comprises a native extracellular domain of TLR4 or a fragment thereof, and a modified extracellular domain of 0X40 or a fragment thereof.

64. The dendritic cell or precursor thereof of claim 63, wherein the native extracellular - 172 - NAI-5007599937vldomain of TLR4 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.

65. The dendritic cell or precursor thereof of claim 63, 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, and the modified extracellular domain of 0X40 or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 60.

66. The dendritic cell or precursor thereof of any one of claims 62-65, wherein the intracellular domain of the CAR is derived from a TLR.

67. The dendritic cell or precursor thereof of any one of claims 62-66, wherein the intracellular domain of the CAR is derived from TLR4.

68. The dendritic cell or precursor thereof of any one of claims 62-67, wherein the transmembrane domain of the CAR is derived from a TLR.

69. The dendritic cell or precursor thereof of any one of claims 62-68, wherein the transmembrane domain of the CAR is derived from TLR4.

70. The dendritic cell or precursor thereof of any one of claims 1-69, wherein the target antigen is a tumor antigen.

71. The dendritic cell or precursor thereof of claim 70, wherein the tumor is a solid tumor.

72. The dendritic cell or precursor thereof of claim 70 or claim 71, 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.

73. The dendritic cell or precursor thereof of claim 70, wherein the tumor is a liquid tumor.

74. The dendritic cell or precursor thereof of claim 70 or claim 73, wherein the target antigen is selected from the group consisting of CD 19, CD20, B-cell maturation antigen - 173 - NAI-5007599937vl(BCMA), G-protein-coupled receptor class 5 member D (GPRC5D), Fc receptor-like 5 (FCRL5), CD22, CD33, CD123, and CD30.

75. The dendritic cell or precursor thereof of claim 70, 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.

76. The dendritic cell or precursor thereof of claim 75, wherein the target antigen is selected from the group consisting of B7H3, EphA2, and Her2.

77. The dendritic cell or precursor thereof of any one of claims 1-76, wherein the target antigen is a cancer antigen.

78. The dendritic cell or precursor thereof of any one of claims 1-77, wherein the target antigen is selected from the group consisting of B7H3, PSMA, CD19, CD20, EGFR, Her2, EphA2, and beta amyloid.

79. The dendritic cell or precursor thereof of any one of claims 1-69, 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.

80. The dendritic cell or precursor thereof of claim 79, 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), CD 177, and keratin.

81. The dendritic cell or precursor thereof of any one of claims 1-80, wherein the extracellular antigen-binding domain comprises a VHH domain.

82. The dendritic cell or precursor thereof of claim 81, wherein the target antigen is B7H3 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 4.84.- 174 - NAI-5007599937vl 83. The dendritic cell or precursor thereof of claim 81, wherein the target antigen is PSMA and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 8.

84. The dendritic cell or precursor thereof of claim 81, wherein the target antigen is CD 19 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 12.

85. The dendritic cell or precursor thereof of claim 81, wherein the target antigen is CD20 and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 16.

86. The dendritic cell or precursor thereof of claim 81, wherein the target antigen is EGFR and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 20.

87. The dendritic cell or precursor thereof of claim 81, wherein the target antigen is beta amyloid and the VHH domain comprises the CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 50.

88. The dendritic cell or precursor thereof of any one of claims 82-87, wherein 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.

89. The dendritic cell or precursor thereof of claim 82 or claim 88, wherein 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.

90. The dendritic cell or precursor thereof of any one of claims 82, 88, and 89, wherein the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 4.

91. The dendritic cell or precursor thereof of claim 83 or claim 88, wherein 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.

92. The dendritic cell or precursor thereof of any one of claims 83, 88, and 91, wherein94.- 175 - NAI-5007599937vl the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

93. The dendritic cell or precursor thereof of claim 84 or claim 88, wherein 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.

94. The dendritic cell or precursor thereof of any one of claims 84, 88, and 93, wherein the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 12.

95. The dendritic cell or precursor thereof of claim 85 or claim 88, wherein 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.

