Single- and multi-chain synthetic antigen receptors for a variety of immune cells

Abstract

The present disclosure provides single and multi-chain synthetic antigen receptors, methods for making such synthetic antigen receptors, and their use for the treatment of diseases and disorders.

Description

[Technical Field]

[0001] Related Application Cross-Reference This application claims priority under 35 USC §119 to U.S. Provisional Application No. 63 / 151,421, filed February 19, 2021, and U.S. Provisional Application No. 63 / 245,181, filed September 16, 2021, the disclosures of which are incorporated herein by reference for all purposes.

[0002] Technical Field The present disclosure relates to the field of biotechnology, and more particularly to single-chain and multi-chain synthetic antigen receptors.

[0003] Incorporation by reference of sequence listing This application is accompanied by a Sequence Listing entitled "NK-DNA-PRT-1-11231_ST25.txt," created on February 21, 2022, containing 34,352,306 bytes of data, and machine formatted for an IBM-PC, MS-Windows operating system. The Sequence Listing is hereby incorporated by reference in its entirety for all purposes. [Background technology]

[0004] CARs are synthetic immune receptors that can redirect T cells to selectively kill tumor cells. Despite the success of CAR-T cells, this approach has several limitations, including toxicities such as "cytokine release syndrome" (CRS) and neurotoxicity.

[0005] In contrast to CAR-T, natural killer (NK) cells have natural cytolytic function and antiviral immunity, but lack the TCR that can cause GvHD. CAR-NK cells, in contrast to CAR-T, are less likely to cause excessive cytokine production.

[0006] No. 2世代 CARs are fusions of several different polypeptides. For example, Kymriah is composed of a mouse scFv (FMC63), the human CD8 hinge and transmembrane domains, the human 4-1BB costimulatory domain, and the human CD3z activation domain. These domains are strung together in a somewhat arbitrary manner, and the resulting constructs suffer from several issues, including nonspecific aggregation, tonic signaling, and a lack of physiological regulation.

[0007] Without wishing to be bound by theory, these problems may be exacerbated when scFvs are used to design CARs with bispecific, bivalent, or biparatopic antigen-binding moieties.

[0008] Another major limitation of most of the above-mentioned next-generation SAR designs is that they are primarily active in T cells and not in other immune cells such as NK cells, monocytes / macrophages, dendritic cells, or neutrophils. To overcome some of the design limitations of conventional second-generation CARs, several alternative designs, collectively referred to as next-generation CARs, have been reported, including Ab-TCRs (International Publication No. 2017 / 070608 A1, incorporated herein by reference), TCR receptor fusion proteins or TFPs (International Publication No. WO 2016 / 187349 A1, incorporated herein by reference), synthetic immune receptors (SIRs) (see International Publication No. WO 2018 / 102795 A1, incorporated herein by reference), and trifunctional T cell antigen couplers (Tri-TACs) (see International Publication No. WO 2015 / 117229 A1, incorporated herein by reference). These alternative CAR designs generally lack a costimulatory domain. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2017 / 070608 [Patent Document 2] International Publication No. 2016 / 187349 [Patent Document 3] International Publication No. 2018 / 102795 [Patent Document 4] International Publication No. 2015 / 117229 Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides next-generation SAR designs that are monospecific, bispecific, multispecific, and general purpose.

[0011] The present disclosure provides synthetic antigen receptors (SARs) with specific configurations of extracellular domains, transmembrane domains, and cytoplasmic domains, which, when expressed in immune cells (e.g., T cells, NK cells, NKT cells, monocytes, macrophages, neutrophils, etc.), exhibit improved immune cell activation, target cell killing, cytokine secretion (e.g., IL-2, interferon-γ, TNFα), and in vivo activity when compared to immune cells expressing conventional chimeric antigen receptors (CARs), such as second-generation CARs expressing a CD3z activation domain and a 41BB or CD28 costimulatory domain.

[0012] The present disclosure also provides novel accessory modules that can be co-expressed with the SARs of the present disclosure. The present disclosure provides vectors containing nucleic acids encoding the following polypeptides: a) low-affinity mutants of cytokines (e.g., IL-2 and / or IL-15) that are anchored to the cell membrane; b) membrane-anchored cytokines with epitope tags; and c) multipurpose gene switches that perform suicide, survival, and marker functions.

[0013] The present disclosure provides methods for producing cells that express any one or more accessory modules with any one or more of the single-chain or multi-chain SARs of the present disclosure. The accessory modules can be expressed in cells without the SARs. The present disclosure also provides optimized vectors with short promoters and internal ribosome sites optimized for expression of the accessory modules and / or SARs of the present disclosure.

[0014] In one aspect, the present disclosure relates to novel single-chain next-generation SAR designs that provide physiological signaling. More importantly, in another aspect, the present disclosure relates to novel multi-chain next-generation SAR designs.

[0015] In aspects, the present disclosure relates to novel next-generation synthetic antigen receptor (SAR) designs that are active in a variety of immune cells, including T cells, NKT cells, NK cells, monocytes / macrophages, and neutrophils. This disclosure relates to a novel SAR design, termed universal TCR-SAR (or uTCR-SAR), that confers T cell receptor-like antigen-binding specificity to any cell.

[0016] The present disclosure also provides non-T cells, including any cells with T cell-like binding properties, including the ability to bind to MHC (or HLA) molecules and bind peptide antigens. The present disclosure provides general methods for generating such cells and their use in treating various diseases.

[0017] The present disclosure also provides a multipurpose switch that can be used in adoptive cell therapy to provide cell survival, detection, tracking, enrichment, selection and removal functions.

[0018] The present disclosure also provides a general method for generating chimeric fusion proteins comprising type I and type II transmembrane proteins, which can be used to generate synthetic antigen receptors incorporating the antigen-binding domain of a type I protein and the cytosolic, transmembrane, hinge, and / or extracellular antigen-binding domains of a type II protein.

[0019] Also provided herein are nucleic acids encoding any of the single-, double-, or multi-chain SARs and / or accessory modules described herein. Also provided herein are sets of nucleic acids that together encode any of the single-, double-, and multi-chain SARs and / or accessory modules described herein.

[0020] In some embodiments, any of the above-described SARs (e.g., isolated SARs) are provided to effector cells (e.g., T cells, NK cells, macrophages, iPSCs, etc.) that display the SAR on their surface, wherein the effector cells comprise one or more vectors having one or more promoters that include one or more nucleic acids encoding one or more polypeptide chains of the SAR and / or any accessory modules.

[0021] Also provided herein are mammalian cells comprising any of the nucleic acids described herein encoding any of the single-, double-, and multi-chain SARs and / or accessory modules described herein. Also provided herein are mammalian cells comprising any of the sets of nucleic acids described herein, which together encode any of the single-, double-, and multi-chain SARs and / or accessory modules described herein.

[0022] In some embodiments, the present disclosure provides that, in contrast to TCRs, the SARs of the present disclosure can be expressed and functionally active in any mammalian cell. In one embodiment, the mammalian cell is a T cell, NK cell, macrophage, granulocyte, etc. In some embodiments of any mammalian cell described herein, the mammalian cell is a CD8 + T cells, CD4 +In some embodiments of any of the mammalian cells described herein, the mammalian cells are selected from the following group: T cells, memory T cells, naive T cells, T stem cells, Treg cells, natural killer T (NKT) cells, iNKT (innate natural killer cells), NK cells, g-NK cells, memory-like NK cells, cytokine-induced killer cells (CIK), iPSC-derived NK cells, α / β T cells, γ / δ T cells, iPSC-derived T cells, B cells, macrophages / monocytes, iPSC. In some embodiments, the mammalian cells are immortalized cell lines such as NK92, NK92MI, YTS, or derivatives thereof. In some embodiments, the mammalian cells are: In some embodiments, the subject is diagnosed or identified as having cancer; In some embodiments, the subject is human; In some embodiments, the cells are allogeneic.

[0023] Also provided herein are single-chain and multi-chain TCRs that can be functionally expressed in cells other than T cells, including, but not limited to, NK cells, monocytes, macrophages, dendritic cells and granulocytes.

[0024] Also provided herein are pharmaceutical compositions comprising any of the mammalian cells described herein and a pharmaceutically acceptable carrier. Also provided herein are kits comprising any of the pharmaceutical compositions described herein.

[0025] Also provided herein is a pharmaceutical composition comprising any of the nucleic acids described herein encoding any of the single-stranded, double-stranded, and multi-stranded SARs and / or accessory modules described herein, or any of the sets of nucleic acids described herein that together encode any of the single-stranded, double-stranded, and multi-stranded SARs and / or accessory modules described herein, and a pharmaceutically acceptable carrier. Also provided herein is a kit comprising any of the pharmaceutical compositions described herein.

[0026] In some embodiments, a method is provided for killing a target cell that presents one or more target antigens, the method comprising contacting the target cell with an effector cell that expresses a SAR according to any of the above-described SARs (such as an isolated SAR), wherein the SAR specifically binds to the one or more target antigens.

[0027] In some embodiments, according to any of the methods of killing target cells described above, the contacting occurs in vivo. In some embodiments, the contacting occurs in vitro.

[0028] Methods are provided for the detection, isolation, purification, expansion, enrichment and removal of cells expressing any of the SARs described herein.

[0029] Also provided herein are methods for producing cells expressing single-, double-, and multi-chain SARs and / or accessory modules, the methods comprising introducing into a mammalian cell any of the nucleic acids described herein encoding any of the SARs and accessory modules described herein, or any of the sets of nucleic acids described herein encoding any of the multi-chain SARs described herein. In some embodiments of any of the methods described herein, the mammalian cell is a human cell. In some embodiments of any of the methods described herein, the mammalian cell is a cell selected from the group consisting of CD8+ T cells, CD4+ T cells, memory T cells, Treg cells, natural killer T cells, B cells, NK cells, and macrophages / monocytes. In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell obtained from a subject. In some embodiments of any of the methods described herein, the subject is diagnosed or identified as having cancer. Some embodiments of any of the methods described herein further comprise culturing the cells in liquid medium after the introducing step. Some embodiments of any of the methods described herein further comprise obtaining the mammalian cells from the subject before the introducing step.

[0030] Also provided herein are methods for treating a disease (e.g., cancer, infectious disease, allergy, immune disorder, etc.) in a subject, comprising administering to the subject a therapeutically effective amount of any of the mammalian cells described herein. Some embodiments of any of the methods described herein further comprise obtaining initial cells from the subject to obtain the mammalian cells to be administered to the subject prior to the administering step, and introducing into the initial cells any of the nucleic acids described herein encoding any of the single-stranded, double-stranded, or multi-stranded SARs and / or accessory modules described herein, or any of the sets of nucleic acids described herein that together encode any of the single-stranded, double-stranded, or multi-stranded SARs and / or accessory modules described herein. Some embodiments of any of the methods described herein further comprise culturing the cells to be administered to the subject in liquid medium between the introducing and administering steps. In some embodiments of any of the methods described herein, the subject is a human.

[0031] In some embodiments of any of the single-chain SARs described herein, the heterologous antigen-binding domain is selected from the group consisting of an antibody, an antibody fragment (vL, vH, Fab, etc.), an scFv, an (scFv)2, a VHH domain, an FHVH (fully human vH domain), a single-domain antibody, a non-immunoglobulin antigen-binding scaffold (e.g., centilin, an affibody, a ZIP domain, an adaptor, etc.), a VNAR domain, a ligand, a TCR, a variable domain of a TCR (Va, Vb, Vg, Vd), and a receptor. In some embodiments of any of the single-chain SARs described herein, the heterologous antigen-binding domain comprises an scFv.

[0032] In some embodiments of any of the single-chain SARs described herein, the heterologous antigen-binding region specifically binds to a single antigen. In some embodiments of any of the single-chain SARs described herein, the single antigen is a tumor antigen. In some embodiments of any of the single-chain SARs described herein, the tumor antigen is selected from the antigens listed in Table B.

[0033] In some embodiments of any of the single-chain SARs described herein, when proceeding from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, the single-chain SAR comprises a non-naturally occurring extracellular antigen-binding domain, an optional linker, an optional extracellular ligand-binding domain of a naturally occurring receptor, an optional hinge domain, a transmembrane domain, an optional cytoplasmic costimulatory domain, and an optional cytoplasmic primary signaling domain that constitutes an ITAM. In some embodiments of any of the single-chain SARs described herein, the transmembrane domain and any cytoplasmic primary signaling domain are immediately adjacent to each other. In some embodiments of any of the single-chain SARs described herein, the transmembrane domain and any cytoplasmic primary signaling domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids). In some embodiments of any of the single-chain SARs described herein, any primary signaling domain and costimulatory domain are immediately adjacent to each other. In some embodiments of any of the single-chain SARs described herein, any primary signaling domain and costimulatory domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids). In some embodiments of any of the single-chain SARs described herein, the costimulatory domain and the ITAM are immediately adjacent to each other. In some embodiments of any of the single-chain SARs described herein, the costimulatory domain and the ITAM are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids).

[0034] In some embodiments of any of the single-chain SARs described herein, when proceeding in the N-terminal to C-terminal or C-terminal to N-terminal direction, the single-chain SAR comprises an optional linker, including the non-naturally occurring extracellular antigen-binding domain(s), any extracellular ligand-binding domain of a naturally occurring receptor, any hinge domain, transmembrane domain, costimulatory domain, primary signaling domain, and ITAM.

[0035] In some embodiments of any of the single-chain SARs described herein, when proceeding in the N-terminal to C-terminal or C-terminal to N-terminal direction, the single-chain SAR comprises a non-naturally occurring extracellular antigen-binding domain(s), a transmembrane domain, a primary signaling domain, an ITAM, and a costimulatory domain.

[0036] In some embodiments of any of the single-chain SARs described herein, when going in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, the single-chain synthetic antigen receptor comprises a non-naturally occurring extracellular antigen-binding domain(s), a transmembrane domain, a second intracellular signaling domain, an ITAM, and a first intracellular signaling domain.

[0037] In some embodiments of any of the single-chain SARs described herein, when going in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, the single-chain synthetic antigen receptor comprises a heterologous extracellular antigen-binding domain(s), a transmembrane domain, an ITAM, a primary signaling domain, and a costimulatory domain.

[0038] In some embodiments of any of the single-chain SARs described herein, when going in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, the single-chain synthetic antigen receptor comprises a non-naturally occurring extracellular antigen-binding domain(s), a transmembrane domain, an ITAM, a second intracellular signaling domain, and a first intracellular signaling domain.

[0039] In some embodiments of any of the single-chain SARs described herein, the extracellular antigen-binding domain and the transmembrane domain are immediately adjacent to each other. In some embodiments of any of the single-chain SARs described herein, the extracellular antigen-binding domain and the transmembrane domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids).

[0040] In some embodiments of any of the single-chain SARs described herein, the primary signaling domain is derived from CD3z. In some embodiments of any of the single-chain SARs described herein, the primary signaling domain is derived from FcRγ. In some embodiments of any of the single-chain SARs described herein, the primary signaling domain is derived from DAP10. In some embodiments of any of the single-chain SARs described herein, the primary signaling domain is derived from DAP12.

[0041] Also provided herein are nucleic acids comprising a nucleotide sequence encoding any of the single-chain SARs described herein. Also provided herein are vectors comprising any of the nucleic acids described herein, including a nucleotide sequence encoding any of the single-chain SARs described herein.

[0042] Also provided herein are mammalian cells comprising any of the vectors described herein. In some embodiments of any of the mammalian cells described herein, the mammalian cells are T cells, NK cells, macrophages, or iPSCs.

[0043] Also provided herein are methods for producing SAR-expressing cells, the methods comprising introducing any of the nucleic acids described herein or vectors described herein into mammalian cells. In some embodiments of any of the methods described herein, the mammalian cells are human cells. In some embodiments of any of the methods described herein, the mammalian cells are cells selected from the group consisting of CD8+ T cells, CD4+ T cells, naive T cells, memory T cells, Treg cells, natural killer T cells, NK cells, B cells, and macrophages / monocytes. In some embodiments of any of the methods described herein, the mammalian cells are cells selected from the group consisting of iPSCs (induced pluripotent stem cells), embryonic stem cells, or hematopoietic stem cells that can give rise to immune effector cells (e.g., T cells, NK cells, or NKT cells). In some embodiments, the mammalian cells are immortalized cell lines such as NK92, NK92MI, or derivatives thereof. In some embodiments of any of the methods described herein, the mammalian cells are mammalian cells obtained from a subject. In some embodiments of any of the methods described herein, the subject is diagnosed or identified as having cancer. Some embodiments of any of the methods described herein further include culturing the cells in liquid medium after the introducing step.

[0044] Also provided herein are methods for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of any of the mammalian cells described herein. Some embodiments of any of the methods described herein further comprise, prior to the administering step, obtaining initial cells from the subject and introducing any of the nucleic acids described herein or any of the vectors described herein into the initial cells to obtain the mammalian cells administered to the subject. Some embodiments of any of the methods described herein further comprise culturing the cells administered to the subject in a liquid medium between the introducing step and the administering step. In some embodiments of any of the methods described herein, the subject is a human.

[0045] Provided herein are multi-chain SARs comprising at least one first polypeptide comprising: an optional hinge domain, a transmembrane domain, and an optional cytosolic domain.

[0046] In some embodiments of any of the multi-chain SARs described herein, the extracellular antigen-binding domain is selected from the group consisting of Vα, Vβ, Vγ, Vδ, vL, vH domains, scFv, a(scFv)2, 、 a VHH domain, FHVH (fully human vH domain), single domain antibody, non-immunoglobulin antigen-binding scaffold, VNAR domain, ligand, and receptor. In some embodiments of any of the multi-chain SARs described herein, the extracellular antigen-binding domain comprises an scFv.

[0047] In some embodiments of any of the multi-chain SARs described herein, at least one first polypeptide comprises an extracellular antigen-binding region that specifically binds to a single antigen. In some embodiments of any of the multi-chain SARs described herein, the single antigen is a tumor antigen.

[0048] In some embodiments of any of the multi-chain SARs described herein, the SAR lacks an ITAM but recruits a signaling protein that comprises an ITAM-containing primary stimulatory domain. In some embodiments of any of the multi-chain SARs described herein, the SAR recruits a signaling protein selected from the group consisting of CD3z, FcRγ, DAP10, and / or DAP10.

[0049] In some embodiments of any of the multi-chain SARs described herein, when proceeding from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, at least the first polypeptide of the multi-chain SAR comprises the heterologous antigen-binding domain(s), any linker, any extracellular domain of a naturally occurring receptor, any hinge domain, a transmembrane domain, any cytoplasmic costimulatory domain, and any cytoplasmic primary signaling domain that constitutes an ITAM. In some embodiments of any of the multi-chain SARs, the transmembrane domain and any cytoplasmic primary signaling domain are immediately adjacent to each other. In some embodiments of any of the multi-chain SARs, the transmembrane domain and any cytoplasmic primary signaling domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids). In some embodiments of any of the multi-chain SARs, the transmembrane domain and any cytoplasmic primary signaling domain are immediately adjacent to each other. In some embodiments of any of at least the first polypeptide of the multi-chain SAR described herein, any primary signaling domain and costimulatory domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids). In some embodiments of any of at least the first polypeptide of the multi-chain SAR described herein, the costimulatory domain and ITAM are immediately adjacent to each other. In some embodiments of any of at least the first polypeptide of the multi-chain SAR described herein, the costimulatory domain and ITAM are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids).

[0050] In some embodiments of at least the first polypeptide of any of the multi-chain SARs described herein, when proceeding in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, the multi-chain SAR includes a heterologous antigen-binding domain, an optional linker, an optional hinge domain, a transmembrane domain, a costimulatory domain, a primary signaling domain, and an ITAM.

[0051] In some embodiments of any of at least the first polypeptide of a multi-chain SAR described herein, when proceeding in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, at least the first polypeptide of the multi-chain SAR includes a heterologous antigen-binding domain, a transmembrane domain, a primary signaling domain, an ITAM, and a costimulatory domain.

[0052] In some embodiments of any of at least the first polypeptide of a multi-chain SAR described herein, when proceeding in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, at least the first polypeptide of the multi-chain SAR includes a heterologous antigen-binding domain, a transmembrane domain, a second intracellular signaling domain, an ITAM, and a first intracellular signaling domain.

[0053] In some embodiments of any of at least a first polypeptide of a multi-chain SAR described herein, when proceeding in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction, the at least first polypeptide of the multi-chain SAR includes an extracellular antigen-binding domain, a transmembrane domain, an ITAM, a primary signaling domain, and a costimulatory domain.

[0054] In some embodiments of any of at least a first polypeptide of a multi-chain SAR described herein, when proceeding in an N-terminal to C-terminal direction or a C-terminal to N-terminal direction, the first polypeptide of the multi-chain SAR includes an extracellular antigen-binding domain, a transmembrane domain, an ITAM, a second intracellular signaling domain, and a first intracellular signaling domain.

[0055] In some embodiments of at least the first polypeptide of the multi-chain SAR described herein, the extracellular antigen-binding domain and the transmembrane domain are immediately adjacent to each other. In some embodiments of at least the first polypeptide of the multi-chain SAR described herein, the extracellular antigen-binding domain and the transmembrane domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids, or 1 to 50 amino acids).

[0056] In some embodiments of at least the first polypeptide of any of the multi-chain SARs described herein, the primary signaling domain is derived from one or more proteins selected from the group of CD3z, FcRγ, DAP10, or DAP12.

[0057] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0058] The invention is further illustrated in the following non-limiting figures. [Brief explanation of the drawings]

[0059] [Figure 1] Figure 1 shows a schematic representation of the monospecific, bispecific, and multispecific SAR of various duplexes.

[0060] [Figure 2] Figure 2 shows a schematic diagram of different double-chain monospecific, bispecific, and multispecific SARs containing different forms of AABDs (e.g., vHH, SVH, aVH, affibody, centuirin, etc.).

[0061] [Figure 3] Figure 3 depicts the various formats that the single- and two-chain CD16-SARs of the present disclosure can have when expressed. These SARs are based on the entire extracellular domain of CD16, including both Ig-like domains (D1 and D2 domains).

[0062] [Figure 4] Figure 4 depicts the various formats that the CD16-SARs of the present disclosure can have upon expression. These SARs are based on the partial extracellular domain of CD16. Because SARs are modular, CD16 modules can be replaced with different modules from NKp44, NKp46, etc. to generate diverse SARs.

[0063] [Figure 5] Figure 5 depicts the various formats that the NKp30 SAR of the present disclosure can have upon expression. Because the SAR is modular, the NKp30 module can be replaced with different modules from NKp44, NKp46, etc. to generate diverse SARs.

[0064] [Figure 6] Figure 6 shows the results of a Matador cytotoxicity assay using NK92 cells expressing the indicated SAR constructs when co-cultured with CD19-expressing RS4;11 GLuc target cells for 2 hours.

[0065] [Figure 7] Figure 7 shows the results of a Matador cytotoxicity assay using NK92 cells expressing the indicated SAR constructs when co-cultured with RS4;11 GLuc target cells for 2 h.

[0066] [Figure 8] Figure 8 shows the results of a Matador cytotoxicity assay using NK92 cells expressing the indicated SAR constructs when co-cultured with L363-GLuc target cells for 2 h.

[0067] [Figure 9] Figure 9 shows the results of a Matador cytotoxicity assay using NK92 cells expressing the indicated SAR constructs when co-cultured with RS4;11 Gluc target cells for 2 hours.

[0068] [Figure 10] Figure 10 shows the results of a Matador cytotoxicity assay using NK92 cells expressing the indicated SAR constructs when co-cultured with RS4;11 GLuc target cells for 2 hours.

[0069] [Figure 11] Figure 11 shows the results of a Matador cytotoxicity assay using NK92 cells expressing the indicated SAR constructs when co-cultured with L363-Gluc target cells for 2 hours.

[0070] [Figure 12] FIG. 12 shows a general description of the generation of a SAR comprising the fusion of an antigen-binding domain to the extracellular domain of a type II membrane protein such as NKG2D.

[0071] [Figure 13] Figures 13A-C show: (A) cell death by primary T cells expressing uTCR-SAR (061621-SCjJ7; SEQ ID NO: 9366) targeting the NY-ESO1 peptide (SEQ ID NO: 10880) when co-cultured with NK92, primary NK cells, and U266 cells (NY-ESO1+ / HLA-A2), (B) upregulation of TNFα, and (C) upregulation of IFNγ by primary T cells expressing uTCR-SAR (061621-SCjJ7; SEQ ID NO: 9366) targeting the NY-ESO1 peptide when co-cultured with control T2 cells or peptide-loaded T2 cells. DETAILED DESCRIPTION OF THE INVENTION

[0072] Detailed Description The present invention will now be further described. In the following passages, various aspects of the present invention will be defined in more detail. Each aspect so defined can be combined with any other aspect, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0073] Unless otherwise stated or implicit from context, the following terms and phrases have the meanings set forth below: Unless expressly stated otherwise or apparent from context, the use of the following terms and phrases does not exclude the meaning that the term or phrase has acquired in the relevant art. Because the scope of the invention is limited only by the claims, definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0075] As used herein, the term "comprising" is used in reference to compositions, methods, and their respective components that are useful for an embodiment but may include unspecified elements, whether or not they are useful. In general, those skilled in the art will understand that the terms used herein are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "including but not limited to," the term "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.).

[0076] In general, the nomenclature used in connection with, and techniques related to, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2013)). The nomenclature used in connection with, and the laboratory procedures and techniques related to, immunology, molecular biology, analytical chemistry, organic synthetic chemistry, and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0077] As used herein, the term "autonomous antigen-binding domain" or "AABD" refers to an antigen-binding domain that can bind to an antigen autonomously, i.e., in the absence of another antigen-binding domain. An exemplary AABD is a single vH domain or autonomous vH domain (aVH), typically a single human vH domain (SVH), that can bind to an antigen in the absence of a vL domain. Another exemplary AABD is a fully human vH domain (FHVH). Another exemplary AABD is a single vL domain or autonomous vL domain, typically a single human vL domain (SVL), that can bind to an antigen in the absence of a vH domain. AABD also refers to other antigen-binding domains that can bind to an antigen autonomously. In certain embodiments, the AABD is a non-scFv antigen-binding domain. Exemplary non-scFV-based autonomous antigen-binding domains include, but are not limited to, vHH domains, humanized vHH domains, single variable domain-TCRs (svd-TCRs), and non-immunoglobulin antigen-binding scaffolds, such as DARPINs, affibodies, ZIP domains (e.g., RZIP, EZIP, E4, R4, etc.), affilins, adnectins, affitins, obodies, lipibodies, fynomers, alphabodies, avimers, atrimers, centilins, pronectins, anticalins, Kunitz domains, armadillo repeat proteins or fragments thereof. Further examples of non-scFV-based autonomous antigen-binding domains include ligand-binding domains of receptors (e.g., CD16-V158A, NKG2D) or fragments thereof, receptor-binding domains of ligands (e.g., APRIL, thrombopoietin, etc.) or fragments thereof, adaptors (e.g., RZIP, EZIP, E4, K4, NKG2D-YA, NKG2D-AF, etc.) or fragments thereof, adaptor-binding proteins (e.g., ULBP2R, ULBP2-S3, etc.) or fragments thereof, epitopes or tags (e.g., Streptag, FLAG tag, etc.), autoantigens or fragments thereof, etc.

[0078] The present disclosure describes the use of AABDs, such as human VH (or vH) domains, such as multiple human VH domains, as building blocks for generating monospecific, bispecific, and multispecific SARs. In certain embodiments, the present disclosure describes the use of AABDs, such as human VH domains, such as multiple human VH domains, as building blocks for generating novel monospecific, bispecific, and multispecific SARs.

[0079] The term "about," when referring to a measurable value, such as an amount, temporal duration, or the like, refers to ±20%, sometimes ±10%, or sometimes ±5%, or sometimes ±1%, or sometimes ±0.1% of the particular value, as such variations are appropriate for carrying out the disclosed methods or describing the compositions herein. Furthermore, any value or range (e.g., less than 20 or similar terminology) explicitly includes any integer between or up to such value. Thus, for example, "1 to 5 variations" explicitly includes variations of 1, 2, 3, 4, and / or 5.

[0080] The term "ABR" or "antigen-binding receptor" as used herein refers to any receptor having an antigen-binding domain. The antigen-binding domain of an ABR can include scFv, vL, vH, VHH, antibody, antibody fragment (e.g., Fab), antibody-like portion, Vα, Vβ, svd-TCR, cytokine, receptor, etc. The ABR has a transmembrane domain or membrane anchor domain that enables expression on the cell surface. Exemplary ABRs include first-generation CARs, second-generation CARs, TFPs, SIRs, STARs, zSIRs, cTCRs, TCRs, Ab-TCRs, TRI-TACs, or TACs, etc. 。 Book 明細 The synthetic antigen receptor (SAR) described in [1] is also an example of an ABR.

[0081] The term "Ab-TCR" or "AbTCR" refers to a next-generation CAR platform as described in WO2017 / 070608 A1, which is incorporated herein by reference. In one embodiment, the Ab-TCR comprises an antibody moiety that specifically binds to a target antigen fused to a TCR module that can recruit at least one TCR signaling module. Exemplary TCR modules that can be used to construct Ab-TCRs are provided in SEQ ID NOs: 6009-6014 (Table 6) and in WO2017 / 070608 A1, which is incorporated herein by reference.

[0082] The term "accessory module" refers to one or more of: PDL1, PDL2, CD80, CD86, crmA, p35, hNEMO-K277A (or NEMO-K277A), hNEMO-K277A-delta-V249-K555, mNEMO-K270A, and K13. -opt, IKK2-S177E-S181E (or IKK2-SS / EE), IKK1-S176E-S180E (or IKK1-SS / EE), MyD88-L265P, TCL-1a, MTCP-1, CMV- 141, 41BBL, CD40L, vFLIP-K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304 -vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4 scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-ipilimumab-scFv, CTLA4-ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, shRNA targeting hTERT, Fx06, Brd4, IgSP-[hTRAC-opt2], IgSP-[hTRBC-opt2], multi-purpose switches (e.g., IL2-tBCMA, IL15-tBCMA, IL2-RQR, IL15-RQR, etc.), NKG2C, CD94, DAP10, DAP12, CD3ε, CD3γ, CD3δ, CD3ζ, FcRγ, and immune cells (e.g., NK cells or T cells (e.g., SAR-NK cells, SAR-T cells, or TCR-T cells) are used to reduce, regulate, or modify the activity of immune cells. In one embodiment, the accessory module is a therapeutic control (e.g., squapase 9). In some embodiments, the accessory module is co-expressed with an immune receptor, such as a SAR or TCR, to increase, reduce, regulate, or modify the expression or activity of the SAR or TCR, or a SAR- or TCR-expressing cell.The accessory module can be co-expressed with the SAR or TCR using a single vector or two or more different vectors. In some embodiments, the accessory module is expressed in an antigen-presenting cell, e.g., a dendritic cell.

[0083] As used herein, "affinity" is meant to represent a measure of binding strength. Affinity generally refers to the "ability" of a binding agent to bind to its target. Numerous methods are used in the art to measure "affinity." For example, methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. As used herein, the term "specific binding" refers to binding that is at least 10 -6 M. In some embodiments, the antibody has a binding affinity of at least about 10 -7 M, typically 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M.

[0084] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be monoclonal or polyclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be "humanized," "chimeric," fully human, or non-human. Antibodies can have a single domain (e.g., a single vH domain).

[0085] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (eg, by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'h), Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (sdAb) such as either vL or vH, camelid vHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDR or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0086] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformations, and which usually determines the class to which the antibody belongs.

[0087] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) light chain and lambda (λ) light chain refer to the two major antibody light chain isotypes.

[0088] "Anticancer agent" refers to an agent that inhibits abnormal cell division and proliferation, inhibits neoplastic cell migration, inhibits invasion, or prevents cancer growth and metastasis. This term includes chemotherapeutic agents, biologic agents (e.g., siRNA, viral vectors such as modified MLV, adenovirus, and herpes viruses that deliver cytotoxic genes), antibodies, and the like.

[0089] The term "anti-cancer effect" or "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in cancer cell number, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with the cancerous condition. An "anti-cancer effect" may also be manifested by the ability of a SAR to prevent the development of cancer in the first place.

[0090] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunologically competent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. As used herein, the term "antigen" generally refers to the binding partner that is specifically recognized by the antigen-binding domains described herein. Non-limiting examples of antigens that can be specifically bound by either the antigen or antigen-binding domain are listed in Table B.

[0091] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., a B cell, a dendritic cell, etc.), that presents foreign antigens complexed with major histocompatibility complexes (MHC) on its surface.

[0092] It refers to a biological effect that can be manifested by a variety of means, including, but not limited to, a reduction in the titer of an infectious agent, a reduction in the colony count of an infectious agent, and amelioration of various physiological symptoms associated with an infectious disease.

[0093] An "antigen-binding domain" or "antigen-binding module" or "antigen-binding segment" or "antigen-specific domain" (ASD) refers to a polypeptide or peptide that binds to an antigen with high specificity due to its primary, secondary, or tertiary sequence, post-translational modification, and / or charge. An ASD can bind to a target with higher affinity than a nonspecific domain. Antigen-binding domains can be derived from different sources, such as antibodies (full-length heavy chains, Fab fragments, single-chain Fv (scFv) fragments, bivalent single-chain antibodies, or diabodies), non-immunoglobulin-binding proteins, ligands, or receptors. In some embodiments, as will be appreciated by those skilled in the art, virtually any molecule that binds to a given cognate or antigen with high affinity can be used as an ASD. In some embodiments, the antigen-binding domain comprises a T cell receptor (TCR) or a portion thereof. In representative embodiments, the target antigens and SEQ ID NOs of various antigen-binding domains are listed in Tables 3-7 herein. In representative embodiments, the target antigens and SEQ ID NOs for vL, vH, scFV, and their CDR regions are set forth in Tables 6A-C of patent application PCT / US18 / 53247 and Tables 3-4 of patent application PCT / US19 / 035096, the entire contents of which are incorporated herein by reference.

[0094] The term "Ka") is defined as the equilibrium constant for the association of receptor and ligand.

[0095] "Autoantibody" refers to an antibody produced by B cells specific for a self-antigen.

[0096] The term "autoantigen" refers to an endogenous antigen that stimulates the production of an autoimmune response, such as the production of autoantibodies. Examples of autoantigens include, but are not limited to, desmoglein 1, desmoglein 3, and fragments thereof.

[0097] "Avidity" refers to the strength of the interaction between a binder and its target (e.g., the strength of the interaction between an antibody and its antigen target, a receptor and its cognate, etc.). Antibody activity in functional assays (e.g., flow cytometry assays or Malibu-Glo assays) also reflects antibody affinity.

[0098] The term "architecture" refers to the organization of different components (e.g., antigen-binding domain, hinge domain, transmembrane domain, signaling domain), including different SARs and generally any optional accessory modules. In one embodiment, the SAR and accessory module are encoded by a single nucleic acid molecule. In another embodiment, the SAR is encoded by a first nucleic acid molecule, and the accessory module is encoded by a second nucleic acid molecule. In some embodiments, the accessory module is encoded by multiple nucleic acid molecules, depending on the number of components in the accessory module. Two or more components of the SAR and accessory module may be separated by a cleavable linker, such as a 2A ribosomal skip sequence (e.g., P2A, T2A, F2A, etc.). Two or more components of the SAR and accessory module may be separated by an internal ribosome entry sequence (IRES). An exemplary IRES is derived from KSHV. Expression of nucleic acids encoding two or more components of the SAR and accessory module may be driven by separate promoters. Examples of promoters include EF1α, EFS, EFS2, CMV, RSV, mutRSV, MNDU3, Hsp70, and Hsp90.

[0099] Table A1: Conventional CAR architecture. First-generation conventional CARs (conventional CAR I) have an intracellular signaling domain (ISD) (e.g., CD3z) and no costimulatory domain. TCR fusion protein (TFP) is another example of a conventional CAR I. Second-generation conventional CARs (conventional CAR 2 or CAR II) have one costimulatory domain (e.g., 41BB or CD28) and an intracellular signaling domain (ISD) (e.g., CD3z). Third-generation conventional CARs (conventional CAR 3 or CAR III) have two costimulatory domains (e.g., 41BB and CD28) and an intracellular signaling domain (ISD) (e.g., CD3z). Ab-TCRs are dual-chain receptors incorporating a vL linker-TCR domain (TCRD) and a vH linker-TCR domain (TCRD), as described in PCT / US2016 / 058305. cTCRs are single-chain, 1.5-chain, or dual-chain receptors consisting of antigen-binding domains derived from vL and vH fragments fused to one or more TCR constant chains (TCR-C), resulting in activation of T cell signaling. The cTCRs are characterized by their type nucleic acid sequences and corresponding wild-type amino acid sequences. Various configurations of cTCRs are described in PCT / US2017 / 064379 or WO 2018 / 102795 A1. Synthetic immunoreceptors are next-generation CARs, as described in PCT / US2017 / 064379 or WO 2018 / 102795 A1. SIRs are single-chain, single-chain, or dual-chain receptors. In one embodiment, the antigen-binding domain of the SIR is derived from a vL and vH fragment fused to one or more TCR constant chains. In some embodiments, the TCR constant chain of the SIR is encoded by a codon-optimized nucleic acid sequence and contains one or more mutations that enhance its expression and chain pairing. zSIR is a dual-chain receptor comprising an antigen-binding domain (e.g., vL, vH, etc.) operably linked to two CD3z chains or fragments thereof using any linker, and is described in PCT / US2019 / 035096.

Table 1

[0100] Tables A1-1 to A1-19 provide exemplary architectures for monospecific, bispecific, and multispecific SARs of the present disclosure. Abbreviations used are as follows: SP (signal peptide). AADB (autonomous antigen-binding domain); L (optional linker); LL (long linker), (AABD-L)n (n copies of AABD with optional linkers, n = 0, 1, 2, 3, 4 or more), AABD1-4 (different AABDs targeting one or more antigens), V1 (vL, vH, Va, Vb, Vg or Vd chain), Ig (Ig linker), TCR-Ig (Ig linker domain from a TCR chain), ConP (connecting peptide), TM (transmembrane domain), CP (cytosolic domain), IC (intracellular domain), Ca (constant chain of TCRα), Cb (constant chain of TCRβ), Cg (constant chain of TCRγ), Cd (constant chain of TCRδ), scFv (single-chain fragment variable), scTFv( a single-chain fragment comprising two variable fragments of the TCR (e.g., Va and Vb), dCa / dCb / dCg / dCd (N-terminally deleted constant chains of TCR α, β, γ, or δ lacking the Ig linker domain), TCR-ConP (connecting peptides of TCR α, β, γ, or δ constant chains), Ca-ConP (connecting peptides of TCR α constant chains), IgCL (Ig linker from immunoglobulin light chains), IgCH1 (Ig linker from immunoglobulin heavy chains), CD3εγδ ECD (extracellular domain of CD3ε, γ, or δ chains), CSD (costimulatory domain), 4-1BB or BB (costimulatory domain of 4-1BB), CD28 or 28 (costimulatory domain of CD28), CD3z or zd or z (CD3z). NKp30-Ig (immunoglobulin-like domain of Nkp30), NKp44-Ig (immunoglobulin-like domain of Nkp44), NKp46-Ig1-Ig2 (immunoglobulin-like domains 1 and 2 of Nkp46), CD16-D1 (domain 1 of CD16), CD16-D2 (domain 2 of CD16), scTCR (single-chain TCR), extracellular domain (ECD), activation domain (AD); Va, Vb, Vg, Vd (variable domains of TCRα, β, γ, δ), FCRG (FcRγ); hinge domain (Hn).

Table 2-1

Table 2-2

Table 3-1

Table 3-2

Table 4-1

Table 4-2

Table 5-1

Table 5-2

Table 6

Table 7

Table 8

Table 9-1

Table 9-2

Table 10-1

Table 10-2

Table 10-3

Table 11-1

Table 11-2

Table 12-1

Table 12-2

Table 13-1

Table 13-2

Table 14-1

Table 14-2

Table 15-1

Table 15-2

Table 16-1

Table 16-2

Table 17

Table 18

Table 19-1

Table 19-2

Table 19-3

Table 20-1

[0101] As used herein, a "beneficial result" or "desired result" includes reducing or alleviating the severity of a disease state, preventing a disease state from worsening, curing a disease state, preventing the onset of a disease state, reducing the likelihood that a patient will develop a disease state, and extending the life or life expectancy of a patient.

[0102] "Binds to the same epitope" refers to the ability of an antibody, scFv, or other antigen-binding domain to bind to a target antigen and have the same epitope as the exemplified antibody, scFv, or other antigen-binding domain. By way of example, the epitopes of the exemplified antibodies, scFv, or other binding agents and other antibodies can be determined using standard epitope mapping techniques. Epitope mapping techniques well known in the art include those described in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, ed., 1996) Humana Press, Totowa, NJ. Exemplary epitopes of human CD20, BCMA, and human MPL antigens bound by the scFvs, SARs, antibodies, and other immunotherapeutics of the present disclosure are provided in SEQ ID NOS: 15149-15154, 15155-15159, and 15160, respectively, of patent application PCT / US18 / 53247, the entire contents of which are incorporated herein by reference.

[0103] When the present disclosure relates to polypeptides, proteins, polynucleotides, antibodies, SARs, or fragments thereof, it is assumed that their equivalents or biological equivalents are intended within the scope of the disclosure, even if not explicitly stated or intended otherwise. As used herein, the terms "biological equivalent thereof" or "variant" or "functional variant" are intended to be synonymous with "equivalent thereof" when referring to a reference protein, antibody, or fragment thereof, or receptor, ligand, or fragment thereof. The non-immunoglobulin antigen-binding domain or fragment thereof, SAR, or fragment thereof, polypeptide, or nucleic acid refers to one that has minimal homology while still maintaining the desired structure or function. Unless otherwise specified herein, all of the above are intended to include equivalents thereof, including alternatively spliced ​​isoforms and equivalents from other animal species. For example, equivalents refer to at least about 70% homology or identity, or at least 80% homology or identity, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 95% homology or identity. Alternatively, it may have at least 98% homology or identity and exhibit substantially the same biological activity as the reference protein, polypeptide, antibody, or fragment thereof, or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement. Alternatively, when referring to a polypeptide or protein, the equivalent is a polypeptide or protein expressed from a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding the reference polypeptide or protein, or its complement.

[0104] It will be appreciated that proteins can share identity or homology with one another and retain similar or identical functions. As used herein, a polypeptide "variant" is a polypeptide that differs from a recited polypeptide only in conservative substitutions and / or modifications such that the therapeutic, antigenic, and / or immunogenic properties of the polypeptide are retained. Polypeptide variants typically exhibit at least about 70%, more typically at least about 90%, and most typically at least about 95% homology to the identified polypeptide. Alternatively, for immunoreactive polypeptides, variants can be identified by modifying the amino acid sequence of any of the above polypeptides and assessing the immunoreactivity of the modified polypeptide. Such modified sequences can be prepared and tested, for example, using the representative procedures described herein. The present disclosure includes SARs and SAR components having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 98.5%, 99%, or 99.9% identity to any of the amino acid sequences described herein (e.g., the extracellular, hinge, transmembrane, and cytoplasmic regions of CD16, CD32, CD64, FcRγ, DAP10, DAP12, DNAM1, OX40, 2B4, KIR2DL1, KIR2DS4, NKp30, NKp44, NKp46, NKG2D, NKG2A, NKG2C, NKG2E, NKG2F, NKG2H, TCRα, TCRβ, TCRγ, TCRδ, CD3z, etc.) while retaining biological activity. The present disclosure also includes antigen-binding domains, extracellular domains, hinge domains, transmembrane domains, cytoplasmic domains, costimulatory domains, and accessory modules having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 98.5%, 99%, or 99.9% identity to any of the sequences described herein while retaining biological activity. Variants include homologs in other species and alternatively spliced ​​isoforms.

[0105] As used herein, the term "CD3 complex" refers to a cell surface molecular assembly that contains multiple proteins for transmembrane signaling of TCR activation.

[0106] As used herein, the term "CDR" or "complementarity-determining region" refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are defined by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 25 262:732-745 (1996), and the definitions herein include overlapping or subsets of amino acid residues relative to each other. Nevertheless, application of either definition to refer to the CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. As used herein, different CDRs of an antibody may also be defined by a combination of different definitions. For example, vHCDR1 can be defined according to Kabat, and VHCDR2 can be defined according to Chothia. The amino acid residues encompassing the CDRs defined by each of the above-cited references are as follows: [Table 21]

[0107] SEQ IDs for the CDRs of exemplary vL and vH segments that may constitute the antigen-binding domains of SARs, bispecific antibodies, and other immunotherapeutics of the present disclosure are provided in SEQ ID NOS: 13204-14121 and 14122-15039 (Tables 6A and 6B) of PCT / US2018 / 053247, Tables 5-6 of PCT / US2017 / 064379, and Table 39 of PCT / US2021 / 022641, respectively, which are incorporated herein by reference. SEQ ID NOS for exemplary vL and vH segments that may constitute the antigen-binding domains of SARs, antibodies, and other immunotherapeutics of the present disclosure are also provided in Table 3 of the present disclosure. Light chain CDR1, CDR2, and CDR3 of the vL fragments and scFvs provided in this disclosure (e.g., Table 3) are provided in SEQ ID NOs: 10882-11118, 11119-11355, and 11356-11592. CDR1, CDR2, and CDR3 of the vL fragments provided in this disclosure (e.g., Table 3) are provided in SEQ ID NOs: 10882-11118, 11119-11355, and 11356-11592. Heavy chain CDR1, CDR2, and CDR3 of the vH fragments and scFvs provided in this disclosure (e.g., Table 3) are provided in SEQ ID NOs: 11593-11829, 11830-12066, 12067-12303, respectively.

[0108] In some embodiments, reference to an antigen binding module (such as a Fab-like or Fv-like antigen binding module) that specifically binds to a target antigen means that the antigen binding module (a) binds to the target antigen with an affinity that is at least about 10-fold (e.g., about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) greater than its binding affinity to other molecules; or (b) a K that is about 10-fold or less. d ( For example, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 175, 1 / 100, 1 / 200, 1 / 300), 1 / 400, 1 / 500, 1 / 750, 1 / 1000 or less) indicates its K for binding to other molecules.d倍である。 Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, Malibu-Glo assay, Topanga assay, or radioimmunoprecipitation assay (RIA).

[0109] "Cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. The terms "tumor" and "cancer" are used interchangeably herein; for example, both terms encompass solid and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors. The term "cancer" is meant to include any type of cancerous growth or oncogenic process, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0110] "Cell therapy" or "cell-based therapy" or "immune cell therapy" or "immune effector cell therapy" refers to therapy involving the use of cells for the prevention or treatment of disease. Non-limiting examples of cell therapy include CAR-T cell therapy, NK cell therapy, recombinant TCR-T cell therapy, and TIL (tumor infiltrating lymphocytes).

[0111] A "chemotherapeutic agent" is a compound known to be useful in the chemotherapy of cancer.

[0112] CARs are artificial (non-natural) immune cell (e.g., T cell) receptors intended for use as cancer treatments using a technique called adoptive cell transfer. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs are specifically constructed to stimulate T cell activation and proliferation in response to the specific antigen to which the CAR binds. Generally, a CAR refers to a set of polypeptides, typically two in the simplest embodiment, that, when expressed within an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some aspects, the set of polypeptides is adjacent to one another. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, OX40, 2B4, and / or CD28. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR into the cell membrane. In various embodiments, the CAR is a recombinant polypeptide comprising an antigen-specific domain (ASD), a hinge region (HR), a transmembrane domain (TMD), an optional costimulatory domain (CSD), and an intracellular signaling domain (ISD). The optional costimulatory domain is generally not present in first-generation CAR constructs. んThe nucleic acid and protein sequences of several exemplary second-generation CARs comprising different antigen-binding domains (e.g., vL and vH fragments, vHH, ligands and receptors, etc.) and incorporating a 41BB costimulatory domain are set forth in SEQ ID NOs: 1455-1703 and 341-7589 of PCT / US2020 / 014237 (Table 8).

[0113] "Codon optimization" or "control of species codon bias" refers to the preferred codon usage of a particular host cell. As will be understood by those skilled in the art, it may be advantageous to modify a coding sequence to enhance its expression in a particular host. Those skilled in the art will recognize that due to the degenerate nature of the genetic code, a variety of DNA compounds differing in nucleotide sequence may be used to encode a given polypeptide of the present disclosure.

[0114] As used herein, "coexpression" refers to the expression of two or more polynucleotides or genes. A gene can be, for example, a nucleic acid encoding a single protein or a chimeric protein as a single polypeptide chain. The SARs described herein (e.g., CAR, SIR, zSIR, or TCR) can be encoded by a single polynucleotide chain and expressed as a single polypeptide chain, which is then cleaved into different polypeptides, each representing a different functional unit. In some embodiments, when a SAR consists of two or more functional polypeptide units, the different functional units are coexpressed using one or more polynucleotide chains. In one embodiment, costimulation is provided by an accessory module that is coexpressed with the SAR or TCR but is not an integral part of the SAR (e.g., CAR, SIR, zSIR, or TCR) polypeptide. In another embodiment, different polynucleotide chains are linked by a nucleic acid sequence encoding a cleavable linker (e.g., T2A, F2A, P2A, E2A, etc.) (Table 20). In another embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 1239 and 1240) is also added upstream of the cleavable linker sequence to increase cleavage efficiency. The nucleic acid and amino acid sequences of exemplary cleavable linkers and furin cleavage sites are provided in Table 20. Polynucleotides encoding different units of the SAR may be linked by an IRES (internal ribosome entry site) sequence. In one embodiment, different functional units of the SAR (e.g., two or more chains) are expressed using a single vector. The various functional units of the SAR can be expressed using a single promoter or multiple promoters. In one embodiment, different functional units of the SAR are expressed using two or more vectors. The nucleic acid and amino acid sequences of exemplary cleavable linkers and furin cleavage sites are provided in Table 20.

[0115] A "conservative substitution" or "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics or function of the encoded protein. For example, a "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics or function of the SAR of the present disclosure (e.g., a conservative change in a constant chain, antibody, antibody fragment, or non-antibody immunoglobulin binding domain). Such conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine). , cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a SAR of the present disclosure can be substituted with other amino acid residues from the same side chain family, and the modified SAR can be tested using the binding and / or functional assays described herein.

[0116] A "costimulatory intracellular signaling domain" or "costimulatory domain" or "CSD" refers to a portion of a SAR that enhances T cell proliferation, survival, and / or development. A SAR of the present disclosure may contain zero, one, or more costimulatory domains. Each costimulatory domain may consist of one or more costimulatory domains, e.g., from any one of the following: a member of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), BAFF-R, HVEM, CD27, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof. Further exemplary costimulatory domains include the signaling domains of 2B4, NKp30, NKp44, NKp46, GITR, CD81, CD160, DAP10, and B7-H3. Other costimulatory domains (e.g., from other proteins) will be apparent to those skilled in the art and can be used in connection with alternative embodiments of the present disclosure. A costimulatory domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof. The SARs of the present disclosure may contain zero, one, or more costimulatory domains.

[0117] The term "costimulatory molecule" or "costimulatory receptor" refers to a cognate binding partner on immune cells (e.g., T cells, NK cells, macrophages, granulocytes, dendritic cells, etc.) that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the immune cell, such as, but not limited to, proliferation, activation, or cytokine secretion. Costimulatory extracellular molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an efficient immune response. Costimulatory molecules include MHC class I molecules, BTLA, Toll ligand receptor, OX40, Dap10, CD27, CD28, CD2, CD5, CD8, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, CD81, and 4-1BB (CD137). Costimulatory receptors may be expressed on cells other than T cells, such as NK cells and macrophages.

[0118] The term "cTCR" refers to a wild-type TCR nucleic acid coding sequence and the corresponding wild-type TCR protein linked to an antigen-binding domain not derived from a TCR. cTCRs are described in (Gross, Waks, and Eshhar, 1989). In some embodiments, cTCRs are used as reference controls. For example, a cTCR with a CD19-binding domain and a CD19-SIR (comprising a mutant TCR chain and a CD19-binding domain) will have different expression and / or different binding affinity for the target antigen.

[0119] The terms "cytosolic" or "cytoplasmic" refer to an agent, such as a protein, that is located in the cytoplasm of a mature cell. Cytosolic proteins can translocate to the nucleus, but they are not transmembrane proteins and are not secreted outside the cell.

[0120] is a complication of cell therapy (e.g., SAR-T, bispecific T-cell binding antibodies, etc.) that manifests with a range of signs and symptoms, including fever, hypotension, shortness of breath, renal dysfunction, respiratory illness, pulmonary dysfunction and / or capillary leak syndrome.

[0121] The term "degenerative disease" refers to illnesses that affect tissues and organs as a result of an ongoing process based on degenerative changes in cells and that become increasingly worse over time, whether due to normal physical wear and tear or lifestyle choices such as exercise and dietary habits. Examples of degenerative diseases include Alzheimer's disease, Creutzfeldt-Jakob disease, diabetes (type II), and atherosclerosis.

[0122] As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first molecule and the second molecule and does not imply or imply any limitations on the process or source by which the first molecule is derived from the second molecule. For example, in the case of an antigen-binding domain derived from an antibody molecule, the antigen-binding domain retains sufficient antibody structure to have the required function, i.e., the ability to bind to an antigen. This does not imply any limitations on the specific process by which the antibody is produced, nor does it imply, for example, that one must start with an antibody sequence and delete undesired sequences or introduce mutations to provide an antigen-binding domain.

[0123] The term "dimerization molecule," as used herein, refers to a molecule that promotes binding between a first switch domain and a second switch domain. In embodiments, the dimerization molecule is not naturally present in a subject or is not present at concentrations that result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001, rimiduside, or AP20187. Rimiduside can be about 0.01-1 mg / kg and has an EC50 of about 0.1 nM in cell culture. AP20187 can be administered in single or multiple doses at about 2-10 mg / kg / day.

[0124] The present invention also includes, but is not limited to, diseases associated with expression of a target antigen described herein or conditions associated with cells expressing the target antigen described herein, including, for example, a proliferative disease such as a cancer or malignant tumor, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or myeloproliferative disorder or preleukemia, or a non-cancer-related indication associated with cells expressing a target antigen described herein.

[0125] As used herein, "disease targeted by genetically modified cells" encompasses the targeting of any cells that are in any way involved in any disease by the genetically modified cells of the present disclosure, regardless of whether the genetically modified cells target diseased cells or healthy cells to achieve a therapeutic beneficial result.

[0126] The term "dissociation constant (Kd)" is defined as the equilibrium constant for dissociation of a receptor-ligand (e.g., binding domain-cognate) interaction. In some embodiments, the SARs of the present disclosure bind to a target antigen with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM.

[0127] As used herein, a "diverse set of non-natural immune receptors" or "diverse set of SARs" refers to a plurality of non-natural immune receptors or SARSs that target an antigen. In embodiments, the diverse set of SARs has the same binding domain linked to a diverse set of signaling chains or "backbones." In one embodiment, the diverse set of SARs may have a diverse range of binding affinities for the target antigen. In one embodiment, the diverse set of SARs may exhibit diverse expression levels.

[0128] As used herein, an "epitope" is defined as a portion of an antigen that is capable of eliciting an immune response or that binds to an antibody or antibody fragment. An epitope can be a protein sequence or subsequence.

[0129] As used herein, the term "engager" refers to an engager that engages immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils) and tumor cells, resulting in immune cell activation. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TRiKEs), or multispecific killer cell engagers, or universal engagers compatible with multiple immune cell types.

[0130] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0131] A "functional portion" ("biologically active portion") of a protein (e.g., SAR, IL-2, IL-15, etc.) refers to a portion of the protein that retains one or more functions of the full-length or mature protein. Such functions of IL-12 or IL-15 include promoting NK cell survival, regulating NK cell and T cell activation and proliferation, and supporting NK cell development from hematopoietic stem cells.

[0132] As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion; for example, digestion of an antibody with the enzyme papain produces two F(ab) fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to an antigen).

[0133] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, each of which is composed of two separate amino acid chains: a portion of a heavy chain and a light (L) chain joined by an S-linkage to bind to the antigen, with the remaining heavy chains joined together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0134] The term "FcRγ" or "FCER1G" or "FCRG" or "FcRy" as used herein refers to the gene represented by Gene ID: 2207, which is a disulfide-linker transmembrane signaling adaptor that is part of the high-affinity IgE receptor and other Fc receptors.

[0135] The term "functional portion," when used with respect to a SAR, refers to any portion or fragment of a SAR that retains the biological activity of the SAR of which it is a part (the parent SAR). A functional portion includes, for example, a portion of a SAR that retains a similar, equal, or greater ability to recognize a target cell or detect, treat, or prevent a disease than the parent SAR. With respect to a parent SAR, a functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent SAR.

[0136] For example, the term "flexible polypeptide linker" refers to a peptide linker comprised of amino acids such as glycine and / or serine residues, used alone or in combination to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, which has the amino acid sequence (Gly-Gly-Gly-Gly-Ser). n , (e.g., SEQ ID NO: 2431), where n is a positive integer equal to the following: for example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linker includes (Gly4Ser)4 or (Gly4Ser) 3が含まれるが、これらに限定されない .