96. The dendritic cell or precursor thereof of any one of claims 85, 88, and 95, wherein the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 16.

97. The dendritic cell or precursor thereof of claim 86 or claim 88, wherein 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.

98. The dendritic cell or precursor thereof of any one of claims 86, 88, and 97, wherein the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 20.

99. The dendritic cell or precursor thereof of claim 87 or claim 88, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 47, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 48, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 49.

100. The dendritic cell or precursor thereof of any one of claims 87, 88, and 99, wherein the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 50.

101. The dendritic cell or precursor thereof of any one of claims 1-80, wherein the extracellular antigen-binding domain comprises a monobody.

102. The dendritic cell or precursor thereof of claim 101, wherein the monobody is based - 176 - NAI-5007599937vlon a fibronectin type III (FN3) domain.

103. The dendritic cell or precursor thereof of claim 101 or claim 102, wherein the monobody comprises a BC loop, a DE loop, and an FG loop.

104. The dendritic cell or precursor thereof of any one of claims 101-103, 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.

105. The dendritic cell or precursor thereof of any one of claims 101-104, wherein the monobody comprises the amino acid sequence set forth in SEQ ID NO: 24.

106. The dendritic cell or precursor thereof of any one of claims 1-105, wherein the cell further comprises a signal peptide.

107. The dendritic cell or precursor thereof of claim 106, wherein the signal peptide is covalently connected to the N-terminus of the extracellular antigen-binding domain.

108. A chimeric antigen receptor (CAR) comprising: a) an extracellular antigen-binding domain that binds to a target antigen, b) a transmembrane domain, and c) an intracellular domain derived from CD40, a Toll-like receptor (TLR), or 0X40, wherein the extracellular antigen-binding domain comprises i) a variable heavy domain of heavy chain (VHH domain) or ii) a monobody, and wherein the intracellular domain is not capable of activating an immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling.

109. The CAR of claim 108, wherein the intracellular domain is derived from a TLR.

110. The CAR of claim 108 or claim 109, wherein the TLR is TLR4.

111. The CAR of any one of claims 108-110, wherein the intracellular domain is derived from TLR4 and comprises amino acids 660 to 835 of SEQ ID NO: 27 or a fragment thereof.

112. The CAR of any one of claims 108-110, wherein the intracellular domain is derived from TLR4 and comprises amino acids 653 to 839 of SEQ ID NO: 28 or a fragment thereof.

113. The CAR of claim 108, wherein the intracellular domain is derived from CD40.116.- 177 - NAI-5007599937vl 114. The CAR of claim 108 or claim 113, wherein the intracellular domain is derived from CD40 and comprises amino acids 216 to 289 of SEQ ID NO: 31 or a fragment thereof.

115. The CAR of claim 108 or claim 113, wherein the intracellular domain is derived from CD40 and comprises amino acids 216 to 277 of SEQ ID NO: 32 or a fragment thereof.

116. The CAR of claim 108, wherein the intracellular domain is derived from 0X40.

117. The CAR of claim 108 or claim 116, wherein the intracellular domain is derived from 0X40 and comprises amino acids 237 to 272 of SEQ ID NO: 29 or a fragment thereof.

118. The CAR of claim 108 or claim 116, wherein the intracellular domain is derived from 0X40 and comprises amino acids 236 to 277 of SEQ ID NO: 30 or a fragment thereof.

119. The CAR of any one of claims 108-118, wherein the transmembrane domain is derived from CD8, a Toll-like receptor, CD40, CD3(^, a FcR, CD166, or 0X40.

120. The CAR of any one of claims 108-119, wherein the transmembrane domain of the CAR is derived from CD 8.

121. The CAR of claim 120, wherein the CD8 is CD8a.

122. The CAR of any one of claims 108-121, wherein the transmembrane domain of the CAR comprises a transmembrane domain of CD8a or a fragment thereof.

123. The CAR of any one of claims 108-122, wherein the transmembrane domain is derived from CD8a and comprises amino acids 197 to 217 of SEQ ID NO: 25 or a fragment thereof.