[0137] As used herein, "genetically modified cells," "redirected cells," "genetically engineered cells," or "modified cells" refer to cells expressing a SAR of the present disclosure. In some embodiments, the genetically modified cells comprise a vector encoding the SAR. In some embodiments, the genetically modified cells comprise vectors encoding the SAR and one or more accessory molecules (e.g., PDL1, PDL2, crmA, MC159, etc.) within the same vector. In some embodiments, the genetically modified cells comprise a first vector encoding the SAR and a second vector encoding an accessory molecule. In some embodiments, the genetically modified cells comprise a first vector encoding the SAR and a second vector encoding two or more accessory molecules. In some embodiments, the genetically modified cells comprise a first vector encoding the SAR, a second vector encoding a first accessory molecule, and a third vector encoding a second accessory molecule.

[0138] As defined herein, an "HLA-independent TCR" or "MHC-independent TCR" is a TCR that can recognize an antigen independently of MHC restriction. In an exemplary embodiment, the HLA-independent TCR can bind to an antigen on the cell surface that is not presented by an MHC complex. In one embodiment, the HLA-independent TCR can bind to an antigen expressed on the cell surface independently of presentation by an MHC complex. The HLA-independent TCR can be a naturally occurring TCR. In an exemplary embodiment, the HLA-independent TCR is MC.7.G5 (MC7G5), which recognizes MR1, a ubiquitously expressed monotypic antigen-presenting molecule. The HLA-independent TCR can be an engineered TCR or a recombinant TCR. In an exemplary embodiment, the HLA-independent TCR is an engineered TCR that can bind to a protein expressed on the cell surface, such as CD19, CD20, mesothelin, PSMS, or BCMA. Methods for engineering TCR variable domains (e.g., CDR grafting, etc.) are known in the art and can be used to generate HLA-independent TCRs capable of binding to proteins (e.g., CD19, MSLN, PSMA, etc.) or proteins. The epitope is expressed extracellularly independent of the MHC complex. The present disclosure provides bispecific, biparatopic, and multispecific SARs having a TCR framework, including HLA-independent TCRs, that include one or more AABDs. The AABD domains of the SARs of the present disclosure having a TCR (e.g., HLA-independent TCR) framework can be fully human, humanized, or non-human. In one embodiment, the present disclosure provides a TCR (e.g., an HLA-independent TCR) that includes one or more fully human vH domains. In one embodiment, the present disclosure provides a TCR (e.g., an HLA-independent TCR) that includes one or more fully human vL domains.

[0139] As defined herein, an "HLA-independent TCR variable domain" is a variable domain of a TCR that can bind to an antigen in an HLA-independent manner. The HLA-independent variable domain can be a variable domain of an HLA-independent TCR alpha, TCR beta, TCR gamma, TCR delta, or pre-TCR alpha. The HLA-independent TCR variable domain can be a single variable domain TCR (i.e., svd-TCR). The HLA-independent TCR variable domain can be a naturally occurring HLA-independent variable domain or an engineered HLA-independent variable domain. In an exemplary embodiment, modified HLA-independent variable domains can be generated against the extracellular domain of proteins (e.g., CD19, CD22, BCMA, MSLN, PSMA) using techniques known in the art (e.g., CDR grafting, screening of phage display libraries, etc.).

[0140] As used herein, "HLA-restricted" or "MHC-restricted" refers to antigen recognition that requires both an MHC molecule and its peptide. It is distinct from "HLA-unrestricted," "HLA-independent," or "MHC-unrestricted" antigen recognition.

[0141] As used herein, the term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene introduced from one species into another. A heterologous gene also includes a gene native to an organism that has been modified in some way (e.g., mutated, added multiple copies, linked to non-native regulatory sequences, etc.). As another example, a heterologous gene includes a gene expressed in a previous or future cell lineage or differentiated state of a cell. Heterologous genes are distinguished from endogenous genes in that they are typically associated with DNA sequences not naturally found in the gene sequence in a chromosome or in a part of a chromosome not naturally found (e.g., a gene expressed at a locus where the gene is not normally expressed).

[0142] As used herein, "hinge region" (HR) refers to the hydrophilic region located between the antigen-binding domain and the transmembrane domain of a SAR. Hinge regions include, but are not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, a CD8a hinge and an artificial spacer consisting of a polypeptide that may be as small as, for example, Gly3 or the CH1 and CH3 domains of an IgG (such as human IgG4). In some embodiments, the hinge region is one or more of: (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8a, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vi) the hinge and CH2 regions of IgG1. Some exemplary hinge regions are provided in Table 29 of the present disclosure. Other hinge regions will be apparent to those of skill in the art and can be used in connection with alternative embodiments of the present disclosure.

[0143] The term "immune disorder" refers to a disease characterized by a malfunction of the immune system. Autoimmune diseases are conditions caused by an abnormal immune response against healthy parts of the body. There are at least 80 different autoimmune diseases.

[0144] For example, it refers to cells involved in promoting immune effector responses. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, monocytes / macrophages, and myeloid cells. Derived phagocytes.

[0145] "Immune effector function" or "immune effector response" refers to a specialized function of a differentiated cell. The effector function of a T cell or NK cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. For example, an immune effector function or immune response refers to a property of a T cell or NK cell that promotes the killing or inhibition of growth or proliferation of a target cell. For T cells, primary stimulation and costimulation are examples of immune effector functions or responses. For antigen-presenting cells (e.g., dendritic cells), antigen presentation and cytokine secretion are examples of effector functions.

[0146] As used herein, "immune response" refers to immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and / or hyperimmunity.

[0147] As used herein, "interleukin-2" ("IL-2") and "interleukin-15" ("IL-15") refer to cytokines that regulate the activation and proliferation of T and NK cells. These cytokines share many biological activities. They are known to bind to common receptor subunits and may compete for the same receptor, thereby negatively regulating each other's activity. Various IL-2 and IL-15 molecule sequences are known in the art. In one embodiment, the IL-2 is wild-type IL-2 or a variant thereof having 70-99.9% amino acid sequence identity (e.g., SEQ ID NOs: 7833-7837). In one embodiment, the IL-15 is wild-type IL-15 or a variant thereof having 70-99.9% amino acid sequence identity (e.g., SEQ ID NOs: 7838-7841). In some embodiments, the IL-2 is mammalian IL-2. In some embodiments, the IL-15 is mammalian IL-15 (e.g., Homo sapiens interleukin-15 (IL15), transcript variant 3, mRNA, NCBI Reference Sequence: NM_000585.4; Canis lupus familiaris interleukin-15 (IL15), mRNA, NCBI Reference Sequence: NM_001197188.1; Felis catus interleukin-15 (IL15), mRNA, NCBI Reference Sequence: NM_001009207.1). In certain embodiments, all or a functional portion of IL-2 or IL-15 is linked to all or a portion of a transmembrane protein. In one embodiment, the NK cell or T cell expresses a fusion protein comprising all or a portion of IL-2 or IL-15 fused to all or a portion of a transmembrane protein. In certain embodiments, the portion of the transmembrane protein comprises all or a portion of the transmembrane domain of the transmembrane protein.

[0148] As used herein, the term "intracellular signaling domain" (ISD) or "activation domain" refers to the intracellular signaling portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of the cell. Examples of immune effector functions include cytolytic activity and helper activity, including cytokine secretion. Examples of domains that transmit effector function signals include, but are not limited to, the z chain of the T cell receptor complex or its homologs, human CD3 zeta chain, CD3 polypeptides (γ, δ, and ε), Syk family tyrosine kinases (e.g., Syk, ZAP 70), Src family tyrosine kinases (e.g., Lck, Fyn, Lyn), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28. Other intracellular signaling domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present disclosure.

[0149] In another embodiment, the intracellular signaling domain can comprise a "primary intracellular signaling domain" or "activation domain." Exemplary primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent stimulation. In another embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include domains derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, the primary intracellular signaling domain can comprise the cytoplasmic sequence of CD3z, and the costimulatory intracellular signaling domain can comprise the cytoplasmic sequence derived from a co-receptor or costimulatory molecule, such as CD28 or 41BB.

[0150] The primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3-zeta, common FcR gamma (FCER1G or FcRγ or FCRG), Fc gamma RIIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0151] The term "isolated" as used herein refers to a molecule, biological material, or cellular material that is substantially free of other substances. In one aspect, the term "isolated" refers to a nucleic acid, such as DNA or RNA, or a protein or polypeptide (e.g., an antibody or its derivative), or a cell or organelle, or a tissue or organ, that has been separated from other DNA or RNA. The term "isolated" also refers to an RNA, protein, or polypeptide, a cell or organelle, or a tissue or organ that is present in a natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" refers to a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in its natural state. The term "isolated" is also used herein to refer to a polypeptide that has been isolated from other cellular proteins, and is intended to encompass both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue that has been isolated from other cells or tissues, and is intended to encompass both cultured and engineered cells or tissues.

[0152] A "long linker" or "long linker domain" is a linker that is 25 to 500 amino acids in length. In one embodiment, a long linker is about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 450, 500 amino acids, and any number of amino acids in between. In one embodiment, a long linker is 25 to 125 amino acids in length. In one embodiment, a long linker is 50 to 150 amino acids in length. In one embodiment, the long linker is between 75 and 175 amino acids in length. In one embodiment, the long linker is 100-200 amino acids in length. In one embodiment, the long linker is 120-220 amino acids in length. In one embodiment, the long linker is 100-300 amino acids in length.

[0153] In one embodiment, the linker encodes or comprises an immunoglobulin (Ig) domain or an Ig-like domain or a fragment thereof. The terms "Ig domain," "Ig linker domain," "Ig-like domain," and "Ig-like linker domain" are used interchangeably in this disclosure. An immunoglobulin domain is a type of protein domain consisting of a two-layer sandwich of seven to nine antiparallel beta strands arranged in two beta sheets with a Greek key topology, and is composed of approximately 125 amino acids. Ig domains can be classified as IgV, IgC1, IgC2, or IgI. IgV domains, with nine beta strands, are generally longer than IgC domains, with seven beta strands. In one embodiment, the linker comprises an IgV domain or a fragment thereof. In one embodiment, the linker comprises an IgC domain or a fragment thereof. Ig domains are found in immunoglobulins, T cell receptor chains, class I MHC, class II MHC, β2-microglobulin, coreceptors (e.g., CD4, CD8, CD19, etc.), antigen receptor accessory molecules (e.g., CD3γ, CD3δ, CD3ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), NK cell receptors (e.g., KIR), leukocyte immunoglobulin-like receptors (LILR), IgSF CAMs (e.g., NCAM, ICAM, CD2, etc.), cytokine receptors (e.g., IL-1R, CSF-1R, etc.), growth factor receptors (e.g., PDGFR), receptor tyrosine kinases and phosphatases, Ig-binding receptors, cytoskeletal proteins (e.g., titin, pancreatin, etc.) and other proteins (e.g., CD147, CD90, etc.). Exemplary Ig linker domains are IgCL (SEQ ID NO: 3536) and IgG1-CH1 (SEQ ID NO: 3537). Additional exemplary Ig linkers are shown in Table 13 (SEQ ID NOs: (PRT): 3538-3569). In one embodiment, the linker has an E-set domain. E-set domains are an "early" Ig-like fold family that may be related to the immunoglobulin and / or fibronectin type III superfamilies.In one embodiment, the linker comprises a fibronectin type III domain.

[0154] In some embodiments, the SAR of the present disclosure comprises an Fv-like or Fc-TCR antigen-binding module comprising: a) a first polypeptide chain comprising a first antigen-binding domain consisting of a vL, Vα, or Vγ domain; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a vH, Vβ, or Vδ domain. In some embodiments, there is a first peptide linker fused to the C-terminus of the vL, Vα, or Vγ domain and / or a second peptide linker fused to the C-terminus of the vL, Vα, or Vγ domain. In some embodiments, the first and second peptide linkers can be linked to each other. In some embodiments, the first and / or second peptide linkers are derived from immunoglobulin heavy and / or light chain constant regions. In some embodiments, the first and / or second peptide linkers comprise a CH3 antibody domain or a variant thereof. In some embodiments, the immunoglobulin heavy chain constant domain (e.g., CH1 or CH3) included in the peptide linker is an IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain, optionally human. In some embodiments, the first and / or second peptide linker is derived from a TCR subunit constant region. For example, in some embodiments, the first and / or second peptide linker is derived from a) a TCR α and β subunit constant domain; or b) a TCR γ and δ subunit constant domain. In some embodiments, the first and / or second peptide linker is synthetic. In some embodiments, all of the CDRs of vL, Vα or Vγ and vH, Vβ or Vδ are derived from the same antibody or TCR moiety. In some embodiments, the vL antibody domain and the vH antibody domain comprise antibody CDRs derived from two or more antibody moieties. In some embodiments, the vL antibody domain comprises an antibody CDR derived from a vH antibody domain and / or the vL antibody domain comprises an antibody CDR derived from a vH antibody domain.In some embodiments, the vL antibody domain comprises a framework region from one antibody and one or more CDRs from another antibody, and / or the vH antibody domain comprises a framework region from one antibody and one or more CDRs from another antibody. In some embodiments, the Vα domain and Vβ domain comprise TCR CDRs from two or more TCRs. In some embodiments, the Vα domain comprises CDRs from a Vβ TCR domain, and / or the Vβ domain comprises CDRs from a Vα domain. In some embodiments, the Vα domain comprises framework regions from one TCR and one or more CDRs from another TCR, and / or the Vβ domain comprises framework regions from one TCR and one or more CDRs from another TCR. In some embodiments, the Vγ domain and Vδ domain comprise TCR CDRs from two or more TCRs. In some embodiments, the Vγ domain comprises CDRs from a Vδ TCR domain, and / or the Vδ domain comprises CDRs from a Vγ domain. In some embodiments, the Vγ domain comprises a framework region from one TCR and one or more CDRs from another TCR, and / or the Vδ domain comprises a framework region from one TCR and one or more CDRs from another TCR. In some embodiments, the first and second polypeptide chains are linked by a covalent bond (e.g., a peptide or other chemical bond) or a non-covalent bond, etc. In some embodiments, the first and second antigen-binding domains are linked by a disulfide bond. In some embodiments, the first and second peptide linkers are linked by a disulfide bond. In some embodiments, the first and / or second peptide linkers are variants containing one or more modifications (e.g., amino acid substitutions, insertions, and / or deletions) compared to the sequences from which they are derived. In some embodiments, the first and / or second peptide linkers contain one or more modifications that do not substantially alter their binding affinity for each other.In some embodiments, the first and / or second peptide linkers contain one or more modifications that increase their binding affinity to each other and / or introduce a non-naturally occurring disulfide bond. In some embodiments, the first and second peptide linkers contain knob-into-hole modifications (see, e.g., Carter P. Immunol Methods. 248:7-15, 2001). In some embodiments, the first and second peptide linkers are modified by electrostatic steering to enhance their association with each other (see, e.g., WO2006106905 and Gunasekaran K et al., J Biol Chem. 285:19637-46, 2010). In some embodiments, the Fv-like or TCR-Fv-like antigen-binding module is human, humanized, chimeric, semi-synthetic, or fully synthetic.

[0155] An exemplary SAR construct comprising an IgCL and an IgG1-CHI linker is represented by CD8SP-hu-mROO5-1-vL-xho-IgCL-Bam-DAP10-opt1-Spe-CD3zCP-opt1-F-P2A-dSPE-IgSP-hu-mROO5-1-vH-Mlu-IgG1-CHI-Kpn-DAP10-opt2-Xba-CD3zCP-opt2-F-F2A-dXBA-Nde-K13-opt (SEQ ID NO: 5869). The IgG1-CHI linker (SEQ ID NO: (DNA): 1143, SEQ ID NO: (PRT): 3537) in this construct can be replaced with other Ig-like linkers shown in Table 13, such as IgG2-IC-CHI1, IgG3-CHI1. , IgG4-CHI1, IgAI-CHI1, IgA2-CHI1, IgD-CHI1, IgE-CHI1, or IgM-CHI1. The IgCL and IgG1-CH1 linkers can also be replaced by Ig-like linkers derived from TCR alpha and TCR beta, respectively (Table 13). Alternatively, the IgCL and IgG1-CH1 linkers can also be replaced by Ig-like linkers derived from the TCR gamma and TCR delta chains (Table 13).

[0156] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In particular, a ligand binds to a receptor on another cell, allowing recognition and / or interaction between the cells.

[0157] As used herein, the term "linker" (also "linker domain" or "linker region") refers to an oligo or polypeptide (or an oligo encoding a polypeptide) that links two or more domains or regions of a SAR polynucleotide or polypeptide, each disclosed herein. A linker can be either 1 to 500 amino acids in length or 3 to 1500 nucleotides in length. In some embodiments, a "linker" is cleavable or non-cleavable. Unless otherwise specified, the term "linker" as used herein refers to a non-cleavable linker. The non-cleavable linker may be composed of flexible residues that allow free movement of adjacent protein domains relative to each other. Non-limiting examples of such residues include glycine and serine. In some embodiments, the linker comprises a non-flexible residue. Examples of cleavable linkers include a 2A linker (e.g., T2A), a 2A-like linker or functional equivalents thereof, and combinations thereof. In some embodiments, the linker includes a picornavirus 2A-like linker, a porcine teschovirus (P2A), a Thesea signavirus (T2A) CHYSEL sequence, or combinations, variants, and functional equivalents thereof. In some embodiments, the linker sequence may include a motif that results in cleavage between 2A glycine and 2B proline (see, e.g., T2A sequence). Nucleic acid sequences of some exemplary cleavable linkers are provided in SEQ ID NOs: 1233 to 1238, and amino acid sequences of some exemplary linkers are provided in SEQ ID NOs: 3627 to 3632. The terms used herein will be readily understood by those skilled in the art. Linker module also refers to the TCR and antibody linkers set forth in Table 13.

[0158] In one embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NO: 3633) is also added upstream of the cleavable linker sequence to enhance cleavage efficiency. A potential drawback of cleavable linkers is that a small 2A tag left at the N-terminal end of the protein may affect protein function or contribute to the protein's antigenicity. To overcome this limitation, in some embodiments, a furin cleavage site (RAKR) (SEQ ID NO: 3635) is added upstream of the SGSG motif to facilitate cleavage of the remaining 2A peptide after translation.

[0159] The term "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. They are one of the most efficient gene delivery vectors because they can deliver large amounts of genetic information into the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses.

[0160] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and specifically includes self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Soc. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX™ vector system. Non-clinical types of lentiviral vectors are also available and would be known to those skilled in the art. Other examples of lentiviral vectors are pLENTI-EF1α (SEQ ID NO: 1), pLENTI-EF1α-DWPRE (SEQ ID NO: 2), pCCLc-MNDU3-WPRE (SEQ ID NO: 4), and pCCLc-MNDU3-Eco-Nhe-Sal-WPRE (SEQ ID NO: 5). In an exemplary embodiment, a SAR, or a SAR plus an accessory module, or a nucleic acid fragment encoding an accessory module, can be cloned between the Nhe I and Sal I sites present in pLENTI-EF1α and pCCLc-EF1α, and the MNDU3-Eco-Nhe-Sal-WPRE vector using methods known in the art.

[0161] As used herein, "killer cell immunoglobulin-like receptor" or "KIR" refers to a family of transmembrane glycoproteins expressed by natural killer cells and a subset of T cells.

[0162] Refers to any member of the class Mammalia.

[0163] A "marker gene" encodes a protein not normally expressed by target cells, allowing for identification of successful transduction. Marker genes can also be used to selectively remove or enrich for transduced cells (e.g., SAR-expressing cells). Exemplary marker genes include tEGFR, CD20, tCD19, tBCMA, and RQR8.

[0164] A "multipurpose switch" or "multipurpose gene" encodes a protein that provides suicide, survival, and marker functions. In one embodiment, all of these functions are provided by a single polypeptide chain. Examples of multipurpose switches include IL2-tBCMA, IL15-tBCMA, IL2-RQR8, and IL2-tHer2.

[0165] As used herein, a "mimotope" is a large molecule, often a peptide, that mimics the structure of an epitope. This property allows it to elicit an antibody response similar to that elicited by the epitope. Antibodies against a particular epitope antigen recognize a mimotope that mimics that epitope. A mimotope is a type of peptide aptamer.

[0166] The term "multi-chain synthetic antigen receptor" or "multi-chain SAR" refers to a synthetic antigen receptor containing two or more polypeptide chains. A multi-chain SAR may be a double-chain SAR. A double-chain SAR contains two membrane-associated domains (e.g., transmembrane domains or membrane anchor domains). An exemplary multi-chain SAR targeting CD19 is CD8SP-CD19-hu-mROO5-1-vL-Xho-CD16-F158V-FL-TMCP-v1-F-P2A-Spe-SP-Bst-CD19-hu-mROO5-1-vH-Mlu-CD16-F158V-S197P-FL-TMCP-v3-F-F2A-Xba-PAC (SEQ ID NO: (DNA): 5451 and SEQ ID NO: (PRT): 6283). In this SAR construct, the hu-mROO5-1 vL fragment is operably linked to the CD16-F158V-FL-TMCP-v1 module, and the hu-mROO5-1-vH fragment is operably linked to CD16-F158V-S197P-FL-TMCP-v3 (TMCP-v3). The two chains of this SAR are separated by a furin (F) and P2A cleavable linker sequence. This SAR construct also expresses a puromycin resistance gene (PAC), separated from the SAR polypeptide by a furin (F) and F2A cleavable linker sequence. Because the SAR is modular, the CD16A-F158V-S197P-FL-v3 and CD16-F158V-FL-TMCP-v1 modules can be replaced with other signaling modules to generate SARs with different signaling chains. Furthermore, the hu-mROO5-1 vL and hu-mROO5-vH fragments can be replaced with antigen-binding domains targeting other antigens (e.g., vL, vH, vHH, FHVH, centtilin, svd-TCR, etc.) to generate SARs targeting different antigens. Examples of such multi-chain SARs are shown in Table 41 of the provisional application. (For example, sequence numbers 5451-5462, 5483-5494, 5515-5526, 5547-5558, 5579-5590, 5611-5622, 5643-5654, etc.).The expression and activity of these novel SARs can be tested using the methods described in this disclosure to select SARs with optimal functional activity.

[0167] As used herein, "MHC" or "major histocompatibility complex" refers to cell surface molecules encoded by multiple genes in mammals. MHC molecules include class I and class II. Class I molecules are also called "HLA" or "human leukocyte antigens" in humans. Due in part to the complexity of HLA molecule expression, HLA is also referred to as the HLA system. Humans express HLA-A, HLA-B, and HLA-C molecules, which are typically involved in presenting processed antigens to CD8+ cells, i.e., HLA-restricted. Class II molecules, such as DR, DQ, and DP, are typically involved in presenting exogenous peptides to CD4+ cells, i.e., MHC class II restriction. Commonly restricted MHCs include both class I and class II, as in transplant (bone marrow) matching.

[0168] As used herein, "native" or "naturally occurring" or "endogenous" refers to a gene, protein, nucleic acid (e.g., DNA, RNA, etc.), or fragment thereof, that is native to a cell or that is naturally expressed in a cell. Thus, a natural or endogenous TCR α chain polypeptide of a T cell consists of a variable domain (Vα) attached to a TCR α constant chain. A natural or endogenous TCR α chain precursor polypeptide also consists of an amino-terminal signal peptide that is cleaved from the mature polypeptide.

[0169] As used herein, "native receptor" or "natural receptor" or "endogenous receptor" or "natural receptor" refers to any receptor that occurs in nature and contains an antigen-binding domain or a ligand-binding domain. This term includes functional variants, isoforms, and homologs from other mammalian species. A native receptor may be a "native signaling receptor" or "natural signaling receptor" if it is capable of transducing a cellular signal upon binding to a target. A naturally occurring receptor or native receptor is endogenous to a cell or naturally expressed within a cell. Examples of naturally occurring signaling receptors or native receptors include, but are not limited to, CD16A, CD16B, NKp30, NKp44, NKp46, KIR2DS4, NKG2D, etc. For purposes of this disclosure, the CD3 signaling chains (CD3ε, CD3γ, CD3δ, and CD3ζ) are not included in the definition of "naturally occurring receptor" and are instead classified as signaling adaptors.

[0170] As used herein, the terms "non-TCR native receptor" or "non-TCR native signaling receptor" or "non-TCR receptor" or "non-TCR signaling receptor" refer to a receptor that is not a T cell receptor (TCR). Non-TCR receptors may be expressed on cells other than T cells. Non-TCR receptors may be expressed on cells that lack expression of the CD3ζ, CD3ε, CD3δ, and / or CD3γ chains. A "non-TCR native receptor" lacks the transmembrane and / or cytosolic domains of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. A "non-TCR native receptor" does not recruit the entire TCR signaling module. In one embodiment, a "non-TCR native receptor" does not include a TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα polypeptide. In one embodiment, a "non-TCR native receptor" does not include the entire coding region of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, a "non-TCR native receptor" does not include the entire constant chain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, a "non-TCR native receptor" does not include the entire hinge domain (or connecting peptide) of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, a "non-TCR native receptor" does not include the entire transmembrane and cytosolic domains of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. Non-TCR receptors may be expressed in cells other than T cells. A "non-TCR signaling receptor" may include a fragment of a TCR, such as a TCR variable domain (e.g., Vα, Vβ, Vγ, Vδ) or an Ig domain (e.g., SEQ ID NOs: 1158-1175). A non-TCR signaling receptor does not include the entire TCR constant chain (i.e., the constant chain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα).

[0171] As used herein, the terms "non-T cell receptor module" or "non-TCR module" or "non-TCR signaling module" or "NTCRM" refer to a module that lacks sequences consisting of a T cell receptor transmembrane domain and may further lack all or a portion of the T cell receptor-linked peptide and / or the intracellular domain. An NTCRM lacks sequences consisting of the transmembrane domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. An NTCRM may further lack all or a portion of the connecting peptide and / or the intracellular domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα.

[0172] As used herein, the term "non-CD3 adaptor module" or "non-CD3 adaptor" or "non-TCR / CD3 adaptor" or "non-TCR / CD3 signaling adaptor" or "NCAM" refers to a signaling adaptor that is not a component of the T cell receptor / CD3 receptor complex. In one embodiment, a "non-TCR / CD3 adaptor" does not include the transmembrane and / or cytoplasmic regions of the CD3ε, CD3ζ, CD3γ, or CD3δ chain or variants thereof.

[0173] As used herein, the term "near the N-terminus" refers to within the N-terminal 30 amino acids. For example, the term "AABD operably linked to or near the N-terminus of a vL and / or vH domain" refers to an AABD operably linked to the N-terminus of a vL or vH fragment, or an AABD operably linked to the N-terminal 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, or 30 amino acids comprising a vL or vH domain. Similarly, the term "AABD operably linked to or near the N-terminus of a Va and / or Vb domain" means an AABD operably linked to the N-terminus of a Va or Vb fragment, or an AABD operably linked to the N-terminal 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 amino acids comprising a Va or Vb domain. An AABD of the present disclosure can be operably linked to or near the N-terminus of another domain, either directly or through an intervening linker sequence.

[0174] As used herein, "natural killer cell receptor" or "NK receptor" refers to a cell surface receptor expressed on natural killer (NK) cells, including functional variants, isoforms, and homologs from other mammalian species. NK receptors can be activating or inhibitory receptors. Exemplary activating NK receptors include NKp30, NKp44, NKp46, NKG2D, and KIR3DS4. Exemplary inhibitory NK receptors include CD94-NKG2A, TIGIT, and CD96.

[0175] As used herein, "natural killer cells" ("NK cells") refer to a type of cytotoxic lymphocyte of the immune system.

[0176] As used herein, "NKp30" or "NCR3" is a gene (Gene ID: 259197) that encodes a protein that is a natural cytotoxicity receptor (NCR) that may assist NK cells in lysing tumor cells. The term includes functional variants, isoforms, and homologs in other mammalian species.

[0177] As used herein, "NKp44" or "NCR2" refers to a gene (Gene ID: 9436) that encodes a protein that is a natural cytotoxicity receptor (NCR). This term includes functional variants, isoforms, and homologs in other mammalian species.

[0178] As used herein, "NKp46" or "NCR1" is a gene (Gene ID: 9437) that encodes a protein that is a natural cytotoxicity receptor (NCR). Five transcript variants encoding different isoforms have been discovered for this gene. The term includes functional variants, isoforms, and homologs in other mammalian species.

[0179] As used herein, "NKG2D" or "KLRK1" refers to a gene (Gene ID: 22914) that encodes a protein that is a member of the C-type lectin family. The encoded transmembrane protein is characterized by type II membrane orientation (with an extracellular C-terminus) and the presence of a C-type lectin domain. The term includes functional variants, isoforms, and homologs in other mammalian species.

[0180] As used herein, "non-natural agent" or "non-natural" or "exogenous" refers to an agent that is not naturally expressed in a cell. In other words, a non-natural agent is "engineered" to be expressed in a cell. A non-natural agent may be a cloned version of a natural agent. Exemplary non-natural agents include SARs (e.g., CARs, SIRs, Ab-TCRs, TFPs, recombinant TCRs). A non-natural agent can be expressed in a cell using gene transfer techniques known in the art, such as lentivirus- or retrovirus-mediated gene transfer. A non-natural agent can be expressed in an immune cell using an exogenous promoter (e.g., the EF1α promoter) or an endogenous promoter (e.g., the TCRα or TRAC promoter). Another example of a non-natural agent is when an endogenous gene (e.g., CD16, NKp30, etc.) is cloned and ectopically expressed in a cell.

[0181] As used herein, "non-natural immunoreceptor" or "exogenous immunoreceptor." A "non-natural receptor" refers to an immunoreceptor that is not naturally expressed in an immune cell. Stated differently, a non-naturally occurring immunoreceptor has been "engineered" to be expressed in an immune cell. A non-naturally occurring immunoreceptor may be a cloned version of a natural immunoreceptor. Alternatively, a non-naturally occurring immunoreceptor may be a chimeric receptor generated using recombinant molecular biology techniques. Exemplary non-naturally occurring immunoreceptors are SARs (e.g., 第2 generation CAR, SIR, cTCR, STAR, zSIR, Ab-TCR, TFP, and recombinant TCR).

[0182] As used herein, a "non-native TCR antigen-binding domain" or "exogenous TCR antigen-binding domain" refers to a binding domain operably linked to a TCR constant region that is chimeric and non-native with respect to a naturally occurring TCR. Stated differently, the non-naturally occurring TCR antigen-binding domain is "engineered" using recombinant molecular biology techniques to operably bind to a TCR, and further, the antigen-binding domain is obtained from or derived from a molecule distinct from a naturally occurring TCR. Antigen-binding domains that are essentially distinct from TCRs include antibody vH and vL fragments, humanized antibody fragments, chimeric antibody fragments, receptor ligands, and the like.

[0183] As used herein, a "non-natural antigen-binding domain" or "non-natural extracellular antigen-binding domain" or "heterologous antigen-binding domain" refers to an antigen-binding domain that is not part of a natural receptor. Stated differently, a non-naturally occurring antigen-binding domain is "engineered" using recombinant molecular biology techniques to be operably linked to a naturally occurring signaling receptor, and further, the antigen-binding domain is obtained or derived from a molecule different from the naturally occurring signaling receptor. Exemplary heterologous antigen-binding domains include antibodies, antibody fragments (e.g., vL, vH, scFv, Fab, F(ab)2, etc.), single-domain antibodies (e.g., sVH, FHVH, vHH, etc.), non-immunoglobulin antigen-binding domains, single variable domain-TCRs (svd-TCRs), recombinant TCRs, HLA-independent TCRs, scTCRs, epitopes, adapters, ligands, and receptors.

[0184] The term "operably linked" or "functionally linked" or "operably linked" refers to a functional linkage or bond between a first component and a second component such that each component can function. For example, operably linked includes the linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. With respect to two operably linked polypeptides, the first polypeptide functions independently of any linkage, and the second polypeptide functions as if there were no linkage between the two. The term "operably linked" as used in the context of different domains of a SAR of the present disclosure refers to domains that are linked via a covalent bond (e.g., a peptide bond or a non-peptide chemical bond). In an exemplary embodiment, SAR comprising a heterologous antigen binding domain (for example, CD19 scFv) operably linked to the N-terminus of the extracellular domain of CD16A refers to a SAR polypeptide encoded by a nucleic acid sequence consisting of CD19 scFv fused in frame to the nucleic acid sequence encoding the extracellular domain, transmembrane domain and cytoplasmic domain of CD16A.In one embodiment, the functional bond between different domains of the SAR polypeptide is achieved through peptide bonds.However, in certain embodiments, different domains of the SAR can be linked through non-peptide bonds, such as disulfide bonds, or chemical bonds.

[0185] "Percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if the two sequences have a specified percentage of the same amino acid residues or nucleotides (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over the specified region, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using one of the sequence comparison algorithms below, or by manual alignment and visual inspection. Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, and more typically over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0186] Two examples of algorithms that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms. These are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0187] The percent identity between two amino acid sequences was calculated using the ALIGN program (version 2.0) using the PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4 (E. Meyers and W. Miller, (1988) Comput. Applied Biosciences. 4:11-17).

[0188] Non-limiting examples of target antigens are listed in Table B. The SARs of the present disclosure can bind to one or more (e.g., 2, 3, 4, 5, or more) target antigens listed in Table B directly or via a SAR adaptor described herein.

[0189] [Table 22-1] [Table 22-2] [Table 22-3]

[0190] In some embodiments, the SARs of the present disclosure comprise one or more antigen-binding domains (e.g., vL, vH, Va, Vb, Vg, Vd, Fv, TCR-Fv, svd-TCR, scTCR, etc.) that specifically bind to a complex comprising a peptide derived from a disease-associated antigen (e.g., a peptide derived from a tumor-associated antigen or a virus-encoded antigen; e.g., NY-ESO-1, MAGE-A3, MAGE-A4, WT1, mutant Ras, HPV16-E7, EBV-LMP2A, AFP, gp100, PSA, mutant p53, HIV-1, etc.) and an MHC class I protein, where the MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the MHC class I protein is HLA-A, HLA-B, or HLA-C. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, the MHC class I protein is HLA-B. In some embodiments, the MHC class I protein is HLA-C. In some embodiments, the MHC class I protein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is any one of HLA-A*02:01-555, such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A*02:010, HLA-A*02:021, HLA-A*02:03, HLA-A*02:011, HLA-A*02:022, HLA-A*02:033, HLA-A*02:012, HLA-A*02:013, HLA-A*02:014,-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A *02: 10, HLA-A*02: 11, HLA-A*02: 12, HLA-A*02: 13, HLA-A*02: 14, HLA-A*02: 15, HLA-A*02 :16, HLA-A*02:17, HLA-A*02:18, HLA-A*02:19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, or HLA-A*02:24. In some embodiments, the MHC class I protein is HLA-A*02:01, which is expressed in 39-46% of Caucasians overall and therefore represents an appropriate choice of MHC class I protein for use in the present disclosure.

[0191] In some embodiments, the SARs of the present disclosure comprise one or more antigen-binding domains (e.g., vL, vH, Va, Vb, Vg, Vd, Fv, TCR-Fv, svd-TCR, scTCR, etc.) that specifically bind to a complex comprising a peptide derived from a disease-associated antigen (e.g., a peptide derived from a tumor-associated or virus-encoded antigen, e.g., NY-ESO-1, MAGE-A3, MAGE-A4, WT1, mutant Ras, HPV16-E7, EBV-LMP2A, AFP, gp100, PSA, mutant p53, HIV-1, etc.) and an MHC class II protein, wherein the MHC class II protein is HLA-DP, HLA-DQ, or HLA-DR.

[0192] The SARs of the present disclosure can also bind to complexes containing peptides derived from disease-associated antigens and MHC class I or class II proteins from other species (e.g., dog, cat, mouse, rat, cow, horse, monkey, etc.).

[0193] As used herein, the term "receptor" refers to a polypeptide or portion thereof present on a cell membrane that selectively binds one or more ligands.

[0194] As used herein, the term "region" or "portion," when used in reference to a nucleic acid molecule, refers to a set of linked nucleotides that is less than the entire length of the molecule, such as the CD3ζ signaling region described herein.

[0195] The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome. Examples of retroviral vectors include MSCVneo, MSCV-pac (or MSCV-puro), and MSCV-hygro, available from Addgene or Clontech.

[0196] As used herein, the term "SAR" or "synthetic antigen receptor" includes traditional CARs and also encompasses newer approaches to confer antigen specificity to cells, such as antibody-TCR chimeric molecules or Ab-TCRs (WO 2017 / 070608 A1, incorporated herein by reference), TCR receptor fusion proteins or TFPs (WO 2016 / 187349 A1, incorporated herein by reference), synthetic immune receptors (SIRs) (WO 2018 / 102795 A1, incorporated herein by reference), STARs (see WO2020 / 029774), HLA-independent TCRs (see WO2019157454 A1), trifunctional T cell antigen couplers (Tri-TACs or TACs) (WO2015 / 117229 A1, incorporated herein by reference). A1) and zSIR (PCT / US2019 / 035096, incorporated herein by reference). Bispecific and multispecific CARs are described in PCT / US2021 / 022641. The term "SAR" encompasses CARs and other antigen-binding receptors (including, but not limited to, recombinant TCRs).

[0197] The term "SAR-T" is typically used to refer to T cells engineered to express a synthetic antigen receptor. Therefore, T lymphocytes with such SAR are commonly referred to as SAR-T lymphocytes. SAR can also be expressed in cells other than T cells, such as hematopoietic stem cells, induced pluripotent stem cells (iPSCs), NK cells, and macrophages. In some embodiments, SAR is expressed in immortalized cell lines such as NK92, NK92MI, or their derivatives. The term "SAR-NK" refers to NK cells engineered to express SAR.

[0198] The term "SAR-NK" refers to T cells (e.g., αβ T cells, γδ T cells, Tregs, TILs, etc.) engineered to express a synthetic antigen receptor. Therefore, T lymphocytes with such SAR are commonly referred to as SAR-T lymphocytes. SAR can also be expressed in cells other than T cells, such as hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), NK cells, NKT cells, monocytes, macrophages, B cells, granulocytes, dendritic cells, and cytokines. In some embodiments, SAR is expressed in immortalized cell lines such as NK92, NK92MI, or their derivatives. The term "SAR-NK" refers to NK (natural killer) cells engineered to express SAR.

[0199] The term "Sleeping Beauty transposon" or "Sleeping Beauty transposon vector" refers to a vector derived from at least a portion of the Sleeping Beauty transposon genome.

[0200] The term "TCR constant chain" or "constant region of a T cell receptor" is defined as the constant chains of TCRα / TCRa, TCRβ1 / TCRb1, TCRβ2 / TCRb2, TCRγ / TCRd, TCRδ / TCRd, and pre-TCRα. Exemplary TCR constant chains are listed in Table 12. TCR constant chains can be divided into several subdomains, such as the Ig-like C1 domain (e.g., SEQ ID NOS: 1168-1175; Table 13), connecting peptide (e.g., SEQ ID NOS: 1177-1184; Table 13), transmembrane domain (SEQ ID NOS: 1187-1190; Table 15), and cytosolic domain (e.g., SEQ ID NOS: 1193-1196; Table 16). The cytosolic domains of the TCRα, TCRβ1 / β2, TCRγ, and TCRδ chains are short and generally not thought to play a significant role in their signaling activity. The present disclosure also provides exemplary deletion mutants and variants of TCR chains (Table 12). These deletion mutants and variants can be used in constructing SARs as long as they retain one or more functional or biological properties of the original TCR chain, such as the ability to pair with a complementary TCR chain, the ability to associate with the TCR / CD3 complex, or the ability to transduce a T cell signal (e.g., activate the NFAT pathway) upon engagement with a target antigen-expressing cell.

[0201] The term "single-chain variable region" or "scFv" refers to a fusion protein comprising at least one antibody fragment consisting of a light chain variable region and at least one antibody fragment consisting of a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short, flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv may have the vL and vH variable regions in either order; for example, with respect to the N- and C-termini of the polypeptide, an scFv may comprise vL-linker-vH or vL-linker-vH. In the present disclosure, an scFv is also referred to as vL-Gly-Ser-Linker-vH. Alternatively, an scFv may be referred to as (vL + vH) or (vH + vL).

[0202] As used herein, the term "specifically binds" or "specific" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody or antibody portion, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. In some embodiments, a SAR or antigen-binding domain that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (e.g., a cell surface antigen or a peptide / MHC protein complex) with a binding affinity that is at least about 10 times greater than the binding affinity for other targets.

[0203] The term "signaling domain" refers to a functional region of a protein that transmits information intracellularly through defined signaling pathways and regulates cellular activity, either by generating second messengers or by functioning as an effector in response to such messengers.

[0204] The term "signaling module" refers to a molecule or molecular complex containing one or more signaling mediators or signaling adaptors that can initiate a cellular signal. Cellular signals include, but are not limited to, activation of cellular signaling pathways such as NFAT, AKT, STAT, or NF-κB pathways. In exemplary embodiments, the signaling module recruits one or more proteins with cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) that are part of the signaling complex. Illustratively, signaling modules that function in NK cells include signaling adaptors such as CD3ζ, FcRγ, DAP10, and DAP12.

[0205] The term "signaling mediator" or "signaling adaptor" refers to a molecule that can initiate or inhibit a cell signal when recruited by a natural or non-natural signaling receptor. In contrast to a signaling receptor, a signaling adaptor does not have its own antigen-binding or ligand-binding domain. Examples of signaling adaptors include CD3ζ (CD3z), FcRγ, DAP10, DAP12, CD3ε, CD3γ, and CD3δ. In one embodiment, the present disclosure provides a SAR in which one or more heterologous antigen-binding domains are operably linked to the hinge domain or transmembrane domain of one or more chains of a signaling adaptor. In one embodiment, the SAR comprises a signaling adaptor that is a component of a TCR complex (e.g., CD3ε, CD3ε, CD3γ, CD3ε, etc.). In one embodiment, the SAR comprises a signaling adaptor that interacts with the TCR α, β, γ, and / or δ chains of the TCR complex. In one embodiment, the SAR comprises a signaling adaptor that does not interact with the TCR α, β, γ, and / or δ chains of the TCR complex. In one embodiment, the SAR comprises a signaling adaptor (e.g., CD3ζ) that has a conserved aspartic acid residue in its transmembrane domain that interacts with a positively charged residue in the TCR α / β transmembrane region. In one embodiment, the SAR comprises a signaling adaptor that lacks a conserved aspartic acid residue in its transmembrane domain. In one embodiment, the SAR comprises a signaling adaptor that is not a component of the TCR complex (e.g., DAP10). In one embodiment, the SAR comprises a signaling adaptor (e.g., CD3ζ) that activates cell signaling. In one embodiment, the SAR comprises a signaling adaptor that inhibits cell signaling. In one embodiment, the SAR comprises a signaling adaptor with one or more ITAM motifs. In one embodiment, the SAR comprises a signaling adaptor with two or more ITAM motifs. In one embodiment, the SAR comprises a signaling adaptor with a single ITAM motif. In one embodiment, the SAR comprises a signaling adaptor that lacks an ITAM motif.In one embodiment, the SAR comprises a signaling adaptor that is a native disulfide linker dimer. In one embodiment, the signaling adaptor is not a disulfide linker dimer in its native form. In one embodiment, the SAR comprises a signaling adaptor that, in its native state, comprises an interchain disulfide bond located in a transmembrane region. In one embodiment, the SAR comprises a native signaling adaptor that comprises an interchain disulfide bond that is not located in a transmembrane region. In one embodiment, the SAR comprises a signaling adaptor that, in its native state, comprises an interchain disulfide bond located in its extracellular region. In one embodiment, the extracellular domain of the signaling adaptor is less than 10 amino acids in length. In one embodiment, the extracellular domain of the signaling adaptor is less than 8 amino acids in length. In one embodiment, the extracellular domain of the signaling adaptor is more than 10 amino acids in length. In one embodiment, the extracellular domain of the signaling adaptor is more than 15 amino acids in length. In one embodiment, the SAR comprises a signaling adaptor that induces protein phosphorylation. In one embodiment, the SAR comprises a signaling adaptor that induces protein dephosphorylation. In one embodiment, the SAR comprises a signaling adaptor that interacts with Zap70. In one embodiment, the SAR comprises a signaling adaptor that does not interact with Zap70. In one embodiment, the two chains of the double-chain SAR comprise identical signaling adaptors (e.g., CD3ζ and CD3ζ). In one embodiment, the two chains of the double-chain SAR comprise non-identical signaling adaptors (e.g., CD3ε and FcRγ, or CD3ε and DAP10, etc.).

[0206] The term "signaling chain" or "signaling fragment" refers to a polypeptide comprising the transmembrane and / or intracellular regions of a cell signaling receptor, and optionally the extracellular hinge / connecting peptide region. Exemplary signaling chains include the constant chains of TCRα, TCRβ, TCRγ, and TCRδ. Further exemplary signaling chains include chains consisting of the transmembrane and / or intracellular regions of CD16, NKp30, NKp44, NKp46, DAP10, DAP12, DNAM-1, NKG2D, CD32, CD64, KIR3DL1, KIR2DS4, FcRγ, and CD3z. In some embodiments, the signaling chain also comprises the hinge domain or connecting peptide of CD16, NKp30, NKp44, NKp46, DAP10, DAP12, DNAM-1, NKG2D, CD32, CD64, KIR3DL1, KIR2DS4, FcRγ, and CD3z.

[0207] The term "synthetic antigen receptor" or "SAR" refers to a non-naturally occurring or synthetic receptor that can be expressed on the surface of a cell and contains at least one heterologous antigen-binding domain and at least one membrane-associated domain, where the membrane-associated domain can be a transmembrane domain or a membrane anchor domain (i.e., a GPI-linked domain). The antigen-binding domain of a SAR is heterologous to its membrane-associated domain, i.e., the antigen-binding domain is derived from a different source than the membrane-associated domain. A SAR may further contain a hinge domain, an extracellular ligand-binding domain, and / or a cytosolic domain. In one embodiment, a SAR comprises a polypeptide or set of polypeptides that, when expressed in an effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. A SAR can be a single chain, two chains, or three or more chains. A SAR can be monospecific, bispecific, or multispecific. A SAR can have one or more heterologous antigen-binding domains. The term "SAR" includes conventional chimeric antigen receptors (e.g., 第2These include first-generation CARs and next-generation CARs (e.g., SIR, cTCR, AbTCR, zSIR, HIT, TFP, TAC, etc.). This disclosure describes CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4 KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2D, NKG2C, NKG2A, NKG2E, NKG2F, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, Novel SAR compositions are described that contain one or more regions from CEACAM, ILT2, KLRG1, LAIR1, CD161, Siglec3, Siglec-7, Siglec-9, CD3ζ, DAP10, DAP12, FcRγ, TCRαβ, and TCRγδ, as well as variants and fragments thereof. The present disclosure also provides SARs that contain functional variants of the above genes and / or proteins, including alternatively spliced ​​isoforms and homologs from other species. Exemplary regions or fragments of the above genes and proteins that can be used to construct the SARs of the present disclosure are provided in Tables 12-18 and Tables 25-31 of the provisional application. Exemplary extracellular domains of native receptors are provided in SEQ ID NOs: 10842-10877. SARs of the present disclosure can also be constructed using polypeptides or fragments having 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to any of the fragments provided in Tables 12-18 and 25-31 of the provisional application. Nucleic acid and amino acid sequences of exemplary additional components (e.g., vL, vH, scFv, vHH, etc.) that can be used in constructing SARs are provided in Tables 2-11.SARs of the present disclosure can also be constructed using polypeptides or fragments having 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to any of the fragments provided in Tables 2-18 and 25-31 of the provisional application. Exemplary SARs of the present disclosure are provided in Tables 32-34 of the provisional application. SARs are modular in design, and additional SARs can be constructed by replacing one module of a SAR with another. The expression and activity of these novel SARs can be tested using the methods described in this disclosure to select SARs with optimal functional activity.

[0208] The term "single-chain synthetic antigen receptor" or "single-chain SAR" refers to a synthetic antigen receptor comprising a single polypeptide chain. An exemplary single-chain SAR targeting CD19 and based on the CD16 signaling chain is CD8SP-CD19-hu-mROO5-1-(vL-vH)-CD16A-F158V-S197P-FL-v3 (SEQ ID NO: 4954). In this SAR, a humanized scFv targeting CD19 (hu-mROO5-1) is functionally linked to the extracellular, transmembrane, and cytosolic domains of a high-affinity, non-cleavable variant of CD16 harboring the F158V and S197P mutations (CD16A-F158V-S197P-FL-v3). Additional exemplary single-chain SARs in which different antigen-binding domains are operably linked to the CD16A-F158V-S197P-FL-v3 module are provided in Tables 36-39 of the provisional application. Further exemplary single-chain SARs are provided in SEQ ID NOs: 5463-5482. Because the SAR is modular in design, the CD16A-F158V-S197P-FL-v3 module (SEQ ID NO: (DNA): 1417 and SEQ ID NO: (PRT): 3811) can be replaced with other modules, such as NKp30-ECDTMCP-. opt2 (SEQ ID NO: 1375), NKp44-ECDTMCP-opt2 (SEQ ID NO: 1389), NKp46-ECDTMCP-opt2 (SEQ ID NO: 1405), CD8-hinge-NKG2D-TM-2B4CP-opt-2 (SEQ ID NO: 1434), CD32-ECDTMCP-opt2 (SEQ ID NO: 1582), CD64-ECDTMCP-opt2 (SEQ ID NO: 1584), 2B4-ECDTMCP-opt2 (SEQ ID NO: 1580), OX40-ECDTMCP-opt2 (SEQ ID NO: 1578), CD28-ECDTMCP-opt2 (SEQ ID NO: 1576), 41BB-ECDTMCP-opt2 (SEQ ID NO: 1574), KIR3DL1 (SEQ ID NO: 9644) and KIR2DS4 (SEQ ID NO: 9653) to generate single-chain SARs with different signaling chains. Exemplary modules derived from naturally occurring receptors that can be used to construct SARs are provided in SEQ ID NOs: 9635-9668.Examples of such SARs are provided in SEQ ID NOS: 9860-9895 and in Table 41 of the provisional application. The expression and activity of these novel SARs can be tested using the methods described in this disclosure to select SARs with optimal functional activity.

[0209] The term "synthetic immunoreceptor" or alternatively "SIR" refers to a set of polypeptides, typically two in some embodiments, that, when expressed in an effector cell, provides the cell with specificity for a target cell, typically a cancer cell. This involves intracellular signal generation. SIRs represent the next-generation CAR platform described in WO 2018 / 102795 A1, incorporated herein by reference. In typical embodiments, the SIR comprises one or more antigen-binding domains (e.g., antibodies or antibody fragments, ligands, or receptors) that bind to an antigen described herein and are linked to one or more T cell receptor constant chains or regions, optionally via linkers. In some embodiments, the sets of polypeptides are adjacent to each other. In some embodiments, the SIR comprises two or more sets of two or more polypeptides. The polypeptides of each set of SIRs are adjacent to each other (functional polypeptide unit 1) but not adjacent to the polypeptides of the other sets (functional polypeptide unit 2). In some embodiments, the T cell receptor constant chain (or region) of the SIR is selected from the constant chain of human T cell receptor alpha (TCR alpha or TCR α or TCRa or hTCR alpha or hTCR α or hTCRa ​​or Cα), human T cell receptor-β1 (TCR-β1 or TCR β1 or TCRb1 or hTCR-β1 or hTCR β1 or hTCRb1 or Cβ1), human T cell receptor-β2 (TCR-β2 or TCR β2 or TCRb2 or hTCR-β2 or hTCR β2 or hTCRb2 or Cβ2, also known as TCR-beta, TCR β, TCRb, or Cβ), human pre-T cell receptor alpha ((pre-TCR alpha or pre-TCR α or pre-TCRa or pre-Cα), human T cell receptor gamma (TCR-gamma or TCR γ or TCRg or hTCR) -gamma or hTCRγ or hTCRg or hTCRγ1 or hTCRgamma1, or Cγ), or human T cell receptor-delta (TCR-delta or TCRd or TCRδ or hTCR-delta or hTCRd or hTCRδ or Cδ).In some embodiments, the TCR constant chains of the SIRs are encoded by their wild-type nucleotide sequences, while in other aspects, the TCR constant chains of the SIRs are encoded by non-wild-type nucleotide sequences. In some embodiments, the TCR constant chains of the SIRs encode wild-type polypeptide sequences, while in other embodiments, the TCR constant chains of the SIRs encode polypeptides with one or more mutations. In some embodiments, the TCR constant chains of the SIRs are encoded by their codon-optimized sequences with one or more mutations. The present disclosure also covers deletion mutants of TCR constant chains that retain at least one of the biological and functional properties of the corresponding full-length TCR chains. SIRs containing an antigen-binding domain (e.g., scFv or vHH) targeting a specific tumor maker "X," such as those described herein, are also referred to as X-SIRs or XS-SIRs. For example, an SIR containing an antigen-binding domain targeting CD19 is referred to as CD19-SIRs or CD19-SIRs. The TCR constant chains / domains of the SIRs may be derived from the same species in which the SIRs will ultimately be used. For example, when used in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or composed of a human TCR constant chain. However, in some cases, it may be beneficial for the TCR constant chain to be derived from the same species in which the SIR will ultimately be used, but modified with amino acid substitutions that enhance TCR constant chain expression. For example, when used in humans, it may be beneficial for the TCR constant chain of the SIR to be derived from or composed of a human TCR constant chain, but with certain amino acids substituted with corresponding amino acids from a mouse TCR constant chain. Such murine TCR constant chains result in increased expression of the SIR. The SIR or functional portion thereof can contain additional amino acids at the amino or carboxy terminus, or both, where these additional amino acids are not found in the amino acid sequence of the TCR or antigen-binding domain that constitutes the SIR. Desirably, the additional amino acids do not interfere with the biological function of the SIR or functional portion, such as target cell recognition, cancer detection, cancer treatment, or prevention.Additional amino acids may be desirable to enhance biological activity.

[0210] As used herein, the term SVH domain refers to a single human VH domain. H Domain antibodies (V H The term SVH refers to an sdAb (single-stranded antibody). Therefore, these terms are used interchangeably. The term SVH is used interchangeably with independent vH domain. SVH is an example of an autonomous antigen-binding domain (AABD). An exemplary SVH is a fully human vH domain (FHVH) set forth in SEQ ID NOs: (DNA):827-828 and (PRT):3221-3222. Another exemplary SVH is a chVH domain set forth in SEQ ID NOs: (DNA):830-831 and (PRT):8223-8224. Another exemplary SVH is an aVH domain set forth in SEQ ID NOs: (DNA):850-851 and (PRT):3244-3245. SEQ ID NOs for other exemplary SVH domains are listed in Table 5. Additional SVH domains that can be used in constructing the SARs of the present disclosure are provided in WO2016062988, WO2016113556, WO2017191476, WO2018039180, WO2019006072, WO2018237037, WO2018.119215, WO2019126756, WO2019055689 and WO2020018922, which are incorporated by reference in their entireties.

[0211] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand (or target antigen), thereby mediating a signaling event, such as, but not limited to, TCR / CD3-mediated signaling. Stimulation may mediate changes in the expression of specific molecules.

[0212] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides a cytoplasmic signal sequence for at least a portion of an immune cell signaling pathway that stimulatorily controls immune cell activation. In certain embodiments, the signal is a primary signal initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, that leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs that contain cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcRγ, CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0213] The term "subject" or "individual" or "animal" or "patient" is used interchangeably herein and refers to any subject, particularly a mammalian subject, to whom administration of a composition or pharmaceutical composition of the present disclosure is desired. Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bovines, etc., with humans being preferred.

[0214] As used herein, a "switch domain" or "dimerization domain" typically refers to a polypeptide-based entity that associates with another switch domain in the presence of a dimerization molecule.

[0215] A "suicide gene," "suicide switch," or "kill switch" encodes a protein capable of inducing cell death. Examples of suicide genes include HSV-TK, iCaspase 9, tEGFR, CD20, tCD19, tHer2, tBCMA, and RQR8. For example, CD20-expressing cells can be selectively eliminated by treatment with the antibody rituximab. Similarly, tBCMA-expressing cells can be selectively eliminated by treatment with belantamab mafodotin, and tHer2-expressing cells can be selectively eliminated by treatment with Herceptin.

[0216] A "survival gene," "survival switch," or "life switch" encodes a protein that provides a pro-survival signal to a cell. Exemplary survival genes include membrane-anchored IL2 and membrane-anchored IL15.

[0217] As used herein, the term "T lymphocyte" or "T cell" refers to a cell that expresses CD3 (CD3+) and a T cell receptor (TCR+). In one embodiment, the T cell is a natural cell (i.e., not a recombinant or engineered cell) that expresses CD3 and a TCR.

[0218] As used herein, the term "TCR" or "T cell receptor" refers to a dimeric heterogeneous cell surface signaling protein that typically forms an alpha-beta or gamma-delta receptor, which is involved in the recognition of antigens presented by MHC molecules (i.e., antigen recognition in the context of MHC molecules). The TCRs of the present disclosure may be non-naturally occurring, purified, and / or engineered. The TCRs of the present disclosure may have multiple mutations present in the α chain variable domain and / or the β chain variable domain compared to a parent TCR. "Modified TCR" and "mutant TCR" are used interchangeably herein and generally refer to a TCR that has one or more mutations introduced, particularly in its Va and / or Vb or Vg and / or Vd domains, compared to a parent TCR. Engineered TCRs may bind antigens in an HLA-dependent or HLA-independent manner.