124. The CAR of any one of claims 108-122, wherein the transmembrane domain of the CAR is derived from CD8a and comprises amino acids 183 to 203 of SEQ ID NO: 26 or a fragment thereof.

125. The CAR of any one of claims 108-119, wherein the transmembrane domain of the CAR is derived from TLR.

126. The CAR of claim 125, wherein the TLR is TLR4.

127. The CAR of any one of claims 108-119, 125, and 126, wherein the transmembrane - 178 - NAI-5007599937vldomain of the CAR comprises a transmembrane domain of TLR4 or a fragment thereof.

128. The CAR of any one of claims 108-119, and 125-127, wherein the transmembrane domain of the CAR is derived from TLR4 and comprises amino acids 639 to 659 of SEQ ID NO: 27 or a fragment thereof.

129. The CAR of any one of claims 108-119, and 125-127, wherein the transmembrane domain of the CAR is derived from TLR4 and comprises amino acids 632-652 of SEQ ID NO: 28 or a fragment thereof.

130. The CAR of any one of claims 108-119, wherein the transmembrane domain of the CAR is derived from 0X40.

131. The CAR of any one of claims 108-119 and 130, wherein the transmembrane domain of the CAR comprises a transmembrane domain of 0X40 or a fragment thereof.

132. The CAR of any one of claims 108-119, 130, and 131, wherein the transmembrane domain of the CAR is derived from 0X40 and comprises amino acids 212 to 236 of SEQ ID NO: 29 or a fragment thereof.

133. The CAR of any one of claims 108-119, 130, and 131, wherein the transmembrane domain is derived from 0X40 and comprises amino acids 215 to 235 of SEQ ID NO: 30 or a fragment thereof.

134. The CAR of any one of claims 108-133, wherein the CAR further comprises a hinge region.

135. The CAR of claim 134, wherein the hinge region has a length of between about 5 and about 100 amino acids or between about 5 and about 50 amino acids.

136. The CAR of claim 134 or claim 135, wherein the hinge region is positioned between the extracellular antigen-binding domain and the transmembrane domain.

137. The CAR of any one of claims 134-136, wherein the hinge region is derived from CD8, a TLR, CD40, CD3< a FcR, CD166, 0X40, IgGl, IgG4, CD28, 4-1BB, or ICOS.

138. The CAR of any one of claims 134-137, wherein the hinge region is derived from CD8.141.- 179 - NAI-5007599937vl 139. The CAR of claim 138, wherein the CD8 is CD8a.

140. The CAR of any one of claims 134-138, wherein the hinge region comprises a native extracellular domain of CD8a or a fragment thereof.

141. The CAR of claim 140, wherein the hinge region is derived from CD8a and comprises amino acids 149 to 196 of SEQ ID NO: 25 or a fragment thereof.

142. The CAR of claim 140, wherein the hinge region is derived from CD8a and comprises amino acids 159 to 196 of SEQ ID NO: 25.

143. The CAR of claim 140, wherein the hinge region is derived from CD8a and comprises amino acids 179 to 196 of SEQ ID NO: 25.

144. The CAR of any one of claims 134-138, wherein the hinge region comprises a modified extracellular domain of CD8a or a fragment thereof.

145. The CAR of claim 144, wherein the hinge region is derived from CD8a and 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.

146. The CAR of any one of claims 134-137, wherein the hinge region is derived from a TLR.

147. The CAR of claim 146, wherein the TLR is TLR4.

148. The CAR of any one of claims 134-137, 146, and 147, wherein the hinge region comprises a native extracellular domain of TLR4 or a fragment thereof.

149. The CAR of any one of claims 134-137, and 146-148, wherein the hinge region is derived from TLR4 and comprises amino acids 619 to 638 of SEQ ID NO: 27 or a fragment thereof.

150. The CAR of any one of claims 134-137, and 146-148, wherein the hinge region is derived from TLR4 and comprises amino acids 622 to 631 of SEQ ID NO: 28 or a fragment thereof.

151. The CAR of any one of claims 134-137, wherein the hinge region is derived from 0X40.154.- 180 - NAI-5007599937vl 152. The CAR of any one of claims 134-137, and 151, wherein the hinge region comprises a native extracellular domain of 0X40 or a fragment thereof.

153. The CAR of any one of claims 134-137, 151, and 152, wherein the hinge region is derived from 0X40 and comprises amino acids 166 to 211 of SEQ ID NO: 29 or a fragment thereof.

154. The CAR of any one of claims 134-137, 151, and 152, wherein the hinge region is derived from 0X40 and comprises amino acids 167 to 214 of SEQ ID NO: 30 or a fragment thereof.

155. The CAR of any one of claims 134-137, and 151, wherein the hinge region comprises a modified extracellular domain of 0X40 or a fragment thereof.

156. The CAR of any one of claims 134-137, 151, and 155, wherein the hinge region is derived from 0X40 and comprises the amino acid sequence set forth in SEQ ID NO: 39.

157. The CAR of any one of claims 134-137, 151, and 155, wherein the hinge region is derived from 0X40 and comprises the amino acid sequence set forth in SEQ ID NO: 60.

158. The CAR of any one of claims 134-137, wherein the hinge region comprises a native or modified extracellular domain of TLR4 or a fragment thereof, and a native or modified extracellular domain of 0X40 or a fragment thereof.

159. The CAR of any one of claims 134-137, and 158, wherein the hinge region comprises a native extracellular domain of TLR4 or a fragment thereof, and a modified extracellular domain of 0X40 or a fragment thereof.

160. The CAR of claim 159, wherein the native extracellular domain of TLR4 or a 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 a fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 39.

161. The CAR of claim 159, wherein the native extracellular domain of TLR4 or a 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 a fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 60.164.- 181 - NAI-5007599937vl 162. The CAR of any one of claims 158-161, wherein the intracellular domain of the CAR is derived from TLR4.

163. The CAR of any one of claims 158-162, wherein the transmembrane domain of the CAR is derived from TLR4.

164. The CAR of any one of claims 108-163, wherein the target antigen is a tumor antigen.

165. The CAR of claim 164, wherein the tumor is a solid tumor.

166. The CAR of claim 164 or claim 165, 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, ROR1 (Receptor Tyrosine Kinase-Like Orphan Receptor 1), GPC3 (Glypican-3), MAGE-A4 (Melanoma-Associated Antigen A4), B7-H4, and Axl.

167. The CAR of claim 164, wherein the tumor is a liquid tumor.

168. The CAR of claim 164 or claim 167, 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.

169. The CAR of claim 164, 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.

170. The CAR of claim 164 or claim 169, wherein the target antigen is selected from the group consisting of B7H3, EphA2, and Her2.

171. The CAR of any one of claims 108-170, wherein the target antigen is a cancer antigen.

172. The CAR of any one of claims 108-171, wherein the target antigen is selected from the group consisting of B7H3, PSMA, CD 19, CD20, EGFR, Her2, EphA2, and beta amyloid.

173. The CAR of any one of claims 108-163, 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 - 182 - NAI-5007599937vlconsisting 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.

174. The CAR of claim 173, 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), CD 177, and keratin.

175. The CAR of any one of claims 108-174, wherein the extracellular antigen-binding domain comprises a VHH domain.

176. The CAR of any one of claims 108-174, wherein the extracellular antigen-binding domain comprises a monobody.

177. The CAR of any one of claims 108-176, wherein the CAR further comprises a signal peptide.

178. The CAR of claim 177, wherein the signal peptide is covalently connected to the N-terminus of the extracellular antigen-binding domain.

179. A nucleic acid molecule encoding the CAR of any one of claims 108-178.

180. The nucleic acid molecule of claim 179, wherein the nucleic acid molecule is a mRNA.

181. A vector comprising the nucleic acid molecule of claim 179.

182. A cell comprising the CAR of any one of claims 108-178, the nucleic acid molecule of claim 179 or claim 180, or the vector of claim 181.