[0219] As used herein, the term "transgene" refers to a heterologous gene that is integrated into the genome of an organism (e.g., a non-human animal) and transmitted to the organism's offspring during sexual reproduction.

[0220] As used herein, the term "T lymphocyte" or "T cell" refers to a cell that expresses CD3 (CD3+) and a T cell receptor (TCR+). In one embodiment, a T cell is a natural cell that naturally expresses CD3 and a TCR (i.e., a cell that has not been engineered to express CD3 or a TCR). The terms "T cell" and "T lymphocyte" are interchangeable and used synonymously herein. Examples include naive T cells ("lymphocyte precursors"), central memory T cells, effector memory T cells, stem memory T cells (T scm These include, but are not limited to, iPSC-derived T cells, synthetic T cells, tumor-infiltrating T cells (TILs), αβ T cells, γδ T cells, regulatory T cells (Tregs), or combinations thereof.

[0221] The term "non-T cell" refers to a cell that is not a T cell. In one embodiment, the non-T cell lacks cell surface expression of CD3 and a T cell receptor. In one embodiment, the non-T cell does not respond to a T cell-activating antibody, such as OKT3. In one embodiment, the non-T cell lacks surface expression of CD3. In one embodiment, the non-T cell lacks expression of one or more CD3 chains selected from the group of CD3ε, CD3γ, and CD3δ. In one embodiment, the non-T cell exhibits a germline configuration of TCR genes and has not undergone T cell gene rearrangement. In one embodiment, the non-T cell lacks the ability to form a functional T cell / CD3 receptor complex. Examples of non-T cells include NK cells, B cells, macrophages, granulocytes, dendritic cells, and epithelial cells. The non-T cell may be an immortalized cell line. In an exemplary embodiment, the non-T cell is an NK cell line, such as NK92, NK92MI, NKG, and YTS. In one embodiment, the non-T cells are iPSC-derived cells that lack expression of CD3 and T cell receptors.

[0222] The term "T cell receptor module" or "TCRM" refers to a heterodimer containing sequences derived from a T cell receptor. A TCRM includes a T cell receptor transmembrane domain and may further include all or part of the T cell receptor connecting peptide and / or the intracellular domain.

[0223] As used herein, the term "TCR-Fv" or "Fv-TCR" of "fragment variable TCR" refers to an antigen-binding module formed by the variable domains of a TCR chain. A TCR-Fv can be formed by Vα and Vβ domains, or Vγ and Vδ domains. A TCR-Fv exhibits some or all of the specific binding affinity for the target antigen (e.g., peptide / MHC complex) of the TCR from which the variable domains are derived. In one embodiment, the SAR of the present disclosure demonstrates the ability to form a TCR-Fv antigen-binding module when Vα / Vβ chains or Vγ / Vδ chains from a TCR bind to the two polypeptides.

[0224] As used herein, the term "Fv" or "Fragment variable" refers to an antigen-binding module formed by the variable domains of an antibody. The Fv is formed by the vL domain and the vH domain. The Fv exhibits some or all of the specific binding affinity for the target antigen of the antibody from which the variable domains are derived. In one embodiment, the SAR of the present disclosure demonstrates the ability of a vL chain and a vH chain from an antibody to form an Fv antigen-binding module when the vL chain and vH chain from the antibody bind to the two polypeptides.

[0225] As used herein, a "transmembrane protein" or "membrane protein" is a protein located on and / or within a membrane, such as the phospholipid bilayer of a biological membrane (e.g., a biological membrane such as a cell membrane). Some proteins are bound only to the membrane surface, while others have one or more regions embedded within the membrane and / or domains on one or both sides of the membrane. Specific examples of transmembrane proteins include CD8a, CD4, CD3ζ, CD16, NKp30, NKp44, NKG2D, etc.

[0226] As used herein, "transmembrane module" or "TMM" refers to a molecule or molecular complex that comprises a transmembrane protein (e.g., CD16A).

[0227] MAM refers to a molecule or molecular complex that contains a membrane-anchored protein (e.g., CD16A) or a membrane-anchored protein (e.g., CD16B). This term includes transmembrane proteins, such as CD16A, and GPI (glycosylphosphatidylinositol)-linked proteins, such as CD16B. MAMs may further include all or part of a hinge domain and / or a cytosolic domain.

[0228] As used herein, the term "therapeutic agent" refers to, for example, an agent used to treat, inhibit, prevent, reduce the effects, reduce the severity, reduce the likelihood of onset, slow progression, and / or cure a disease. Diseases targeted by therapeutic agents include, but are not limited to, infectious diseases, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematological and / or inflammatory diseases.

[0229] As used herein, the term "therapeutic control" refers to an element used to control the activity of SAR-expressing cells. In some embodiments, the therapeutic control for controlling the activity of SAR-expressing cells of the present disclosure comprises one or more of a truncated epidermal growth factor receptor (tEGFR), a truncated epidermal growth factor receptor viii (tEGFRviii), a truncated CD30 (tCD30), a truncated epidermal growth factor receptor viii (tEGFRviii), a truncated CD30 (tCD30), and a truncated epidermal growth factor receptor (tEGFRviii). BCMA (tBCMA), truncated CD19 (tCD19), thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyl transferase, human caspase 8, human caspase 9, inducible caspase 9, purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), gamma-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), selectable markers, and combinations thereof. Exemplary therapeutic controls are listed in Table 24 of the provisional application.

[0230] The term "therapeutic effect" refers to a biological effect that can be manifested by various means, including, but not limited to, reduction in tumor volume, reduction in cancer cell number, reduction in metastasis number, extension of life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival, reduction in infectious agent titer, reduction in infectious agent colony count, amelioration of various physiological symptoms associated with a disease state, etc. A "therapeutic effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the onset of disease in the first place or to prevent the recurrence of disease.

[0231] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical composition comprising one or more peptides disclosed herein or mutants, variants, analogs or derivatives thereof to reduce at least one or more symptoms of a disease or disorder, and relates to a quantity of a pharmaceutical composition sufficient to provide the desired effect. As used herein, the phrase "therapeutically effective amount" means that amount of the composition sufficient to treat a disorder, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0232] The term "TCR receptor fusion protein" or "TFP" refers to the next-generation SAR platform described in WO2016 / 187349A1, which is incorporated herein by reference. In one embodiment, the TFP comprises an antibody moiety that specifically binds to a target antigen fused to a TCR chain, such as CD3ε, CD3γ, CD3δ, TCRα, or TCRβ. Exemplary TCR chains that can be used to construct TFPs are represented by SEQ ID NOs: 11903-11906 of the present disclosure and are provided in WO2017 / 070608A1, which is incorporated herein by reference. A TFP incorporating a CD3ε chain is referred to as CD3ε TFP or TFPε. A TFP incorporating a CD3γ chain is referred to as CD3γ TFP or TFPγ. A TFP incorporating a CD3δ chain is referred to as CD3δ TFP or TFPδ. TFPs incorporating CD3ε, CD3γ, or CD3δ chains are collectively referred to as CD3ε / γ / δ TFPs or TFPε / γ / δ.

[0233] The term "transfer vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adenoviral vectors, retroviral vectors, lentiviral vectors, etc.

[0234] For example, the term "SAR" refers to a region of a membrane-spanning protein (e.g., a type I or II transmembrane protein). The transmembrane domain of a SAR of the present disclosure is a transmembrane region of a membrane-spanning protein (e.g., a type I or II transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains will be apparent to those skilled in the art and can be used in connection with alternative embodiments of the present disclosure. In some embodiments, the TMD-encoded SAR is selected from the group consisting of a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD3 gamma, CD3 epsilon, CD3 delta, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f , ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244), 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO- 3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. Exemplary transmembrane domains are provided in Table 28 of the provisional application. The transmembrane domain of the SAR of the present disclosure may be native to the receptor or non-native.Because SAR has a modular design, in some embodiments, the transmembrane domain of one SAR can be replaced by the transmembrane domain of another SAR, as long as it retains its biological and functional properties.Therefore, the NKp30 transmembrane domain in NKp30-based SAR can be replaced by the transmembrane domain of NKp44.The obtained SAR with non-natural transmembrane domain can be tested for its cell surface expression and functional activity using assays known in the art and described in the present disclosure.

[0235] As used herein, "trifunctional T cell antigen coupler" or "Tri-TAC" or "TAC" refers to the next-generation SAR platform described in WO 2015 / 117229 A1, which is incorporated herein by reference. Tri-TACs targeting different antigens can be constructed using techniques known in the art and antigen-binding domains described in this disclosure (e.g., vL and vH fragments, scFv, vHH, ligands and receptors, etc.).

[0236] As used herein, the terms "treat," "treat," or "ameliorate" refer to therapeutic treatment, the purpose of which is to reverse, alleviate, improve, inhibit, slow, or halt the progression or severity of the condition associated with a disease or disorder.

[0237] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0238] A vector is a vehicle by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and facilitate expression (e.g., transcription and translation of the introduced sequence). Vectors include plasmids, phages, and viruses.

[0239] The term "viral vector" refers to a vector obtained from or derived from a virus. Typically, the virus is a retrovirus, including, but not limited to, lentivirus and gammaretrovirus. The viral vector of the present disclosure may be a retroviral vector, such as a gammaretroviral vector. The viral vector may be based on a human immunodeficiency virus. The viral vector of the present disclosure may be a lentiviral vector. The vector may be based on a non-primate lentivirus, such as equine infectious anemia virus (EIAV). The viral vector of the present disclosure comprises a mitogenic T cell activating transmembrane protein and / or a cytokine-based T cell activating transmembrane protein within the viral envelope. The mitogenic T cell activating transmembrane protein and / or cytokine-based T cell activating transmembrane protein is derived from the host cell membrane, as described above.

[0240] The terms "zeta", alternatively "zeta chain", "CD3-zeta", "TCR-zeta", "CD3ζ", and "TCR-zeta" are defined as the protein provided as GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "zeta stimulatory domain", alternatively "CD3-zeta stimulatory domain" or "TCR zeta stimulatory domain", are defined as amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, sufficient to functionally transduce the initial signal required for T cell activation.

[0241] "HLA-deficient," including HLA class I-deficient, or HLA class II-deficient, or both, refers to cells that lack, no longer maintain, or have reduced levels of surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, such that the reduced or decreased levels are lower than those naturally detectable by other cells or synthetic methods.

[0242] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that have been further modified by introducing genes expressing proteins associated with, but not limited to, improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL1 1b, NOTCH, RUNX1, IL15, 41BB, DAP 10, DAP 12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1. "Modified HLA-deficient" cells also include cells other than iPS cells.

[0243] CD16, an FcγR receptor, has been identified as having two isoforms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). Unless otherwise specified, CD16 refers to both the CD16a and CD16b isoforms, as well as other alternatively spliced ​​variants derived from human or non-human species. CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and promoting antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high-affinity CD16," "non-cleavable CD16," or "high-affinity non-cleavable CD16 (hnCD16)" refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and is prone to exodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes during NK cell activation. F176V (or F158V or V158) is an exemplary CD16 polymorphic variant with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) has been altered or removed are not susceptible to shedding. The cleavage site and membrane proximal region are described in detail in International Publication No. 2015148926, the entire disclosure of which is incorporated herein by reference. CD16 S197P or S197R variants are engineered non-cleavable versions of CD16. CD16 variants containing both F158V and S197P (or S197R) have high affinity and are not cleaved. Another exemplary high-affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 containing an extracellular domain derived from one or more of the three exons of the CD64 extracellular domain. The CD16 SAR of the present disclosure may comprise a wild-type CD16 sequence, or a naturally occurring or non-naturally occurring variant thereof, such as F158V and S197P (or S197R).

[0244] Although immune cells and iPSCs expressing CD16 or CD16 variants are known in the art, in one embodiment, the present disclosure provides a SAR comprising an extracellular Fc-binding region of CD16 or a CD16 variant. In one embodiment, the CD16-SAR retains the ability to bind to the Fc region of an antibody or antibody fragment, but its non-natural antigen-binding domain (e.g., AABD, scFv, vHH, FHVH, Fv, etc.). In one embodiment, the antigen-binding domain of the CD16 SAR is located at or near the N-terminal region of the D1 domain (SEQ ID NO: 3836) of CD16 or a CD16 variant, i.e., near the N-terminal region of the extracellular domain of CD16 or a CD16 variant. In one embodiment, an optional linker is present between the antigen-binding domain of the SAR and the D1 domain of CD16 or a CD16 variant. Exemplary linkers are provided in Table 11 of the provisional application.

[0245] In one embodiment, the CD16-SAR lacks the ability to bind to the Fc region of an antibody or antibody fragment, but has the ability to bind to an antigen via its non-natural antigen-binding domain (e.g., AABD, scFv, vHH, FHVH, Fv, etc.). In an exemplary embodiment, the CD16-SAR comprises one or more non-natural antigen-binding domains (e.g., AABD, scFv, vHH, FHVH, Fv, etc.) operably linked to the transmembrane domain and optionally the cytoplasmic domain of CD16 via an optional hinge domain. In one embodiment, the CD16-SAR has the ability to recruit signaling adaptors such as CD3z and / or FceRy1 upon binding to its target antigen. The binding domain of the SAR binds to an epitope or antigen of interest. For example, the epitope recognized by the SAR is determined from the epitope recognized by the scFv used as the binding domain of the SAR. For example, the antigen-specific domain of the CD19-targeting SAR CD8SP-CD19-hu-mROO5-1-vL-Xho-IgCL-DAP10-opt1- FP 2A-Spe-IgSP-Bst-CD19-hu-mROO5-1-vH-Mlu-Ig1CH1-DAP10-opt2- FF 2A-Xba-PAC (SEQ ID NO: 2275) is composed of fragments of the vL (SEQ ID NO: 2543) and vH (SEQ ID NO: 2785) derived from the hu-mROO5-1 scFv (SEQ ID NO: 3027). Therefore, the SAR is expected to target the same epitope as the scFv and / or the parent antibody from which the scFv is derived. The epitopes recognized by several scFvs and / or their parent antibodies used in constructing the SARs and backbones of the present disclosure are known in the art. Alternatively, the epitope targeted by a SAR can be determined by generating a series of mutants of the target antigen and testing the ability of the mutants to bind to SAR-expressing cells using techniques known in the art, such as the Topanga Assay (Gopalakrishnan, R, Sci Reports, 2019).As an example, the epitope recognized by the CD19-targeting SAR CD8SP-CD19-hu-mROO5-1-vL-Xho-IgCL-DAP10-opt1- FP 2A-Spe-IgSP-Bst-CD19-hu-mROO5-1-vH-Mlu-Ig1CH1-DAP10-opt2- FF 2A-Xba-PAC (SEQ ID NO: 2275) can be determined by generating a panel of deletion and point mutants of CD19-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac (DNA SEQ ID NO: 1282). The mutant constructs are transfected into an appropriate cell line (e.g., 293FT cells), and the supernatant containing the fusion protein is collected and assayed for NLuc activity to confirm that the different mutant CD19-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis fusion proteins are secreted into the supernatant. The fusion proteins are then tested for their ability to bind to cells (e.g., Jurkat cells or T cells) expressing the CD8SP-CD19-hu-mROO5-1-vL-Xho-IgCL-DAP10-opt1-F-P2A-Spe-IgSP-Bst-CD19-hu-mROO5-1-vH-Mlu-Ig1CH1-DAP10-opt2-F-F2A-Xba-PAC (SEQ ID NO: 2275) construct. Mutants that do not bind to SAR-expressing cells are candidates for containing the epitope targeted by the CD19-specific SAR. Another approach to determining the epitope recognized by a particular SAR is to conduct a functional competition assay using different test antibodies. For example, T cells expressing the CD8SP-CD19-hu-mROO5-1-vL-Xho-IgCL-DAP10-opt1-F-P2A-Spe-IgSP-Bst-CD19-hu-mROO5-1-vH-Mlu-Ig1CH1-DAP10-opt2-F-F2A-Xba-PAC (SEQ ID NO: 2275) SAR can be co-cultured with a CD19-expressing cell line (e.g., RAJI cells) in the absence and presence of increasing concentrations of different test CD19 antibodies.If the epitope recognized by the test CD19 antibody overlaps with the epitope recognized by SAR CD8SP-CD19-hu-mROO5-1-vL-Xho-IgCL-DAP10-opt1-F-P2A-Spe-IgSP-Bst-CD19-hu-mROO5-1-vH-Mlu-Ig1CH1-DAP10-opt2-F-F2A-Xba-PAC (SEQ ID NO: 2275), the test antibody is identified as CD8SP-CD19-hu-mROO5-1-vL-Xho-IgCL-DAP10-opt1-F-P2A-Spe-IgSP-Bst-CD19-hu-mROO5-1-vH-Mlu-Ig1CH1-DAP10-opt2-F-F2A-Xba-PAC (SEQ ID NO: 2276). 2275) is expected to dose-dependently block target cell killing and cytokine production induced by SAR-expressing T cells. A nonspecific antibody of the same isotype as the test antibody is included as a control and is expected to have no effect on target cell killing and cytokine production induced by SAR-expressing T cells. Similarly, a specific SAR can be expressed in Jurkat-NFAT-EGFP cells, and the ability of the test antibody to inhibit EGFP induction by the SAR-expressing Jurkat-NFAT-GFP cells upon co-culture with a target cell line can be used to determine whether the epitope recognized by the test antibody overlaps with the epitope recognized by the SAR.

[0246] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6]

[0247] Table 24

Table 25-1

Table 25-2

Table 28-1

Table 28-2

Table 28-3

Table 28-4

Table 29-1

Table 29-2

Table 29-3

Table 29-4

Table 30-1

Table 30-2

[0248] [Table 33-1] [Table 33-2] [Table 33-3] [Table 33-4]

[0249] In one embodiment, the present disclosure provides a SAR (such as an isolated SAR) comprising one or more heterologous antigen-binding domains operably linked to a naturally occurring (i.e., native or endogenous) receptor or a variant thereof via an optional linker. In one embodiment, the SAR retains the binding ability and function of the native receptor, but also acquires the binding ability and function conferred by one or more heterologous antigen-binding domains. In one embodiment, the SAR partially or completely retains the binding ability and function of the native receptor. In one embodiment, the SAR acquires the binding ability and function conferred by one or more heterologous antigen-binding domains.

[0250] In one embodiment, the naturally occurring receptor is any receptor expressed on the surface of a cell (e.g., an immune cell, e.g., an immune effector cell). In an exemplary embodiment, the immune cell is selected from, but is not limited to, a T cell, an NK cell, a monocyte / macrophage, a granulocyte, and a B cell. In one embodiment, the naturally occurring signaling receptor is expressed on the surface of an immune cell (e.g., a T cell, an NK cell, an NKT cell, a macrophage, a dendritic cell, etc.).

[0251] In one embodiment, the naturally occurring receptor induces cell signaling, i.e., it is a naturally occurring signaling receptor. The naturally occurring signaling receptor can be an activating receptor (i.e., induces cell activation) or an inhibitory receptor (i.e., blocks cell activation). In one embodiment, the naturally occurring receptor is an NK cell receptor, for example, an NK activating receptor or an NK inhibitory receptor. In one embodiment, the naturally occurring signaling receptor can be a receptor that induces cytotoxicity. In another embodiment, the naturally occurring signaling receptor can be a receptor that provides costimulation.

[0252] In one embodiment, a naturally occurring receptor that can be used to construct a SAR of the present disclosure has a transmembrane domain. In one embodiment, a naturally occurring receptor can recruit a transmembrane adaptor protein. In one embodiment, a naturally occurring receptor can recruit a transmembrane adaptor protein selected from the group consisting of, but not limited to, CD3ε, FcRγ, DAP10, DAP12, or variants or fragments thereof. Examples of such receptors include CD16A, NKp30, NKp44, NKp46, and NKG2D. In one embodiment, a naturally occurring receptor can recruit a transmembrane adaptor protein that contains a negatively charged residue (e.g., aspartic acid) in its transmembrane region. In one embodiment, a naturally occurring receptor has a transmembrane domain that contains a positively charged residue (e.g., lysine or arginine) that interacts with a negatively charged residue (e.g., aspartic acid) in the transmembrane region of a signaling adaptor protein. In one embodiment, a naturally occurring receptor has a transmembrane domain and a cytoplasmic domain. In one embodiment, a naturally occurring receptor has a hinge (spacer) domain and a transmembrane domain. In one embodiment, naturally occurring receptor has hinge (spatial) domain, transmembrane domain and cytosol domain.An example of such receptor is CD16A.In one embodiment, naturally occurring receptor has hinge (spatial) domain, membrane anchor domain (e.g., GPI-linked domain), but lacks cytosol domain.An example of such receptor is CD16B.

[0253] In exemplary embodiments, naturally occurring receptors that can be used to construct SARs of the present disclosure include, but are not limited to, CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1. , KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2D, NKG2C, NKG2A, NKG2E, NKG2F, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, CEACAM, ILT2, KLRG1, LAIR1, CD161, Siglec3, Siglec-7, Siglec-9, TCRαβ and TCRγδ, etc., and their variants and fragments.

[0254] In one embodiment, the naturally occurring receptor that can be used to construct the SAR of the present disclosure is not a T cell receptor. In one embodiment, the naturally occurring receptor that can be used to construct the SAR of the present disclosure is not TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the SAR does not comprise the constant chain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the SAR does not comprise the transmembrane domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the SAR does not comprise the entire extracellular domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the naturally occurring non-T cell receptor that can be used to construct the SAR of the present disclosure comprises an intracellular activation domain. In one embodiment, the activation domain comprises one or more ITAMs. In one embodiment, the activation domain comprises one or more ITIMs. In one embodiment, the naturally occurring non-T cell receptor that can be used to construct the SAR of the present disclosure comprises a costimulatory domain.

[0255] In exemplary embodiments, the SAR comprises an intracellular activation domain derived from CD3ε, FcRγ, DAP10, or DAP12.

[0256] In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the entire extracellular domain of a non-TCR native receptor, i.e., a receptor that is not TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to a partial extracellular domain of a non-TCR native receptor. In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the entire or partial extracellular domain of an NTCRM (non-T cell receptor module). In one embodiment, the native signaling receptor that can be used to construct the SAR of the present disclosure is not CD4, CD8, CD28, CD27, CD16A, or NKG2D.

[0257] In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to the entire or partial extracellular domain of a naturally occurring receptor, wherein the receptor is not part of a TCR / CD3 receptor complex. In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to the entire or partial extracellular domain of a naturally occurring receptor polypeptide chain, wherein the receptor polypeptide chain is not part of a TCR / CD3 receptor complex. In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to the entire or partial extracellular domain of a naturally occurring receptor, or a variant or fragment thereof, wherein the receptor is not associated with a TCR / CD3 receptor complex.

[0258] In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the entire or partial antigen (or ligand)-binding domain of a naturally occurring receptor or a variant or fragment thereof.

[0259] In certain embodiments, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the whole or partial antigen (or ligand)-binding domain of a naturally occurring non-TCR signaling receptor or NTCRM, or a variant or fragment thereof. In certain embodiments, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the whole or partial antigen (or ligand)-binding domain of a non-TCR signaling receptor or NTCRM, or a variant or fragment thereof. In certain embodiments, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the whole or partial antigen (or ligand)-binding domain of a naturally occurring signaling receptor or a variant or fragment thereof, wherein the naturally occurring signaling receptor is not TCRα, TCRβ, TCRγ, TCRδ, or preTCRα. In certain embodiments, the SAR of the present disclosure retains at least a portion of the antigen-binding properties of the naturally occurring non-TCR signaling receptor. In certain embodiments, the SAR of the present disclosure acquires novel antigen-binding properties conferred by one or more of the heterologous antigen-binding domains. In certain embodiments, the SARs of the present disclosure retain at least some of the antigen-binding properties of a naturally occurring non-TCR signaling receptor and acquire novel antigen-binding properties conferred by one or more of the heterologous antigen-binding domains.

[0260] In one embodiment, a double-chain SAR is provided that comprises a heterologous antigen-binding domain derived from a variable domain of a TCR (i.e., Vα, Vβ, Vδ, or Vγ) via an optional linker. Naturally occurring signaling receptor. In one embodiment, the naturally occurring signaling receptor is an NTCRM (non-T cell receptor module).

[0261] provides a double-chain SAR comprising a heterologous antigen-binding domain derived from the variable domain of a TCR (i.e., Vα, Vβ, Vδ, or Vγ) operably linked via an optional linker to the hinge domain of an NTCRM (T cell receptor module) and / or a signaling adaptor (e.g., CD3ε, FcRγ, DAP10, DAP10, etc.) or a variant or fragment thereof.

[0262] In one embodiment, the SAR does not comprise the entire extracellular domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the SAR does not comprise the transmembrane domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα.

[0263] Schematic diagrams of the SAR in this disclosure are shown in Figures 1-5 and Tables A1-1 to A1-19.

[0264] In one embodiment, a naturally occurring receptor that can be used to construct a SAR of the present disclosure can comprise a single polypeptide chain or multiple polypeptide chains. A naturally occurring receptor can be a component of a multi-chain receptor complex (e.g., a T cell receptor complex).

[0265] In one embodiment, the SAR comprises two or more antigen-binding domains. In one embodiment, the SAR comprises one or more heterologous (or non-natural) antigen-binding domains. Exemplary heterologous antigen-binding domains that can be used in constructing the SARs of the present disclosure include autonomous antigen-binding domains (e.g., fully human vH domains, vHH domains, single-chain TCRs, recombinant TCRs, or svd-TCRs, etc.), scFvs, antibodies, antibody fragments (vL, vH, Fab, etc.), non-immunoglobulin antigen-binding domains (e.g., centilin, affibody, DARPIN, ZIP domain, adapter, etc.), ligands, extracellular domains of receptors (e.g., CD16A extracellular domain, NKp30 extracellular domain, etc.), autoantigens, TCRs, TCR variable fragments (e.g., Va, Vb, Vg, Vd, etc.), and variants and fragments thereof. In an exemplary embodiment, one or more heterologous antigen-binding domains comprise scTCRs, svd-TCRs, or TCR-mimetic scFvs, or fragments thereof. In some embodiments, the SAR acquires TCR-like binding ability, eg, the ability to bind to a peptide / MHC complex.

[0266] Second-generation chimeric antigen receptor constructs currently in clinical use (e.g., axicabtagene ciloleucel, lysocabtagene malareucel, etc.) contain heterologous antigen-binding domains (e.g., scFvs) operatively linked to stalk (hinge), transmembrane, costimulatory, and cytoplasmic domains, which in turn are derived from multiple different natural receptors. In another aspect, SARs (e.g., next-generation CARs) are provided that consist of the native transmembrane and cytoplasmic domains of naturally occurring signaling receptors (e.g., CD16, NKp30, NKp44, NKp46, NKG2D, KIR2DS4, etc.) or signaling adaptors (e.g., CD3ζ, FcRγ, DAP10, DAP12, etc.). Such SARs exhibit superior physiological cell signaling and regulation compared to SARs that contain non-native transmembrane and cytoplasmic domains of naturally occurring signaling receptors or signaling adaptors. In another aspect, the present disclosure provides that SARs comprising the native configuration of transmembrane and cytoplasmic domains of naturally occurring signaling receptors or signaling adaptors exhibit superior efficacy in vitro and in vivo compared to SARs comprising non-native configurations.

[0267] In one embodiment, a SAR comprises one or more heterologous antigen-binding domains operably linked via an optional linker to the entire extracellular domain, transmembrane domain, and cytoplasmic domain of a naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof. In one embodiment, a SAR comprises one or more heterologous antigen-binding domains operably linked via an optional linker to the entire extracellular antigen-binding domain, transmembrane domain, and cytoplasmic domain of a naturally occurring signaling receptor or a variant or fragment thereof. In one embodiment, a SAR comprises one or more heterologous antigen-binding domains operably linked via an optional linker to a portion or the entire extracellular antigen-binding domain of one naturally occurring receptor and the transmembrane domain and cytoplasmic domain of another naturally occurring signaling receptor or a variant or fragment thereof. In one embodiment, a SAR comprises one or more heterologous antigen-binding domains operably linked via an optional linker to the entire transmembrane domain and cytoplasmic domain, rather than a partial extracellular domain, of a naturally occurring signaling receptor, signaling adaptor, or variant or fragment thereof. In one embodiment, a SAR comprises one or more heterologous antigen-binding domains operably linked, via an optional linker and / or spacer (e.g., hinge domain), to the transmembrane domain and optionally the cytoplasmic domain of a naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof. In one embodiment, a SAR consists of one or more heterologous antigen-binding domains operably linked, via an optional linker, to the hinge domain, transmembrane domain, and optionally the cytoplasmic domain of a naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof.

[0268] In one embodiment, the different domains of the SAR are operably linked via peptide bonds, ie, they are part of a polypeptide chain.

[0269] In one embodiment, the SAR comprises an intracellular activation domain. In an exemplary embodiment, the SAR comprises an intracellular activation domain derived from a signaling adaptor. In an exemplary embodiment, the SAR comprises an intracellular activation domain derived from CD3ε, FcRγ, DAP10, or DAP12. In one embodiment, the activation domain of the SAR comprises one or more ITAM motifs. In one embodiment, the SAR comprises an activation domain with one or more ITAM motifs. In one embodiment, the SAR comprises an activation domain with two or more ITAM motifs. In one embodiment, the SAR comprises an activation domain with a single ITAM motif. In one embodiment, the SAR comprises an activation domain lacking an ITAM motif. In one embodiment, the SAR comprises an activation domain comprising a tyrosine-based motif (YINM). In one embodiment, the SAR comprises an activation domain that recruits the p85 subunit of PI3K and / or Grb2. In one embodiment, the SAR comprises an activation domain that activates one or more of the NFAT, PI3K, NF-κB, and ERK signaling pathways.

[0270] In one embodiment, the SAR comprises an intracellular inhibitory domain. In an exemplary embodiment, the SAR comprises an intracellular inhibitory domain derived from PD1. In one embodiment, the inhibitory domain of the SAR comprises one or more ITIM motifs.

[0271] In one embodiment, the SAR can recruit a signaling adaptor. In an exemplary embodiment, the SAR can recruit one or more signaling adaptors selected from the group consisting of, but not limited to, CD3ε, FcRγ, DAP10, and DAP12. In an exemplary embodiment, the SAR can recruit one or more signaling adaptors selected from the group consisting of, but not limited to, CD3ζ, FcRγ, DAP10, and DAP12. In one embodiment, the SAR can recruit a signaling adaptor through interaction with its hinge, transmembrane, or cytosolic domain. In one embodiment, the SAR can recruit a signaling adaptor through interaction with one or more of the hinge, transmembrane, and cytosolic domains. In one embodiment, immune cells (e.g., T cells, NK cells, macrophages, granulocytes, dendritic cells, etc.) expressing a SAR of the present disclosure recruit a signaling adaptor when its one or more heterologous antigen-binding domains bind to a target antigen. In one embodiment, immune cells (e.g., T cells, NK cells, macrophages, granulocytes, dendritic cells, etc.) expressing a SAR of the present disclosure activate target cells, proliferate, secrete cytokines, and / or modulate (induce or suppress) target cell killing, and have MHC-restricted and / or MHC-unrestricted antibody-type specificity.

[0272] In some embodiments, the SAR lacks a cytosolic domain. In some embodiments, the SAR comprises a cytosolic domain less than 100 amino acids in length (i.e., less than 90, 80, 70, 60, 50, 50, 40, 30, 25, 20, 15, 10, 5, or 2 amino acids in length). In some embodiments, the SAR comprises a cytosolic domain less than 50 amino acids in length. In some embodiments, the SAR comprises a cytosolic domain less than 25 amino acids in length. In some embodiments, the SAR comprises a cytosolic domain less than 10 amino acids in length. In some embodiments, the SAR comprises a cytosolic domain less than 5 amino acids in length. In some embodiments, the SAR lacks an intracellular activation domain. In some embodiments, the SAR lacks an intracellular domain containing an ITAM motif. In some embodiments, the SAR lacks an intracellular signaling domain. In some embodiments, the SAR lacks an intracellular costimulatory domain. In some embodiments, the SAR lacks an intracellular domain derived from one or more of CD3z, 2B4, 4-1BB, CD28, ICOS, CD2, CD40, DAP10, and DAP12. In some embodiments, the SAR lacks an intracellular signaling domain capable of directly recruiting a protein kinase or protein phosphatase.

[0273] In some embodiments, the SAR lacks a costimulatory domain inserted between the transmembrane and cytoplasmic domains of a naturally occurring signal transduction receptor or signal transduction adaptor, or a variant or fragment thereof. In contrast to second-generation CARs, in one embodiment, the transmembrane and cytoplasmic domains of the SAR of the present disclosure are derived from a single naturally occurring signal transduction receptor or signal transduction adaptor, or a variant or fragment thereof. In another embodiment, the transmembrane and cytoplasmic domains of the SAR of the present disclosure are derived from a single naturally occurring signal transduction receptor or signal transduction adaptor, or a variant or fragment thereof, and are not interrupted by a heterologous costimulatory domain derived from a costimulatory receptor. In another embodiment, the transmembrane and cytoplasmic domains of the SAR of the present disclosure are derived from a single naturally occurring signal transduction receptor or signal transduction adaptor, and are adjacent to each other. In another embodiment, the hinge, transmembrane, and cytoplasmic domains of the SAR of the present disclosure are derived from a single naturally occurring signal transduction receptor or signal transduction adaptor, or a variant thereof, and are adjacent to each other, i.e., they exist in a continuous chain without interruption.

[0274] In some embodiments, the SAR further comprises one or more costimulatory domains. In an exemplary embodiment, the SAR comprises one or more costimulatory domains derived from CD28, 4-1BB, CD27, OX40, CD2, CD40, CD81, or 2B4, or variants thereof. Other costimulatory domains are known in the art and can be used in other embodiments of the present disclosure. In some embodiments, one or more costimulatory domains are located in the juxtamembrane region of the SAR. In some embodiments, one or more costimulatory domains are located C-terminal to the transmembrane region of a type I transmembrane SAR. In some embodiments, one or more costimulatory domains are located N-terminal to the transmembrane region of a type II transmembrane SAR. In another embodiment, the SAR lacks a costimulatory domain.

[0275] In one embodiment, the present disclosure provides a SAR comprising one or more costimulatory domains. The costimulatory domain is inserted between the transmembrane domain and the cytoplasmic domain derived from a naturally occurring signaling receptor (e.g., CD16A or NKp30, etc.) or signaling adaptor (e.g., CD3ζ, FcRγ, etc.) or a variant or fragment thereof. In an exemplary embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operatively linked to an extracellular domain derived from a naturally occurring signaling receptor (e.g., CD16A, CD16B, CD64, NKp30, etc.), which in turn includes a hinge domain (e.g., a CD8 hinge or a CD28 hinge), a transmembrane domain (e.g., a CD8 or CD28 transmembrane domain), a costimulatory domain (e.g., a 4-1BB or CD28 costimulatory domain), and an activation domain (e.g., a CD3ζ or FcRγ activation domain). Exemplary such SARs are represented by SEQ ID NOs: 10818 and 10821. In another exemplary embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operatively linked to an extracellular hinge domain derived from a signaling adaptor (e.g., CD3ζ, FcRγ, etc.). The hinge domain is then linked to a transmembrane domain (e.g., CD3ζ or FcRγ transmembrane domain), a costimulatory domain (e.g., 4-1BB, CD28, 2B4, or OX40 costimulatory domain), and an activation domain (e.g., CD3ζ or FcRγ activation domain, etc.).

[0276] In some embodiments, the SAR comprises one or more heterologous antigen-binding domains (e.g., scFv, vHH, FHVH, centilin, scTCR, svd-TCR, etc.). These domains are operatively linked, via an optional linker, to the amino terminus or near the amino terminus of the extracellular domain of a naturally occurring (or native) signaling receptor or signaling adaptor, or a variant or fragment thereof. In some embodiments, the one or more heterologous antigen-binding domains of the SAR are operatively linked, via an optional linker, to the amino terminus or near the amino terminus of the hinge (or spacer) domain of a naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof. In some embodiments, the one or more heterologous antigen-binding domains of the SAR are operatively linked, via an optional linker, to the amino terminus or near the amino terminus of the transmembrane domain of a naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof. In certain embodiments, one or more heterologous antigen-binding domains of the SAR are operatively linked, via an optional linker, to a hinge (spacer) domain, transmembrane domain and cytoplasmic domain of a naturally occurring signaling receptor, or a signaling adaptor, or a variant or fragment thereof. In certain embodiments, one or more heterologous antigen-binding domains of the SAR are operatively linked, via an optional linker, to a transmembrane domain and cytosolic domain of a naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof.

[0277] In exemplary embodiments, naturally occurring (or endogenous) signaling receptors comprising SARs are type I (or group 1) transmembrane proteins with an N-terminus on the extracellular side and a C-terminus on the cytosolic side. Examples of such type I (or group 1) endogenous receptors include CD16A, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, and KIR2DS. 4, KIR2DS5, KIR3DS1, CRTAM, TIGIT, CD96, 2B4, SLAMF6, SLAMF7, CD27, CD100, CD160, ILT2 / LILRB1, CD33, SIGLEC-7, SIGLEC-9, CD32, CD64, etc.

[0278] In one embodiment, the present disclosure provides a synthetic antigen receptor (SAR) comprising one or more heterologous antigen-binding domains operably linked to or near the amino terminus of a type I transmembrane native (or naturally occurring) signaling receptor via an optional linker. In one embodiment, the SAR retains the binding ability and function of the naturally occurring signaling receptor but further acquires binding ability conferred by one or more heterologous antigen-binding domains. In one embodiment, the one or more heterologous antigen-binding domains comprise an scTCR, an svd-TCR, or a TCR mimetic antibody or fragment thereof. In some embodiments, the SAR acquires TCR-like binding ability. In one embodiment, the SAR comprises one or more heterologous antigen-binding domains operably linked to or near the amino terminus of an endogenous receptor that is a type I transmembrane protein. In one embodiment, the SAR comprises a heterologous (non-natural) antigen-binding domain operably linked to or near the N-terminus of CD16A, CD16B, NKp30, NKp44, NKp46, KIR2DL1, or KIR2DL2 via an optional linker. , KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, CRTAM, TIGIT, CD96, 2B4, SLAMF6, SLAMF7, CD27, CD100, CD160, ILT2 / LILRB1, CD33, SIGLEC-7, SIGLEC-9, CD32, or CD64. In one embodiment, the SAR also comprises an N-terminal signal peptide. In one embodiment, the SAR comprises one or more antigen-binding domains located at the C-terminus of the signal peptide.

[0279] In one embodiment, the SAR comprises one or more heterologous antigen-binding domains operably linked via one or more optional linkers to a polypeptide comprising the hinge (spacer), transmembrane, and cytoplasmic domains of CD16A, CD16B, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, CRTAM, TIGIT, CD96, 2B4, SLAMF6, SLAMF7, CD27, CD100, CD160, ILT2 / LILRB1, CD33, SIGLEC-7, SIGLEC-9, CD32, or CD64.

[0280] In an exemplary embodiment, the naturally occurring (or endogenous) signaling receptor is a type II (or group 2) transmembrane protein with its C-terminus on the extracellular side and its N-terminus on the cytoplasmic side. The present disclosure provides a general method for generating a fusion protein between a type I membrane protein and a type II membrane protein, which is a fusion in which one or more modules of a type I membrane protein are operably linked to the entire region or a partial region of a type II membrane protein. In an exemplary embodiment, the N-terminus of the antigen-binding domain of a type I protein lacking a signal peptide sequence is operably linked to the C-terminus of a type II membrane protein via an optional linker. In one embodiment, a SAR consists of one or more heterologous antigen-binding domains operably linked to the carboxy-terminus or near the carboxy-terminus of a naturally occurring (or endogenous) signaling receptor via one or more optional linkers. Examples of such type II (or group 2) endogenous receptors include, but are not limited to, NKG2D, NKG2A, NKG2C, NKG2F, NKG2E, NKG2H, KLRG1, CD161, and CD94. In one embodiment, one or more heterologous antigen-binding domains (e.g., vHH, FHVH, centilin, scTCR, svd-TCR) are operably linked in-frame via one or more optional linkers to the C-terminus or near the C-terminus of an endogenous receptor that is a type II (group 2) transmembrane protein. In one embodiment, the SAR comprises one or more heterologous antigen-binding domains operably linked via an optional linker to the C-terminus or near the C-terminus of NKG2D, NKG2A, NKG2C, NKG2F, NKG2E, NKG2H, KLRG1, CD161, or CD94. Examples of such SARs comprising Her2 and Her3 vHH domains linked to the C-terminus of NKG2D are represented by SEQ ID NOs: (DNA): 7696-7697 and (PRT): 8388-8389, respectively. In one embodiment, one or more heterologous antigen-binding domains are operably linked, via an optional linker, to the hinge domain of an endogenous receptor that is a type II (group 2) transmembrane protein.In one embodiment, the one or more heterologous antigen-binding domains are operably linked to the hinge domain of NKG2D, NKG2A, NKG2B, NKG2C, NKG2F, NKG2E, NKG2H, KLRG1, CD161, or CD94 via an optional linker.

[0281] Efficient expression of NKG2D on the cell surface requires the presence of DAP10. Provided herein is a strategy for obtaining effector cells that stably overexpress NKG2D-SAR (i.e., a SAR containing the transmembrane and / or cytoplasmic domain of NKG2D) alone or together with DAP10 by genetically engineering cells (e.g., iPSCs) to introduce NKG2D-SAR, and optionally DAP10, into cells (e.g., iPSCs), followed by directed differentiation of iPSCs to induce effector cells, including NK cells and T cells. The present disclosure also provides a strategy for efficient expression of NKG2D-SAR in cells lacking DAP10 or expressing low levels of DAP10 by ectopic expression of DAP10 as an accessory module together with a SAR containing the NKG2D transmembrane domain.

[0282] CD94 / NKG2C is a heterodimeric receptor that binds to HLA-E and associates with DAP12, a protein containing an immunoreceptor tyrosine-based activation motif. The presence of DAP12 is required for efficient cell surface expression of CD94 / NKG2C, and the transmembrane domain of DAP12 and the charged amino acids of NKG2C mediate this interaction. Provided herein is a strategy for obtaining effector cells that stably overexpress NKG2C-SAR (i.e., a SAR containing the transmembrane domain of NKG2C) alone or together with DAP12 by genetically engineering cells (e.g., iPSCs) to introduce NKG2C-SAR, and optionally, by introducing NKG2C-SAR. DAP12 is introduced into cells (e.g., iPSCs), and then effector cells, including NK cells and T cells, are derived from the induced iPSC differentiation. In some embodiments, NKG2C-SAR is further co-expressed with CD94 or CD94-SAR.

[0283] In iPSCs or effector cells derived therefrom comprising overexpressed NKG2C-SAR, the cells further comprise overexpressed CD94 or CD94-SAR and / or DAP12. In one embodiment, NKG2C-SAR and CD94 (or CD94-SAR) and / or DAP12 are expressed in separate constructs. In another embodiment, NKG2C-SAR and CD94 (or CD94-SAR) and / or DAP12 are coexpressed in a bicistronic or tricistronic construct and linked by a self-cleaving 2A coding sequence. In another embodiment, NKG2C-SAR, CD94 (or CD94-SAR) and DAP12 are expressed in separate constructs.

[0284] , provided that it can be expressed equally efficiently by co-expression of known CD94 and / or signaling adaptors (e.g., DAP10, DAP12, etc.) that interact with them.

[0285] In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to the carboxy terminus or near the carboxy terminus of a type II transmembrane native signaling receptor. In one embodiment, the SAR retains the binding ability and function of the naturally occurring signaling receptor, but additionally acquires the binding ability conferred by the heterologous antigen-binding domain. In one embodiment, the present disclosure provides one or more heterologous antigen-binding domains operably linked in-frame via an optional linker to the hinge domain or transmembrane domain of a type II transmembrane native signaling receptor to generate a synthetic antigen receptor. In one embodiment, the resulting SAR retains the binding ability and function of the naturally occurring signaling receptor, but additionally acquires the binding ability conferred by the heterologous antigen-binding domain. In one embodiment, the SAR of the present disclosure retains the binding properties and physiological regulation of the naturally occurring receptor while acquiring additional antigen-binding ability capable of responding to target antigens in addition to those to which the naturally occurring receptor can respond.

[0286] The present disclosure also provides single-chain SARs comprising one or more heterologous antigen-binding domains operably linked to the extracellular domain of a native signaling chain or signaling adaptor or a variant thereof via an optional linker. The present disclosure also provides single-chain SARs comprising one or more heterologous antigen-binding domains operably linked to the hinge (spacer) domain of a native signaling chain or signaling adaptor or a variant thereof via an optional linker. The present disclosure also provides single-chain SARs comprising one or more heterologous antigen-binding domains operably linked to the transmembrane domain of a native signaling chain or signaling adaptor or a variant thereof via an optional linker. Exemplary signaling chains or signaling adaptors include, but are not limited to, CD3ζ, FcRγ, DAP10, or DAP12, or variants thereof. In one embodiment, the signaling chain / adaptor further comprises a costimulatory domain.

[0287] In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked to the entire or partial extracellular domain, hinge domain, or transmembrane domain of NCAM (non-CD3 adaptor module) via an optional linker.

[0288] In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen binding domains operably linked via an optional linker to the entire or partial extracellular domain, hinge domain, or transmembrane domain of a signaling adaptor, wherein the signaling adaptor (or signaling chain) is not CD3ε, CD3γ, CD3δ, or CD3ζ. In one embodiment, the signaling adaptor is not FcRγ.

[0289] In one embodiment, one or more heterologous antigen-binding domains of the single-chain SAR comprise an autonomous antigen-binding domain (AABD), such as a single-domain antibody, a single vH domain, FHVH, vHH domain, svd-TCR, or a non-immunoglobulin antigen-binding scaffold (e.g., DARPIN, affibody, affilin, adnectin, afftin, body, repebody, phynomer, alphabody, avimer, atrimer, centilin, pronectin, anticalin, Kunitz domain, Armadillo repeat protein, or a fragment thereof). In one embodiment, one or more heterologous antigen-binding domains of the single-chain SAR comprise an antibody or antibody fragment (e.g., vL, vH, Fab, Fab'2, scFv, scTCR, etc.).

[0290] Because SARs are modular, their different domains can be replaced with other domains to generate new SARs with diverse biological activities and properties. Thus, the extracellular domain, hinge domain, transmembrane domain, and / or cytoplasmic domain of one SAR can be replaced with the extracellular domain, hinge domain, transmembrane domain, and / or cytoplasmic domain of another SAR, as long as the resulting SAR retains at least one of the biological activities (e.g., antigen binding, cell signaling, etc.) of the original SAR. In one embodiment, the present disclosure provides that a new module (costimulatory domain) can be inserted into a SAR. In one embodiment, the present disclosure provides a SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to the entire or partial extracellular domain of one naturally occurring signaling receptor, which is then operably linked to the transmembrane and cytoplasmic domains of a different naturally occurring receptor or variant thereof. In an exemplary embodiment, the extracellular domain of a SAR consisting of the CD16A extracellular domain, transmembrane domain, and cytoplasmic domain is replaced with the extracellular domain of CD64 to generate a new SAR (SEQ ID NO: 4722) consisting of the CD20 vHH domain, the CD64 extracellular domain, the CD16A transmembrane domain, and the CD16A cytoplasmic domain.

[0291] In one embodiment, the different domains of the SAR are derived from a single natural receptor or signaling adaptor. In one embodiment, the different domains of the SAR are derived from two or more naturally occurring receptors or signaling adaptors. In one embodiment, the SAR consists of part or all of the antigen-binding domain of one naturally occurring receptor and one or more heterologous antigen-binding domains operably linked via an optional linker to hinge, transmembrane, and cytoplasmic domains from one or more different receptors or their mutants or fragments. In an exemplary embodiment, the SAR consists, from N-terminus to C-terminus, of a CD19 scFv, a CD16 antigen-binding domain (D1 and D2), a CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z activation domain. The amino acid sequence of such a SAR is set forth in SEQ ID NO: 10836. In another exemplary embodiment, the SAR consists of, from N-terminus to C-terminus, a CD19 scFv, a CD16A antigen-binding domain (D1 and D2), a CD16A hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z activation domain. In another exemplary embodiment, the SAR comprises, from N-terminus to C-terminus, a CD19 scFv, a CD64 antigen-binding domain, a CD16A hinge domain, a CD16A transmembrane domain, and a CD16A cytosolic domain. The amino acid sequence of such a SAR is set forth in SEQ ID NO: 10832.

[0292] In one embodiment, the SAR comprises one or more heterologous antigen-binding domains operably linked via an optional linker to the hinge domain, transmembrane domain, and cytosolic domain of a naturally occurring signaling receptor or a variant thereof. All of the cytosolic domains are derived from a single naturally occurring signaling receptor or signaling adaptor, or a variant or fragment thereof. In one embodiment, the hinge domain, transmembrane domain, and cytosolic domain comprising the SIR are derived from a single naturally occurring signaling receptor (e.g., CD16A) or signaling adaptor (e.g., CD3ζ) or a variant or fragment thereof. In another embodiment, the hinge domain, transmembrane domain, and cytoplasmic domain comprising the SIR are derived from one or more naturally occurring signaling receptors (e.g., the hinge domain and transmembrane domain of CD16A are operably linked to the cytoplasmic domain of NKp30, etc.) or signaling adaptors (e.g., the hinge domain and transmembrane domain of CD16A are operably linked to the cytoplasmic domain of FcRγ, etc.). In one embodiment, a SAR consists of one or more heterologous antigen-binding domains operably linked via an optional linker to the hinge domain, transmembrane domain, and cytoplasmic domain of a naturally occurring signaling receptor, wherein the hinge, transmembrane domain, and cytoplasmic domain are derived from one or more naturally occurring signaling receptors, signaling adaptors, or variants or fragments thereof.

[0293] In one embodiment, the disclosure provides a method for producing a non-native protein (i.e., a synthetic protein) comprising two or more chains having the following general formula from amino (N) terminus to carboxy (C) terminus: Chain 1: SP1-A1-L1-H1-M1-(C1)n Chain 2: SP2-A2-L2-H2-M2-(C2)n

[0294] wherein SP1 and SP2 are optional signal peptides cleaved from the mature polypeptide chain; A1 and A2 are two protein domains that can interact with each other, L1 and L2 are optional linkers, H1 and H2 are optional hinge or spacer domains, M1 and M2 are membrane anchor or transmembrane domains, and C1 and C2 are optional cytoplasmic domains. In one embodiment, the A1 and A2 domains are not derived from an antibody. In one embodiment, the A1 and A2 domains are not antibody fragments. In one embodiment, the A1 and A2 domains are heterologous to the M1 and M2 domains, i.e., the A1 and M1 domains are derived from different proteins, and similarly, the A2 and M2 domains are derived from different proteins. In an exemplary embodiment, the A1 and A2 domains are derived from a TCR (e.g., the Vα and Vβ domains of a TCR), and the M1 and M2 domains are derived from CD3ε. In one embodiment, the A1 and A2 domains are not autonomous domains. In one embodiment, the A1 and A2 domains have greater affinity for each other than for an unrelated protein. In one embodiment, the A1 and A2 domains can associate with each other to generate an antigen-binding domain. In one embodiment, the non-natural protein is a synthetic antigen receptor. In one embodiment, the L1 and L2 linkers are long linkers. In one embodiment, the L1 and L2 linkers are Ig-like linkers. In one embodiment, the L1 and L2 linkers are linked by one or more disulfide bonds. In one embodiment, the M1 and M2 domains are transmembrane domains. In one embodiment, the M1 and M2 domains are derived from the same protein (e.g., CD3ζ). In one embodiment, the M1 and M2 domains are derived from different proteins (e.g., CD3ε and FcRγ). In one embodiment, the M1 and M2 domains are sequence identical and / or share 70% or greater (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, etc.) amino acid sequence homology. In one embodiment, the M1 and M2 domains are associated with each other. In one embodiment, the M1 and M2 domains are linked by a disulfide bond.In one embodiment, the M1 and / or M2 domains can recruit one or more signaling adaptors. In one embodiment, the C1 and C2 domains are cell signaling domains (e.g., activation domains or costimulatory domains). In one embodiment, each chain can have multiple cytosolic domains. In one embodiment, both chains are expressed on the cell surface with the A1-L1-H1 and A2-L2-H2 segments located extracellularly. The present disclosure provides nucleic acids encoding the synthetic proteins, amino acid sequences, one or more vectors encoding the synthetic proteins, and cells expressing the synthetic proteins.

[0295] The present disclosure also provides a double-chain SAR (e.g., an isolated double-chain SAR). In one embodiment, the present disclosure provides a double-chain SAR comprising one or more heterologous antigen-binding domains operably linked, optionally via a linker, to at least one module consisting of the entire or partial extracellular domain of a signaling receptor, a transmembrane domain, and optionally a cytoplasmic domain, a signaling adaptor, or a variant or fragment thereof. In one embodiment, the signaling receptor is a non-TCR signaling receptor. In one embodiment, the signaling adaptor is a non-CD3 adaptor. In one embodiment, the signaling adaptor is not CD3ε.

[0296] In one embodiment, the present disclosure provides a double-chain SAR consisting of two chains, each comprising at least one antigen-binding domain (e.g., vL, vβ, Vα, Vβ, Vγ, or Vδ, etc.), operably linked via an optional peptide linker (e.g., IgCL, IgCH1, etc.) to a membrane-associated module (MAM) consisting of a transmembrane or membrane-associated domain of a signaling receptor (e.g., CD16A, CD16B, NKp30, etc.), a signaling adaptor (e.g., CD3ζ, FcRγ), or a variant or fragment thereof. In some embodiments, the MAM further comprises a whole or partial extracellular antigen-binding domain, a hinge domain and / or cytosolic domain of the signaling receptor, and / or a hinge domain and / or cytosolic domain of the signaling adaptor. In one embodiment, the signaling receptor is a non-TCR signaling receptor. In one embodiment, the module is an NTCRM. In one embodiment, the signaling adaptor is a non-CD3 adaptor (i.e., NCAM). In one embodiment, the signaling adaptor is not CD3ε. In one embodiment, the MAM does not comprise a transmembrane domain of TCRα, β, γ, δ, pre-TCRα, CD3ε, CD3ε, CD3γ, or CD3δ. In some embodiments, at least one antigen-binding domain of a first chain (e.g., vL, Vα, or Vγ) associates with at least one complementary antigen-binding domain of a second chain (e.g., vH, Vβ, or Vδ) to form an antigen-binding module (e.g., Fv or Fc-TCR) that specifically binds to a target antigen. In some embodiments, the first and second polypeptide chains are linked via one or more disulfide bonds. In some embodiments, the first and second peptide linkers are linked via one or more disulfide bonds. In some embodiments, the first and / or second peptide linkers are each about 5 to about 500 amino acids in length. In one embodiment, the first and second antigen-binding domains comprise complementary chains (e.g., vL and vH, Vα and Vβ, or Vγ and Vδ).In some embodiments, the first polypeptide chain and the second polypeptide chain further comprise one or more autonomous antigen-binding domains (AABDs) attached to or near the N-terminus of the first antigen-binding domain (e.g., vL, Vα, or Vγ) and / or the second antigen-binding domain (e.g., vH, Vβ, or Vδ). In some embodiments, the target antigen is a cell surface antigen. In some embodiments, the cell surface antigen is selected from the group consisting of proteins, carbohydrates, and lipids. In exemplary embodiments, the target antigen is one or more of the antigens listed in Table B. In exemplary embodiments, the cell surface antigen is selected from one or more of the following groups, but is not limited to: CD2, CD5, CD19, CD20, CD22, CD33, CD70, CD123, CD138, CD179b, CLL-1, FLT3, claudin 18.2, BCMA, GCC, MPL, SLAMF7, ROR1, ROR2, GPRC5D, FCRL5, MSLN, EGFR, EGFRviii, PSMA, PSCA, KLK2, IL13Ra2, TROP2, PTK7, DLL3, Mucl, Mucl6, or Her2. In some embodiments, the target antigen is a complex comprising a peptide and a major histocompatibility complex (MHC) protein. In exemplary embodiments, the peptide antigen is one or more of the antigens listed in Table B. In exemplary embodiments, the peptide / MHC complex comprises a peptide derived from one or more of NY-ESO-1, MAGE-A2, MAGE-A3, MAGE4, ​​WT1, AFP, TERT, MART-1, pp66-CMV, HPV16-E7, PRAME, EBV-LMP2A, HIV-1, PSA, or gp100.