183. The cell of claim 182, wherein the cell is an immune cell.

184. The cell of claim 182 or claim 183, wherein the cell is a dendritic cell or a precursor thereof.187.- 183 - NAI-5007599937vl 185. The cell of any one of claims 1-107 and 182-184, wherein the cell is a conventional dendritic cell (cDC).

186. The cell of any one of claims 1-107 and 182-185, wherein the cell is a conventional type 1 dendritic cell (cDCl).

187. The cell of any one of claims 1-107 and 182-186, wherein the cell is a murine cDCl or human cDCl.

188. The cell of any one of claims 1-107 and 182-184, wherein the cell is a monocyte-derived dendritic cell (MoDC).

189. A composition comprising an effective amount of the cells of any one of claims 1-107 and 182-188.

190. The composition of claim 189, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

191. 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 1-107 and 182-188, or the composition of claim 189 or claim 190.

192. The method of claim 191, wherein the disease or disorder is tumor.

193. The method of claim 191 or claim 192, wherein the administration results in elimination of a tumor cell expressing the target antigen.

194. The method of any one of claims 191-193, wherein the administration results in elimination of a tumor cell that does not express the target antigen through epitope spreading.

195. The method of any one of claims 191-194, wherein the disease or disorder is cancer.

196. The method of any one of claims 191-195, wherein the cells are dendritic cells and the dendritic cells are capable of cross priming a T cell.

197. The method of claim 196, wherein the T cell is reactive to a target cell expressing the target antigen.

198. The method of claim 196 or claim 197, wherein the T cell is a CD8+T cell.201.- 184 - NAI-5007599937vl 199. The method of claim 191, wherein the disease or disorder is an autoimmune disease.

200. The method of claim 199, wherein the autoimmune disease is a T-cell mediated autoimmune disease.

201. The method of claim 191 or claim 199, 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, p sori asis / p sori atic arthritis, and autoimmune vasculitis.

202. The method of any one of claims 199-201, wherein the cells are capable of cross priming a T cell.

203. The method of claim 202, wherein the T cell is a regulatory T cell (Treg).

204. The method of claim 191, wherein the disease or disorder is a neurodegenerative disease.

205. The method of claim 191, wherein the disease or disorder is associated with beta amyloid accumulation.

206. The method of claim 205, wherein 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).

207. The method of claim 191, wherein the disease or disorder is an infectious disease.

208. 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 1-107 and 182-188, or the composition of claim 189 or claim 190.

209. The method of claim 208, wherein the immune response is a T cell-mediated immune response.

210. The method of claim 209, wherein the T cell is reactive to a target cell expressing the target antigen.213.- 185 - NAI-5007599937vl 211. The method of claim 209 or claim 210, wherein the T cell is reactive to a tumor cell that does not express the target antigen through epitope spreading.

212. The method of any one of claims 209-211, wherein the T cell is a CD8+T cell.

213. The method of any one of claims 209-211, wherein the T cell is a regulatory T cell (Treg).

214. The method of any one of claims 208-213, wherein the dendritic cells or precursors thereof or composition increase expression of one or more surface molecules expressed on DCs.

215. The method of claim 214, wherein the one or more surface molecules are selected from the group consisting of CD86, CD70, OX40L, CD40, CD40L, 4-1BBL, MHC-I, MHC-II, CCR7, and ICOSL.

216. The method of claim 215, wherein the one or more surface molecules are selected from the group consisting of CD86, CD70, MHC-I, CD40, 4-1BBL, and CCR7.

217. A method of producing the dendritic cell or precursor thereof of any one of claims 1-107 and 182-188, comprising: introducing into a cell a messenger RNA (mRNA), wherein the mRNA comprises a nucleic acid encoding the CAR.

218. The method of claim 217, wherein the mRNA further comprises a second nucleic acid encoding a self-cleaving peptide.

219. The method of claim 217 or claim 218, wherein the mRNA is introduced into the cell through electroporation.222.- 186 - NAI-5007599937vl

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