[0297] In one embodiment, the present disclosure provides a double-chain SAR consisting of two chains, each of which comprises one or more heterologous antigen-binding domains operably linked, optionally via a linker, to a module consisting of the hinge, transmembrane domain, and optionally the cytoplasmic domain of a signaling receptor, or a signaling adaptor, or a variant or fragment thereof. In one embodiment, the present disclosure provides a double-chain SAR consisting of two chains, each of which comprises one or more heterologous antigen-binding domains operably linked, via an optional linker, to a module consisting of the transmembrane domain of a signaling receptor and optionally the cytoplasmic domain, signaling adaptor, or a variant or fragment thereof. In one embodiment, the present disclosure provides a double-chain SAR consisting of two chains, each of which comprises one or more heterologous antigen-binding domains operably linked, via an optional linker, to a module consisting of the transmembrane domain of a signaling receptor, or a signaling adaptor, or a variant or fragment thereof. In one embodiment, the present disclosure provides a double-chain SAR consisting of two chains, each of which comprises one or more heterologous antigen-binding domains operably linked, via an optional linker, to a module consisting of the cytoplasmic domain of a signaling receptor, a signaling adaptor, or a variant or fragment thereof. In one embodiment, the signaling receptor is a non-TCR signaling receptor. In one embodiment, the module is NTCRM. In one embodiment, the signaling adaptor is a non-CD3 adaptor (i.e., NCAM). In one embodiment, the signaling adaptor is not CD3ε.

[0298] In one embodiment, the present disclosure provides a double-chain SAR, wherein each chain is comprised of one or more heterologous antigen-binding domains operably linked via an optional linker to a module (e.g., NTCRM) comprising the extracellular, transmembrane, or cytoplasmic domain of a signaling receptor (e.g., a native receptor), a signaling adaptor (e.g., NCAM), or a variant or fragment thereof. In one embodiment, the signaling receptor and signaling adaptor comprising the double-chain SAR are naturally occurring. In one embodiment, the signaling receptor and signaling adaptor comprising the double-chain SAR are not naturally occurring. In one embodiment, the signaling receptor and signaling adaptor comprising the double-chain SAR are a non-T cell receptor and a non-CD3 adaptor. In one embodiment, the signaling receptor and signaling adaptor comprising the double-chain SAR are a naturally occurring non-T cell receptor and a non-CD3 adaptor.

[0299] In one embodiment, the present disclosure provides a duplex SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to at least one module (e.g., NTCRM) consisting of the extracellular, transmembrane, or cytoplasmic domain of a naturally occurring signaling receptor (e.g., CD16A), signaling adaptor (e.g., FcRγ), or a variant or fragment thereof.

[0300] In one aspect, the present application provides a construct (such as an isolated construct) comprising one or more heterologous antigen-binding domains fused to a non-T cell receptor module (NTCRM). In exemplary embodiments, the NTCRM is derived from one or more of the following non-TCR receptors, including, but not limited to, CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, ILT2.

[0301] In some embodiments, the SAR comprises one or more heterologous antigen-binding domains that specifically bind to a target antigen and a non-T cell receptor module (NTCRM) that can recruit at least one signaling adaptor. In one embodiment, the signaling adaptor is a non-CD3 adaptor (i.e., NCAM). In some embodiments, the target antigen is a complex comprising a peptide and an MHC protein (e.g., an MHC class I protein or an MHC class II protein). In some embodiments, the target antigen is a cell surface antigen.

[0302] In some embodiments, a SAR (e.g., an isolated SAR) that specifically binds to a target antigen is provided, wherein the SAR consists of: a) a first polypeptide chain comprising a first antigen-binding domain consisting of a vL, Vα, or Vγ domain and a first membrane-associated module (MAM); and b) a second polypeptide chain consisting of a second antigen-binding domain consisting of a vH, Vβ, or Vδ domain and a second membrane-associated module (MAM), wherein the vL, Vα, or Vγ domain of the first antigen-binding domain and the complementary vH, Vβ, or Vδ domain of the second antigen-binding domain form an Fv or TCR-Fv-like antigen-binding module that specifically binds to the target antigen, and the first MAM and the second MAM form a non-T cell receptor module (NTCRM), which is capable of activating at least one signaling pathway and / or recruiting at least one signaling adaptor. In one embodiment, the first MAM and the second MAM do not comprise a transmembrane domain of a TCR chain selected from TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the first MAM or the second MAM do not comprise a transmembrane domain of a TCR chain selected from TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. In one embodiment, the NTCRM does not comprise a transmembrane domain of two TCR chains selected from i) TCRα and TCRβ, ii) TCRγ and TCRδ, or iii) pre-TCRα and TCRβ. In one embodiment, the first MAM and the second MAM do not comprise a transmembrane domain of a CD3 chain selected from CD3ε, CD3γ, CD3δ, or CD3ε. In one embodiment, the first MAM and the second MAM do not comprise a transmembrane domain of a TCR chain and a CD3 chain. In one embodiment, the first MAM and the second MAM do not comprise a transmembrane domain of CD3ε.In exemplary embodiments, the NTCRM is derived from one or more of the following non-TCR receptors, including, but not limited to, CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, ILT2. In some embodiments, the signaling adaptor is selected from, but not limited to, one or more of CD3ε, FcRγ, DAP10, and / or DAP12, or variants or fragments thereof. In some embodiments, the signaling adaptor is a non-CD3 adaptor (NCAM). In some embodiments, the signaling adaptor is not CD3ε. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked via one or more disulfide bonds. In some embodiments, the first polypeptide chain further comprises a first peptide linker between the first antigen-binding domain and the first MAM. In some embodiments, the second polypeptide chain further comprises a second peptide linker between the second antigen-binding domain and the second MAM. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked via one or more disulfide bonds. In some embodiments, the first peptide linker and / or the second peptide linker are each about 5 to about 500 amino acids in length. In some embodiments, the first and / or second peptide linker individually comprises a constant domain or fragment thereof derived from an immunoglobulin or T cell receptor subunit, hi some embodiments, the first and / or second peptide linker individually comprises a CH1, CH2, CH3, CH4, or CL antibody domain, or fragment thereof.In some embodiments, the first and / or second peptide linker individually comprises a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In exemplary embodiments, the first and / or second linker individually comprises an immunoglobulin-derived Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3560, SEQ ID NOs: 3536-3551), or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.), or a variant or fragment thereof. In some embodiments, the vL domain is linked to an IgCL linker, and the vH domain is linked to an IgCH1 linker. In some embodiments, the vL domain is linked to an IgCH1 linker, and the vH domain is linked to an IgCL linker. In some embodiments, the Vα domain is linked to a Cα-derived linker (e.g., TCRa-Ig3), and the Vβ domain is linked to a Cβ-derived linker (e.g., TCRb-Ig3). In some embodiments, the Vβ domain is linked to a Cα-derived linker (e.g., TCRa-Ig3), and the Vα domain is linked to a Cβ-derived linker (e.g., TCRb-Ig3). In some embodiments, the Vγ domain is linked to a Cγ-derived linker (e.g., TCRg-Ig3), and the Vδ domain is linked to a Cδ-derived linker (e.g., TCRd-Ig3). In some embodiments, the Vγ domain is linked to a Cδ-derived linker (e.g., TCRd-Ig3), and the Vδ domain is linked to a Cγ-derived linker (e.g., TCRg-Ig3). In some embodiments, other configurations of variable domains and linkers are contemplated. In some embodiments, the first and / or second peptide linker comprises mutations that increase expression, affinity, and pairing of the two polypeptide chains. In one embodiment, the first and second antigen-binding domains comprise complementary chains (e.g., vL and vH, Vα and Vβ, or Vγ and Vδ).In some embodiments, the first polypeptide chain and the second polypeptide chain further comprise one or more autonomous antigen-binding domains (AABDs) attached to or near the N-terminus of the first antigen-binding domain (e.g., vL, Vα, or Vγ) and / or the second antigen-binding domain (e.g., vH, Vβ, or Vδ). In some embodiments, the AABDs are selected from, but are not limited to, one or more of a single vH domain (SVH), a single vL domain (SVL), a vHH domain, a single domain antibody, a single variable domain of a TCR (svd-TCR), a non-immunoglobulin antigen-binding scaffold, a ligand-binding domain of a receptor, a receptor-binding domain of a ligand, an autoantigen, an adaptor-binding domain, an Fc-binding domain, or a fragment or variant thereof.

[0303] In some embodiments, the SAR binds to the target antigen with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM. In some embodiments, the target antigen is a cell surface antigen. In exemplary embodiments, the target antigen is one or more of the antigens listed in Table B. In some embodiments, the cell surface antigen is selected from the group consisting of proteins, carbohydrates, and lipids. In some embodiments, the cell surface antigen is CD2, CD5, CD19, CD20, CD22, CD33, CD70, CD123, CD138, CD179b, CLL-1, FLT3, claudin 18.2, BCMA, GCC, MPL, SLAMF7, ROR1, ROR2, GPRC5D, FCRL5, MSLN, EGFR, EGFRviii, PSMA, PSCA, KLK2, IL13Ra2, TROP2, PTK7, DLL3, Mucl, Mucl6, or Her2. In some embodiments, the target antigen is a complex comprising a peptide and a major histocompatibility complex (MHC) protein. In exemplary embodiments, the peptide / MHC complex comprises a peptide derived from one or more of NY-ESO-1, MAGE-A2, MAGE-A3, MAGE4, ​​WT1, AFP, TERT, MART-1, pp66-CMV, HPV16-E7, PRAME, EBV-LMP2A, HIV-1, PSA, or gp100.

[0304] In one embodiment, the vL and vH domains of the SAR are derived from a TCR mimetic antibody capable of recognizing intracellular peptides in an MHC-dependent manner. In one embodiment, the Vα and Vβ domains of the SAR are derived from an HLA-independent TCR capable of recognizing cell surface proteins. In one embodiment, the SAR is bispecific or multispecific. In one embodiment, the present disclosure provides a SAR capable of binding to two or more MHC-restricted antigens. In one embodiment, the SAR can bind to two or more MHC-restricted and / or MHC-unrestricted antigens. In one embodiment, the SAR can bind to a peptide / MHC complex via its Fv or TCR-Fv domain and one or more svd-TCRs bound at or near the N-terminus of its vL and vH, Vα and Vβ, or Vγ and Vδ domains. In one embodiment, the SAR can bind to one or more peptide / MHC complexes via its Fv, TCR-Fv domain and / or svd-TCR domain, and can bind to one or more surface antigens via one or more AABDs (e.g., vHH, FHVH, centilin, etc.) bound to or near the N-terminus of its Vα and Vβ or Vγ and Vδ domains.

[0305] In some embodiments, according to any of the above SARs (e.g., isolated SARs), the first polypeptide further comprises a first hinge domain (or connecting peptide) or a fragment thereof (e.g., a transmembrane domain) N-terminal to the first MAM, and / or the second MAM further comprises a second hinge domain (or connecting peptide) or a fragment thereof (e.g., a transmembrane domain) N-terminal to the second MAM. In some embodiments, the SAR comprises a disulfide bond between a residue in the first MAM and a residue in the second MAM, and / or a disulfide bond between a residue in the first hinge domain and a residue in the second hinge domain. In some embodiments, according to any of the above SARs (e.g., isolated SARs), the first MAM further comprises a first homologous antigen-binding domain or a fragment thereof N-terminal to the first hinge domain, and / or the second polypeptide further comprises a second homologous antigen-binding domain or a fragment thereof N-terminal to the second hinge domain. In one embodiment, the two homologous antigen-binding domains are derived from the same non-T cell receptor as the two hinge domains. In some embodiments, the first MAM further comprises a first cytosolic domain C-terminal to the first transmembrane domain. In some embodiments, the second MAM further comprises a second cytosolic domain C-terminal to the second transmembrane domain. In one embodiment, the first and / or second cytosolic domain is an activation domain comprising one or more ITAMs. In some embodiments, the SAR binds to the target antigen with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM.

[0306] In some embodiments, according to any of the above SARs (e.g., isolated SARs), the first polypeptide chain further comprises a first costimulatory domain C-terminal to the first transmembrane domain. In some embodiments, the second polypeptide chain further comprises a second costimulatory domain C-terminal to the second transmembrane domain. In some embodiments, according to any of the above SARs (e.g., isolated SARs), the first polypeptide chain comprises one or more costimulatory domains C-terminal to the first transmembrane domain, and / or the second polypeptide chain comprises one or more costimulatory domains C-terminal to the second transmembrane domain. In some embodiments, the first polypeptide chain further comprises a first signaling peptide N-terminal to the first antigen-binding domain. In some embodiments, the second polypeptide chain further comprises a second signaling peptide N-terminal to the second antigen-binding domain.

[0307] In some embodiments, the first and / or second MAM and NTCRM are comprised of a transmembrane / membrane-anchoring domain, an optional cytoplasmic domain, an optional costimulatory domain, an optional hinge domain, and / or an optional extracellular domain of a non-T cell receptor and / or signaling adaptor.

[0308] In some embodiments, the first and / or second MAM and NTCRM are comprised of a transmembrane / membrane anchor domain, optional cytoplasmic domain, optional costimulatory domain, optional hinge domain, and / or optional extracellular domain, all derived from a single non-T cell receptor and / or a single signaling adaptor or variant thereof. In an exemplary embodiment, the first polypeptide chain comprises a hinge domain, transmembrane domain, and cytosolic domain all derived from CD3ε, and the second polypeptide chain comprises a hinge domain, transmembrane domain, and cytosolic domain all derived from FcRγ or CD16A.

[0309] In some embodiments, the first and / or second MAM and NTCRM comprise transmembrane / membrane anchor domains, optionally cytoplasmic domains, optionally hinge domains, and / or optionally extracellular domains derived from one or more different non-T cell receptors and / or signaling adaptors or variants thereof. In an exemplary embodiment, the first polypeptide chain comprises a CD3ζ hinge domain, a CD3ζ transmembrane domain, and an FcRγ cytoplasmic domain, and the second polypeptide chain comprises a DAP10 transmembrane domain linked to a cytoplasmic domain comprised of a DAP10 hinge domain, a 41-BB costimulatory domain, and a CD3ζ activation domain.

[0310] In some embodiments, the two transmembrane / membrane anchor domains, any cytosolic domain, any costimulatory domain, any hinge domain, and / or any extracellular domain are identical in sequence and are derived from the same protein. In some embodiments, the two transmembrane / membrane anchor domains, any cytoplasmic domain, any costimulatory domain, any hinge domain, and / or any extracellular domain are different in sequence and / or are derived from different proteins.

[0311] In some embodiments, the two transmembrane / membrane anchor domains, the optional cytosolic domain, the optional costimulatory domain, the optional hinge domain, and / or the optional extracellular domain differ in sequence and / or are derived from different proteins.

[0312] In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to a target antigen, comprising: a) a first chain consisting of one or more heterologous antigen-binding domains operably linked to the extracellular domain of a signaling receptor or a variant thereof via one or more optional linkers; and b) a second chain consisting of one or more heterologous antigen-binding domains operably linked to the extracellular domain of a second signaling receptor or a variant thereof via one or more optional linkers. In one embodiment, one or both signaling receptors are naturally occurring. In one embodiment, one or both signaling receptors are naturally occurring non-T cell receptors. In one embodiment, at least one heterologous antigen-binding domain (e.g., vL, Vα, or Vγ) present on the first chain associates with at least one heterologous antigen-binding domain (e.g., vH, Vβ, or Vδ) present on the first chain. The second chain forms an antigen-binding module (e.g., Fv or TCR-Fv) that specifically binds to the target antigen. In some embodiments, the target antigen is a cell surface antigen. In some embodiments, the target antigen is a complex comprising a peptide and an MHC protein (e.g., an MHC class I protein or an MHC class II protein). In some embodiments, the SAR binds to the target antigen with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked via one or more disulfide bonds. In some embodiments, the first peptide linker and the second peptide linker are linked via one or more disulfide bonds. In some embodiments, a disulfide bond exists between a residue of any first linker in the first polypeptide chain and a residue of any second linker in the second polypeptide chain. In some embodiments, the first linker and / or the second linker are each about 5 to about 500 amino acids in length. In some embodiments, the first and / or second peptide linkers, individually, comprise a constant domain or fragment thereof derived from an immunoglobulin or a T cell receptor subunit.In some embodiments, the first and / or second peptide linker individually comprises a CH1, CH2, CH3, CH4 or CL antibody domain, or a fragment thereof. In some embodiments, the first and / or second peptide linker individually comprises a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In some embodiments, the first and / or second linker individually comprise an immunoglobulin-derived Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3560, SEQ ID NOs: 3536-3551), or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.), or a variant or fragment thereof. In some embodiments, the first and / or second peptide linker comprise mutations that increase their expression, affinity, and chain pairing. In some embodiments, the first and / or second signaling receptor form a non-T cell receptor module (NTCRM) that can recruit at least one signaling adaptor (e.g., CD3ζ or NCAM, etc.). In some embodiments, the signaling adaptor is selected from the group consisting of CD3ε, FcRγ, DAP10, and / or DAP12.

[0313] In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a first antigen-binding domain consisting of a vL (variable domain of an antibody light chain), Va (variable domain of a TCR or Vα), or Vg (variable domain of a TCR or Vγ) domain, operably linked to the entire or a portion of the extracellular antigen-binding domain of a non-TCR signaling receptor chain or a variant thereof via an optional linker; and b) a second antigen-binding domain that is a vH (variable domain of an antibody heavy chain), Vb (variable domain of a TCR or Vβ), or Vd domain (variable domain of a TCR or Vδ), operably linked to the entire or a portion of the extracellular antigen-binding domain of a second non-TCR signaling receptor chain or a variant thereof via an optional linker, wherein the vL, Vα, or Vγ domain of the first antigen-binding domain and the vH, Vβ, or Vδ domain of the second antigen-binding domain form an Fv or TCR-Fv-like antigen-binding module that specifically binds to a target antigen. In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a vL domain operably linked to the whole or part of the extracellular antigen-binding domain of a non-TCR signaling receptor chain or a variant thereof via an optional peptide linker; and b) a vH domain operably linked to the whole or part of the extracellular antigen-binding domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the vL and vH domains form an Fv-like antigen-binding module that specifically binds to a target antigen in an MHC-dependent and / or MHC-independent manner. In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a Vα domain operably linked to all or part of the extracellular antigen-binding domain of a non-TCR signaling receptor or a variant thereof via an optional peptide linker; and b) a Vβ domain operably linked to all or part of the extracellular antigen-binding domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the Vα and Vβ domains form a TCR-Fv-like antigen-binding module that specifically binds to a peptide / MHC complex in an MHC-dependent manner.In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a Vγ domain operably linked to the entire or a portion of the extracellular antigen-binding domain of a non-TCR signaling receptor or a variant thereof via an optional peptide linker; and b) a Vδ domain operably linked to the entire or a portion of the extracellular antigen-binding domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the Vγ and Vδ domains form a TCR-Fv-like antigen-binding module that specifically binds to an antigen in an MHC-dependent or MHC-independent manner. In exemplary embodiments, the non-TCR signaling receptor is selected from, but is not limited to, one or more of the following: CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, and ILT2. In some embodiments, the first and / or second peptide linker individually comprise a constant domain or fragment thereof derived from an immunoglobulin or T cell receptor subunit. In some embodiments, the first and / or second peptide linker individually comprise a CH1, CH2, CH3, CH4, or CL antibody domain, or fragment thereof. In some embodiments, the first and / or second peptide linker individually comprise a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof.In some embodiments, the first and / or second linker individually consists of an immunoglobulin-derived Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3560, SEQ ID NOs: 3536-3551), or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.), or a variant or fragment thereof. In some embodiments, the first and / or second peptide linker comprises mutations that increase their expression, affinity, and chain pairing. In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) attached at or near the N-terminus of the first antigen-binding domain (e.g., vL, Vα, or Vγ) and / or the second antigen-binding domain (e.g., vH, Vβ, or Vδ).

[0314] In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a first antigen-binding domain consisting of a vL (variable domain of an antibody light chain), Va (variable domain of a TCR or Vα), or Vg (variable domain of a TCR or Vγ) domain, operably linked via an optional peptide linker to the whole or partial hinge domain of a non-TCR signaling receptor chain or a variant thereof; and b) a second antigen-binding domain, a vH (variable domain of an antibody heavy chain), Vb (variable domain of a TCR or Vβ), or Vd domain (variable domain of a TCR or Vδ), operably linked via an optional peptide linker to the whole or partial hinge domain of a second non-TCR signaling receptor chain or a variant thereof, wherein the vL, Vα, or Vγ domain of the first antigen-binding domain and the vH, Vβ, or Vδ domain of the second antigen-binding domain form an Fv or TCR-Fv-like antigen-binding module that specifically binds to a target antigen. In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a vL domain operably linked to the whole or partial hinge domain of a non-TCR signaling receptor chain or a variant thereof via an optional peptide linker; and b) a vH domain operably linked to the whole or partial hinge domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the vL and vH domains form an Fv-like antigen-binding module that specifically binds to a target antigen in an MHC-dependent and / or MHC-independent manner. In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a Vα domain operably linked to the entire or partial hinge domain of a non-TCR signaling receptor or a variant thereof via an optional peptide linker; and b) a Vβ domain operably linked to the entire or partial hinge domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the Vα and Vβ domains form a TCR-Fv-like antigen-binding module that specifically binds to a peptide / MHC complex in an MHC-dependent manner.In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a Vγ domain operably linked to the entire or partial hinge domain of a non-TCR signaling receptor or a variant thereof via an optional peptide linker; and b) a Vδ domain operably linked to the entire or partial hinge domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the Vγ and Vδ domains form a TCR-Fv-like antigen-binding module that specifically binds to an antigen in an MHC-dependent or MHC-independent manner. In exemplary embodiments, the non-TCR signaling receptor is selected from, but is not limited to, one or more of the following: CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, and ILT2. In some embodiments, the first and / or second peptide linker individually comprise a constant domain or fragment thereof derived from an immunoglobulin or T cell receptor subunit. In some embodiments, the first and / or second peptide linker individually comprise a CH1, CH2, CH3, CH4, or CL antibody domain, or fragment thereof. In some embodiments, the first and / or second peptide linker individually comprise a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof.In some embodiments, the first and / or second peptide linker individually comprise an immunoglobulin-derived Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3560, SEQ ID NOs: 3536-3551), or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.), or a fragment thereof. In some embodiments, the first and / or second peptide linker comprise mutations that increase their expression, affinity, and chain pairing. In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) attached at or near the N-terminus of the first antigen-binding domain (e.g., vL, Vα, or Vγ) and / or the second antigen-binding domain (e.g., vH, Vβ, or Vδ).

[0315] In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a first antigen-binding domain consisting of a vL (variable domain of an antibody light chain), Va (variable domain of TCR or Vα), or Vg (variable domain of TCR or Vγ) domain, operably linked to the whole or part of a transmembrane / membrane-anchoring domain of a non-TCR signal receptor chain or a variant thereof via an optional peptide linker; and b) a second antigen-binding domain, consisting of a vL (variable domain of an antibody light chain), Va (variable domain of TCR or Vα), or Vg (variable domain of TCR or Vγ) domain, operably linked to the whole or part of a transmembrane / membrane-anchoring domain of a non-TCR signal receptor chain or a variant thereof via an optional peptide linker. a vH (variable domain of an antibody heavy chain), Vb (variable domain of TCRb or Vβ), or Vd domain (variable domain of TCRd or Vδ) operably linked to the whole or part of the transmembrane / membrane anchoring domain of a second non-TCR signaling receptor chain or a variant thereof via a VL, Vα, or Vγ domain of the first antigen-binding domain and a VH, Vβ, or Vδ domain of the second antigen-binding domain form an Fv or TCR-Fv-like antigen-binding module that specifically binds to a target antigen. In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a vL domain operably linked to all or part of the transmembrane / membrane anchoring domain of a non-TCR signaling receptor chain or a variant thereof via an optional peptide linker; and b) a vH domain operably linked to all or part of the transmembrane / membrane anchoring domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the vL and vH domains form an Fv-like antigen binding module that specifically binds to a target antigen in an MHC-dependent and / or MHC-independent manner.In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a Vα domain operably linked to the entire or partial transmembrane / membrane anchoring domain of a non-TCR signaling receptor or a variant thereof via an optional peptide linker; and b) a Vβ domain operably linked to the entire or partial transmembrane / membrane anchoring domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the Vα and Vβ domains form a TCR-Fv-like antigen-binding module that specifically binds to a peptide / MHC complex in an MHC-dependent manner. In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a Vγ domain operably linked to the entire or partial transmembrane / membrane anchoring domain of a non-TCR signaling receptor or a variant thereof via an optional peptide linker; and b) a Vδ domain operably linked to the entire or partial transmembrane / membrane anchoring domain of a second non-TCR signaling receptor or a variant thereof via an optional peptide linker, wherein the Vγ domain and the Vδ domain form a TCR-Fv-like antigen binding module that specifically binds to an antigen in an MHC-dependent or MHC-independent manner. In exemplary embodiments, the non-TCR signaling receptor is selected from, but is not limited to, one or more of the following: CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, and ILT2. In some embodiments, the first and / or second peptide linker individually comprise a constant domain or fragment thereof derived from an immunoglobulin or a T-cell receptor subunit.In some embodiments, the first and / or second peptide linker individually comprises a CH1, CH2, CH3, CH4 or CL antibody domain, or a fragment thereof. In some embodiments, the first and / or second peptide linker individually comprises a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In some embodiments, the first and / or second peptide linkers individually consist of an immunoglobulin-derived Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3560, SEQ ID NOs: 3536-3551), or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.) or fragments thereof. In some embodiments, the first and / or second peptide linkers contain mutations that increase their expression, affinity, and chain pairing. In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) attached at or near the N-terminus of the first antigen-binding domain (e.g., vL, Vα, or Vγ) and / or the second antigen-binding domain (e.g., vH, Vβ, or Vδ).

[0316] In one embodiment, the present disclosure provides a double-chain SAR that specifically binds to an antigen, comprising: a) a first antigen-binding domain operably linked to the entire or partial transmembrane / membrane anchoring domain of a non-TCR signaling receptor chain or signaling adaptor, or a variant or fragment thereof, via an optional peptide linker; and b) a second antigen-binding domain operably linked to the entire or partial transmembrane / membrane anchoring domain of a second non-TCR signaling receptor chain or signaling adaptor, or a variant or fragment thereof, via an optional peptide linker. In some embodiments, the first and / or second peptide linkers individually comprise a constant domain or fragment thereof derived from an immunoglobulin or T cell receptor subunit. In some embodiments, the first and / or second peptide linkers individually comprise a CH1, CH2, CH3, CH4, or CL antibody domain, or a fragment thereof. In some embodiments, the first and / or second peptide linkers individually comprise a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In one embodiment, the first antigen-binding domain and the second antigen-binding domain specifically bind to their respective target antigens. In one embodiment, one or both of the antigen-binding domains are autonomous antigen-binding domains (e.g., AABDs, e.g., vHH, FHVH, SVH, centilin, DARPIN, svd-TCR, adaptors, adaptor-binding domains, ligand-binding domains of receptors, receptor-binding domains of ligands, etc.). In one embodiment, one or both of the antigen-binding domains comprise an antibody, an antibody fragment (e.g., Fab), scFv, TCR, or scTCR. In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) bound at or near the N-terminus of the first and / or second antigen-binding domain. In one embodiment, the first and / or second signaling chains comprise a cytosolic domain comprising an activation domain and an optional costimulatory domain.

[0317] In one embodiment, the double-chain SAR retains all or part of the binding properties of the original signaling receptor (e.g., a non-T cell receptor) and also acquires binding properties conferred by one or more heterologous antigen-binding domains. In one embodiment, the double-chain SAR retains all or part of the binding properties of the original signaling receptor. In one embodiment, the double-chain SAR further acquires binding properties conferred by one or more heterologous antigen-binding domains. In one embodiment, the double-chain SAR retains the signaling properties of two signaling receptors. In one embodiment, the double-chain SAR acquires new signaling properties that are not exhibited by either of the two signaling receptors when activated alone. In one embodiment, the double-chain SAR acquires new signaling properties that are additive to the signaling properties of the two signaling receptors when activated alone. In one embodiment, the double-chain SAR acquires new signaling properties that are synergistic of the signaling properties of the two signaling receptors when activated alone.

[0318] In one embodiment, the signaling receptor comprising one or both chains of the double-chain SAR is a type I membrane protein having a single transmembrane domain. In one embodiment, the signaling receptor is a naturally occurring signaling receptor. In one embodiment, the signaling receptor is a non-TCR signaling receptor. In one embodiment, one or both chains of the double-chain SAR can recruit a signaling adaptor (e.g., CD3ζ, FcRγ, DAP10, or DAP12, etc.). In one embodiment, one or both chains of the double-chain SAR can recruit a signaling adaptor comprising an activation domain. In one embodiment, one or both chains of the double-chain SAR can recruit a signaling adaptor comprising one or more ITAMs. In one embodiment, one or both chains of the double-chain SAR can recruit a signaling adaptor comprising one or more ITIMs. In one embodiment, one or both chains of the double-chain SAR can recruit a signaling adaptor comprising a costimulatory domain. In one embodiment, the signaling adaptor is naturally occurring. In one embodiment, the signaling adaptor is non-naturally occurring. In one embodiment, one or both chains of the double-stranded SAR can recruit a signaling adaptor that activates an intracellular signaling pathway (e.g., NFAT, NF-κB, ERK, PI3K, etc.). In one embodiment, one or both chains of the double-stranded SAR can recruit a signaling adaptor that inhibits an intracellular signaling pathway (e.g., NFAT, NF-κB, ERK, PI3K, etc.).

[0319] In one embodiment, one or both chains of the double-chain SAR comprise a costimulatory domain. In one embodiment, one or both chains of the double-chain SAR comprise an activation domain and a costimulatory domain. In one embodiment, one or both chains of the double-chain SAR comprise an intracellular activation domain. In an exemplary embodiment, one or both chains of the double-chain SAR comprise an intracellular activation domain derived from a signaling adaptor. In an exemplary embodiment, one or both chains of the double-chain SAR comprise an intracellular activation domain derived from CD3ε, FcRγ, DAP10, or DAP12. In one embodiment, the activation domain present in one or both chains of the double-chain SAR comprises one or more ITAMs. In one embodiment, one or both chains of the double-chain SAR comprise an activation domain comprising one or more ITAMs. In one embodiment, one or both chains of the double-chain SAR comprise an activation domain comprising two or more ITAM motifs. In one embodiment, one or both chains of the double-chain SAR comprise an activation domain comprising a single ITAM. In one embodiment, one or both chains of the double-chain SAR lack an ITAM. In one embodiment, one or both chains of the double-chain SAR comprise an activation domain containing a tyrosine-based motif (YINM). In one embodiment, one or both chains of the double-chain SAR comprise an activation domain that recruits the p85 subunit of PI3K and / or Grb2. In one embodiment, one or both chains of the double-chain SAR comprise an activation domain that activates one or more of the NFAT, PI3K, NF-κB and / or ERK signaling pathways.

[0320] In one embodiment, the SAR comprises an intracellular inhibitory domain. In an exemplary embodiment, the SAR comprises an intracellular inhibitory domain derived from PD1. In one embodiment, the inhibitory domain of the SAR comprises one or more ITIM motifs.

[0321] In one embodiment, the SAR can recruit signaling adaptors. In an exemplary embodiment, the SAR can recruit one or more signaling adaptors selected from the group consisting of, but not limited to, CD3ζ, FcRγ, DAP10, and DAP12.

[0322] In one embodiment, the present disclosure provides a double-chain SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to two polypeptide chains, at least one of which is capable of recruiting a signaling adaptor. In one embodiment, the present disclosure provides a double-chain SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to two polypeptide chains, each of which is capable of recruiting a signaling adaptor. In one embodiment, one or both polypeptides comprise a hinge domain, a transmembrane domain, and a cytosolic domain. In one embodiment, at least one polypeptide comprises a transmembrane domain. In one embodiment, both polypeptides comprise a transmembrane domain. In one embodiment, at least one polypeptide comprises a cytosolic domain. In one embodiment, both polypeptides comprise a cytosolic domain. In one embodiment, the present disclosure provides a double-chain SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to two polypeptide chains, at least one of which is capable of recruiting a signaling adaptor. In one embodiment, the present disclosure provides a double-chain SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to two polypeptide chains comprising one or more intracellular activation domains. In one embodiment, the present disclosure provides a double-chain SAR comprising one or more heterologous antigen-binding domains operably linked via an optional linker to two polypeptide chains, at least one of which comprises an intracellular activation domain.

[0323] In one embodiment, the signaling adaptor is a non-TCR / CD3 signaling adaptor. In one embodiment, the signaling adaptor is not a component of the TCR / CD3 signaling complex. In one embodiment, the signaling adaptor is not CD3ε. In one embodiment, the signaling adaptor is a non-native signaling adaptor, i.e., a signaling adaptor that does not occur in nature. In one embodiment, the signaling adaptor comprises one or more ITAMs. In one embodiment, the signaling adaptor comprises one or more ITIMs. In one embodiment, the signaling adaptor is a disulfide-bonded dimeric protein. In one embodiment, the signaling adaptor is a type I transmembrane protein. In one embodiment, the signaling adaptor is capable of recruiting a signaling protein, e.g., a protein kinase, e.g., ZAP70. In one embodiment, the signaling adaptor is capable of activating one or more cell signaling pathways, e.g., NFAT, NF-κB, ERK, PI3K, etc.

[0324] In one embodiment, the two non-TCR signaling receptor chains constituting the two chains of the double-chain SAR are of the same type and sequence (e.g., both signaling chains consist of the extracellular, transmembrane, and cytoplasmic domains of CD16A, NKp46, or NKp30, etc.). Exemplary such double-chain SARs are represented by SEQ ID NOs: 6383-6293, 4675, 4696, and 8344. In one embodiment, the two signaling chains of the double-chain SAR are of different types (e.g., one chain consists of the extracellular, transmembrane, and cytoplasmic domains of CD16A, and the second chain consists of the extracellular, transmembrane, and cytoplasmic domains of NKp30, or the hinge, transmembrane, and cytoplasmic domains of CD16A, and the second chain consists of the hinge, transmembrane, and cytoplasmic domains of CD3ζ, etc.). Examples of such SARs are represented by SEQ ID NOs: 4695 and 4670.

[0325] In one embodiment, the two signaling chains of the double-chain SAR are derived from the same receptor (e.g., both chains are derived from CD16A). An example of such a SAR is represented by SEQ ID NO: 4676. In one embodiment, the two signaling chains of the double-chain SAR are derived from different receptors (e.g., one chain is derived from NKp44 and the second chain is derived from NKp30). An example of such a SAR is represented by SEQ ID NO: 4713.

[0326] In one embodiment, the present disclosure provides a dual-chain SAR comprising a first chain derived from a non-TCR receptor signaling chain (e.g., CD16A) and a second chain derived from a signaling adaptor (e.g., CD3ε or FcRγ). An example of such a SAR is represented by SEQ ID NO: 4670.

[0327] In one embodiment, a double-chain SAR can be composed of a first chain derived from a non-TCR receptor signaling chain (e.g., CD16A) and a second chain derived from a TCR signaling chain (e.g., TCRα, TCRβ, TCRγ, TCRδ, preTCRα constant chain, or a variant or fragment thereof). In some embodiments, the present disclosure provides a double-chain SAR comprising one non-TCR module (or NTCRM) and one TCR module (or TCRRM). Examples of such SARs are represented by SEQ ID NOs: 4708-4710.

[0328] In one embodiment, the optional linker between the vL / vH, Vα / Vβ, Vγ / Vδ chains of the heterologous antigen-binding domain and the non-TCR signaling chain is an Ig-like linker depicted in Table 13 (SEQ ID NOs(DNA)1142-1175 and SEQ ID NOs(PRT):3536-3569).

[0329] In one embodiment, the signal transduction receptor used in the construction of the double-chain SAR is any receptor expressed on the surface of immune cells.In one embodiment, the signal transduction receptor is the signal transduction chain of a naturally occurring signal transduction receptor expressed on the surface of immune cells.In an exemplary embodiment, the immune cells are selected from, but not limited to, T cells, NK cells, monocytes / macrophages, granulocytes and B cells. Exemplary signal receptors that can be used in the construction of duplex SARs of the present disclosure include CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2D, NKG2C, NKG2A, NKG2E, NKG2F, DNAM-1, 2B4, OX40, CD28, 4-1BB, CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, CEACAM, ILT2, KLRG1, LAIR1, CD161, Siglec3, Siglec-7, Siglec-9, etc., or their mutants. In further embodiments, autonomous antigen-binding domains (e.g., fully human vH domains, vHHs, single-chain TCRs, etc.), non-immunoglobulin antigen-binding domains (e.g., centilin, affibodies, etc.), ligands (e.g., APRIL, TPO, NKG2D-YA-G4Sx3-NKG2D-YA, etc.), and extracellular domains of receptors (e.g., NKp30, NKp44, NKp46, NKG2D, CD16A, etc.), adaptor-binding domains (e.g., EZip, RZip, E4, R4, etc.) can be operably linked to or near the amino terminus of the vL, vH, Vα, Vβ, Vγ, or Vδ chain of the SAR to confer additional antigen-binding capability to the SAR.

[0330] In some embodiments, a SAR (e.g., an isolated SAR) that specifically binds to a target antigen is provided, wherein the SAR comprises: a) a first polypeptide chain comprising a first antigen-binding domain consisting of a vL, Vα, or Vγ domain and a first membrane-associated module (MAM); and b) a second polypeptide chain comprising a second antigen-binding domain consisting of a vH, Vβ, or Vδ domain and a second membrane-associated module (MAM), wherein the vL, Vα, or Vγ domain of the first antigen-binding domain and the complementary vH, Vβ, or Vδ domain of the second antigen-binding domain form an Fv or TCR-Fv-like antigen-binding module that specifically binds to the target antigen, and the first MAM and / or the second MAM form a non-T cell receptor module (NTCRM). In one embodiment, the SAR is capable of activating at least one signaling pathway and / or recruiting at least one signaling adaptor. In one embodiment, the first and / or second MAM is derived from one or more of the following signaling adaptors, including, but not limited to, CD3ζ, FcRγ, DAP10, or DAP12, or a variant or fragment thereof. In one embodiment, the NTCRM is composed of one or more of the following signaling adaptors, including, but not limited to, CD3ζ, FcRγ, DAP10, or DAP12. In some embodiments, the signaling adaptor is a non-CD3 adaptor (NCAM). In some embodiments, the signaling adaptor is not CD3ε. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked via one or more disulfide bonds. In some embodiments, the first polypeptide chain further comprises a first peptide linker between the first antigen-binding domain and the first MAM. In some embodiments, the second polypeptide chain further comprises a second peptide linker between the second antigen-binding domain and the second MAM. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked via one or more disulfide bonds. In some embodiments, the first peptide linker and the second peptide linker are linked via one or more disulfide bonds.In some embodiments, the first peptide linker and / or the second peptide linker are each about 5 to about 500 amino acids in length. In some embodiments, the first and / or second peptide linker individually comprise a constant domain derived from an immunoglobulin or a T cell receptor subunit, or a fragment thereof. In some embodiments, the first and / or second peptide linker individually comprise a CH1, CH2, CH3, CH4, or CL antibody domain, or a fragment thereof. In some embodiments, the first and / or second peptide linker individually comprise a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In some embodiments, the first and / or second peptide linker comprise mutations that increase their affinity and chain pairing. In some embodiments, the first and / or second linker individually comprise an immunoglobulin-derived Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3560, SEQ ID NOs: 3536-3551), or a TCR-Ig-like linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.) or fragments thereof. In some embodiments, the first and / or second peptide linkers comprise mutations that increase their expression, affinity, and chain pairing. In one embodiment, the first and second antigen-binding domains comprise complementary chains (e.g., vL and vH, Vα and Vβ, or Vγ and Vδ). In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) attached to or near the N-terminus of the first antigen-binding domain (e.g., vL, Vα, or Vγ) and / or the second antigen-binding domain (e.g., vH, Vβ, or Vδ). In some embodiments, the SAR binds to the target antigen with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM.In some embodiments, the target antigen is a cell surface antigen. In exemplary embodiments, the target antigen is one or more of the antigens listed in Table B. In some embodiments, the cell surface antigen is selected from the group consisting of proteins, carbohydrates, and lipids. In some embodiments, the cell surface antigen is CD19, CD20, CD22, CD33, CD70, CD123, CD138, CLL-1, FLT3, claudin 18.2, BCMA, GCC, MPL, SLAMF7, ROR1, ROR2, GPRC5D, FCRL5, MSLN, EGFR, EGFRviii, PSMA, PSCA, KLK2, IL13Ra2, TROP2, PTK7, DLL3, Mucl, Mucl6, or Her2. In some embodiments, the target antigen is a complex comprising a peptide and a major histocompatibility complex (MHC) protein. In exemplary embodiments, the peptide / MHC complex comprises a peptide derived from one or more of NY-ESO-1, MAGE-A2, MAGE-A3, MAGE4, ​​WT1, AFP, TERT, MART-1, pp66-CMV, HPV16-E7, PRAME, EBV-LMP2A, HIV-1, PSA, or gp100.

[0331] In some embodiments, according to any of the above-described SARs (e.g., isolated SARs), the first MAM further comprises a first hinge domain or a fragment thereof N-terminal to the first transmembrane domain, and the second MAM further comprises a second hinge domain or a fragment thereof N-terminal to the second transmembrane domain. In some embodiments, the NTCRM comprises a disulfide bond between a residue in the first hinge domain and a residue in the second hinge domain. In some embodiments, the first MAM further comprises a first cytosolic domain C-terminal to the first transmembrane domain. In some embodiments, the second MAM further comprises a second cytosolic domain C-terminal to the second transmembrane domain. In one embodiment, the first and / or second cytosolic domain is an activation domain comprising one or more ITAMs. In some embodiments, the SAR binds to a target antigen with an equilibrium dissociation constant (Kd) of about 0.1 pM to about 500 nM.

[0332] In some embodiments, according to any of the above SARs (e.g., isolated SARs), the first polypeptide chain further comprises a first costimulatory domain C-terminal to the first transmembrane domain. In some embodiments, the second polypeptide chain further comprises a second costimulatory domain C-terminal to the second transmembrane domain. In some embodiments, the first polypeptide chain further comprises a first signaling peptide N-terminal to the first antigen-binding domain. In some embodiments, the second polypeptide chain further comprises a second signaling peptide N-terminal to the second antigen-binding domain.

[0333] In one embodiment, the present disclosure provides an Fv or TCR-Fv-like antigen binding module comprising: a) a vL (variable domain of an antibody light chain), Va (variable domain of TCR alpha or Valpha), or Vg (variable domain of TCRg or Vgamma) domain operably linked via any peptide linker to the whole or partial hinge domain of a signaling adaptor or variant thereof; and b) a second antigen binding domain, a vH (variable domain of an antibody heavy chain), Vb (variable domain of TCRb or Vbeta), or Vd domain (variable domain of TCRd or Vdelta), operably linked via any peptide linker to the whole or partial hinge domain of the second signaling adaptor or variant thereof, wherein the vL, Valpha, or Vgamma domain of the first antigen binding domain and the complementary vH, Vbeta, or Vdelta domain of the second antigen binding domain are formed. In one embodiment, the present disclosure provides a) a vL domain operably linked via an optional peptide linker to a whole or partial hinge domain of a signaling adaptor or variant thereof; and b) a vH domain operably linked via an optional peptide linker to a whole or partial hinge domain of a second signaling adaptor or variant thereof, wherein the vL and vH domains form an Fv-like antigen binding module that specifically binds to detect a target antigen in an MHC-dependent and / or MHC-independent manner. In one embodiment, the present disclosure provides a) a Vα domain operably linked via an optional peptide linker to a whole or partial hinge domain of a signaling adaptor or variant thereof; and b) a Vβ domain operably linked via an optional peptide linker to a whole or partial hinge domain of a second signaling adaptor or variant thereof, wherein the Vα and Vβ domains form a TCR-Fv-like antigen binding module that specifically binds to a peptide / MHC complex in an MHC-dependent manner.In one embodiment, the present disclosure provides a signaling adaptor comprising: a) a Vγ domain operably linked via an optional peptide linker to a full or partial hinge domain of a signaling adaptor or variant thereof; and b) a Vδ domain operably linked via an optional peptide linker to a full or partial hinge domain of a second signaling adaptor or variant thereof, wherein the Vγ and Vδ domains form a TCR-Fv-like antigen binding module that specifically binds to an antigen in an MHC-dependent or MHC-independent manner. In exemplary embodiments, the signaling adaptor is selected from one or more of the following, but is not limited to: CD3ε, FcRγ, DAP10, or DAP10, or a variant or fragment thereof. In some embodiments, the signaling adaptor is a non-CD3 adaptor (NCAM). In some embodiments, the signaling adaptor is not CD3ε. In some embodiments, the first and / or second peptide linkers independently comprise a constant domain or fragment thereof derived from an immunoglobulin or a T cell receptor subunit. In some embodiments, the first and / or second peptide linker individually comprises a CH1, CH2, CH3, CH4, or CL antibody domain, or a fragment thereof. In some embodiments, the first and / or second peptide linker individually comprises a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In some embodiments, the first and / or second linker individually comprises an Ig-like linker (e.g., IgCL, IgCH1, etc.) or a TCR-Ig-like linker derived from an immunoglobulin (e.g., SEQ ID NOs: 3536-3551) (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.) or variant, or fragment thereof. In some embodiments, the first and / or second peptide linker comprises mutations that increase their expression, affinity, and chain pairing. In some embodiments, the signaling adaptor further comprises one or more costimulatory domains.In exemplary embodiments, the signaling adaptor comprises a costimulatory domain, or a variant or fragment thereof, from CD28, 4-1BB, OX40, 2B4, CD27, CD81, CD2, CD5, BAFF-R, CD30, CD40, HVEM, or ICOS. In some embodiments, the first and / or second peptide linker individually comprises an Ig-like linker derived from an immunoglobulin (e.g., SEQ ID NOs: 3536-3551) or a TCR-Ig-like linker (e.g., IgCL, IgCH1, etc.), a linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.) or a fragment thereof. In some embodiments, the first and / or second peptide linker comprises mutations that increase their expression, affinity, and chain pairing. In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) attached at or near the N-terminus of the first polypeptide chain (e.g., vL, Vα, or Vγ) and / or the second (e.g., vH, Vβ, or Vδ) antigen-binding domain.

[0334] In one embodiment, the present disclosure provides a duplex SAR that specifically binds to an antigen comprising: a) a first antigen-binding domain comprising a vL, Vα, or Vγ domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a signaling adaptor or variant thereof; and b) a second antigen-binding domain comprising a vH, Vβ, or Vδ domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a second signaling adaptor or variant thereof, wherein the vL, Vα, or Vγ domain of the first antigen-binding domain and the complementary vH, Vβ, or Vδ domain of the second antigen-binding domain form an Fv or TCR-Fv-like antigen-binding module that specifically binds to a target antigen. In one embodiment, the present disclosure provides a TCR-Fv-like antigen binding module comprising: a) a vL domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a signaling adaptor or variant thereof; b) a vH domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a second signaling adaptor or variant thereof, wherein the vL and vH domains form an Fv-like antigen binding module that specifically binds to a target antigen in an MHC-dependent and / or MHC-independent manner. In one embodiment, the present disclosure provides a TCR-Fv-like antigen binding module comprising: a) a Vα domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a signaling adaptor or variant thereof; and b) a Vβ domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a second signaling adaptor or variant thereof, wherein the Vα and Vβ domains form an Fv-like antigen binding module that specifically binds to a peptide / MHC complex in an MHC-dependent manner.In one embodiment, the present disclosure provides a Vδ domain comprising: a) a Vγ domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a signaling adaptor or a variant thereof; and b) a Vδ domain operably linked via an optional peptide linker to a full or partial transmembrane / membrane anchoring domain of a second signaling adaptor or a variant thereof, wherein the Vγ and Vδ domains form a TCR-Fv-like antigen binding module. The Vδ domain specifically binds to an antigen in an MHC-dependent or MHC-independent manner. In exemplary embodiments, the signaling adaptor is selected from one or more of the following, but is not limited to: CD3ε, FcRγ, DAP10, or DAP12, or variants or fragments thereof. In some embodiments, the signaling adaptor is a non-CD3 adaptor (NCAM). In some embodiments, the signaling adaptor is not CD3ε. In some embodiments, the signaling adaptor further comprises one or more costimulatory domains. In exemplary embodiments, the signaling adaptor comprises a costimulatory domain from CD28, 4-1BB, OX40, 2B4, CD27, CD81, CD2, CD5, BAFF-R, CD30, CD40, HVEM, or ICOS, or a variant or fragment thereof. In some embodiments, the first and / or second peptide linker individually comprise a constant domain or fragment thereof derived from an immunoglobulin or T cell receptor subunit. In some embodiments, the first and / or second peptide linker individually comprise a CH1, CH2, CH3, CH4, or CL antibody domain, or a fragment thereof. In some embodiments, the first and / or second peptide linker individually comprise a Cα, Cβ, Cγ, or Cδ TCR domain, or a variant or fragment thereof. In some embodiments, the first and / or second peptide linker individually comprises an immunoglobulin (e.g., SEQ ID NOs: 3536-3551) or an Ig-like linker derived from a TCR-Ig-like (e.g., IgCL, IgCH1, etc.).a linker (e.g., TCRb-Ig3, SEQ ID NO: 3560; TCRa-Ig3, SEQ ID NO: 3562; TCRg-Ig3, SEQ ID NO: 3566; or TCRd-Ig3, SEQ ID NO: 3568, etc.) or a variant or fragment thereof. In some embodiments, the first and second polypeptide chains further comprise one or more autonomous antigen-binding domains (AABDs) attached at or near the N-terminus of the first polypeptide chain (e.g., vL, Vα, or Vγ) and / or the second (e.g., vH, Vβ, or Vδ) antigen-binding domain.

[0335] In one embodiment, the present disclosure provides a SAR comprising: a) one or more heterologous antigen-binding domains operably linked via an optional linker to at or near the amino terminus of one chain of a signaling adaptor (or signaling chain) or a variant thereof; and b) one or more heterologous antigen-binding domains operably linked via an optional linker to at or near the amino terminus of a second chain of the signaling adaptor (or signaling chain) or a variant thereof. In one embodiment, such a SAR retains the signaling capability of the original signaling adaptor (or signaling chain). In one embodiment, the SAR also acquires the binding capability conferred by the heterologous antigen-binding domain. In an exemplary embodiment, the signaling adaptor is any signaling adaptor (or signaling chain) that can be expressed on the plasma membrane of a cell (e.g., an immune cell, e.g., an immune effector cell). In an exemplary embodiment, the immune cell is selected from, but not limited to, a T cell, a NK cell, a monocyte / macrophage, a granulocyte, or a B cell. Exemplary signaling adaptors (or signaling chains) that can be used in constructing the SARs of the present disclosure include, but are not limited to, CD3z (CD3ζ), FcRγ (FcεRIγ), DAP10 and DAP12, etc., or variants thereof.

[0336] The present disclosure provides SARs in which one or more heterologous antigen-binding domains are operably linked to the extracellular domain (e.g., hinge or spacer domain) of one or more chains of a signaling adaptor. In one embodiment, the SAR comprises a signaling adaptor that is a component of a TCR complex (e.g., CD3ε, CD3ε, CD3γ, CD3ε, etc.). In one embodiment, the SAR comprises a signaling adaptor (e.g., CD3ζ) that interacts with the TCR α, β, γ, and / or δ chains of the TCR complex. In one embodiment, the SAR comprises a signaling adaptor that does not interact with the TCR α, β, γ, and / or δ chains of the TCR complex. In one embodiment, the SAR comprises a signaling adaptor that has a conserved aspartic acid residue in its transmembrane domain that interacts with a positively charged residue (lysine or arginine) in the transmembrane region or TCR α, TCR β, TCR γ, or TCR δ. In one embodiment, the SAR comprises a signaling adaptor that lacks a conserved aspartic acid residue in its transmembrane domain. In one embodiment, the SAR comprises a signaling adaptor that is not a component of the TCR complex. In one embodiment, the signaling adaptor is a non-CD3 signaling adaptor (NCAM). In one embodiment, the signaling adaptor is not CD3ε or a variant thereof.

[0337] In some embodiments, according to any of the SARs described above (e.g., isolated SARs), the SAR comprises a signaling adaptor that activates cell signaling (e.g., CD3ζ). In one embodiment, the SAR comprises a signaling adaptor that inhibits cell signaling. In one embodiment, the SAR comprises a signaling adaptor having one or more ITAM motifs (e.g., CD3ζ). In one embodiment, the SAR comprises a signaling adaptor having two or more ITAM motifs. In one embodiment, the SAR comprises a signaling adaptor having a single ITAM motif (e.g., FcRγ). In one embodiment, the SAR comprises a signaling adaptor that lacks an ITAM motif. In one embodiment, the SAR comprises a signaling adaptor that comprises a tyrosine-based motif (YINM) (e.g., DAP10). In one embodiment, the SAR comprises a signaling adaptor that recruits the p85 subunit of PI3K and / or Grb2 (e.g., DAP10). In one embodiment, the SAR comprises a signaling adaptor that is a native disulfide linker dimer. In one embodiment, the signaling adaptor is not a disulfide linker dimer in its native form. In one embodiment, the SAR comprises a signaling adaptor (e.g., CD3ζ) that contains an interchain disulfide bond located in a transmembrane region in its native state. In one embodiment, the SAR comprises a signaling adaptor (e.g., DAP10 and DAP12) that contains an interchain disulfide bond that is not located in a transmembrane region in its native state. In one embodiment, the SAR comprises a signaling adaptor (e.g., DAP10 and DAP12) that contains an interchain disulfide bond located in its extracellular region in its native state.

[0338] In some embodiments, according to any of the above SARs (e.g., isolated SARs), the extracellular domain of the signaling adaptor is less than 10 amino acids in length. In one embodiment, the extracellular domain of the signaling adaptor is less than 8 amino acids in length. In one embodiment, the extracellular domain of the signaling adaptor is more than 10 amino acids in length. In one embodiment, the extracellular domain of the signaling adaptor is more than 15 amino acids in length.

[0339] In some embodiments, according to any of the above SARs (e.g., isolated SARs), the SAR comprises a signaling adaptor that induces protein phosphorylation. In one embodiment, the SAR comprises a signaling adaptor that induces protein dephosphorylation. In one embodiment, the SAR comprises a signaling adaptor that interacts with Zap70. In one embodiment, the SAR comprises a signaling adaptor that does not interact with Zap70. In one embodiment, the two chains of the double-chain SAR comprise identical signaling adaptors (e.g., CD3ζ and CD3ζ). In one embodiment, the two chains of the double-chain SAR comprise non-identical signaling adaptors (e.g., CD3ε and FcRγ, or CD3ε and DAP10, or DAP10 and DAP12, etc.).

[0340] In some embodiments, according to any of the above-described double-chain SARs (e.g., isolated SARs) comprising a signaling adaptor, one or both chains of the double-chain SAR comprise a signaling adaptor comprising a costimulatory domain (e.g., a costimulatory domain derived from 4-1BB, CD28, 2B4, OX40, etc.). In one embodiment, one or both chains of the double-chain SAR comprise a signaling adaptor comprising a costimulatory domain (e.g., a costimulatory domain derived from 4-1BB, CD28, 2B4, OX40, etc.) operably linked to an activation domain (e.g., a CD3ζ activation domain). Exemplary CD3ζ signal adaptors linked to the costimulatory domains of CD28 and 4-1BB are shown in SEQ ID NOs: 3493 and 3494, respectively. Exemplary SARs consisting of the costimulatory domain of OX40 fused to the activation domain of CD3ζ are represented by SEQ ID NOs: 4460 and 4479. In one embodiment, one or both chains of the dual-chain SAR comprise a signaling adaptor comprising a fusion of the cytoplasmic domains of two different signaling adaptors. An exemplary such SAR comprising a chain comprising a fusion of the cytoplasmic domains of DAP10 and CD3ζ is represented by SEQ ID NO: 4460. In one embodiment, the SAR comprises a signaling adaptor comprising mutants of CD3z (or CD3ζ), FcRγ, DAP10, and DAP12 with mutations that eliminate interchain disulfide bonds. Exemplary such signaling adaptors are represented by SEQ ID NOs: 3747, 3753, 3760, 3817, and 3820. In one embodiment, the signaling chain comprises mutants of CD3z, FcRγ, DAP10, and DAP12 (e.g., an IXX variant of CD3z) with one or more mutations in their ITAM motifs. An exemplary such signaling adaptor is represented by SEQ ID NO: 9824.

[0341] Exemplary antigen-binding domains that can be used in constructing the two-chain SARs of the present disclosure include antibody variable domains (e.g., vL, vH), TCR variable domains (e.g., Va, Vb, Vg, or Vd chains), etc.), antibodies, antibody fragments (e.g., Fab, Fab2), autonomous antigen-binding domains (e.g., fully human vH domains, vHH, single-chain TCRs, svd-TCRs, etc.), scFv, non-immunoglobulin antigen-binding domains (e.g., centuirin, affibodies, ZIP domains, adaptors, etc.), ligands, extracellular domains of receptors, autoantigens, TCRs, HLA-independent TCRs, TCR variable domains (e.g., Va, Vb, Vg, etc.). In further embodiments, autonomous antigen-binding domains (e.g., fully human vH domains, vHH, single-chain TCRs, etc.), non-immunoglobulin antigen-binding domains (e.g., centuirin, affibodies, etc.). ), ligands (e.g., APRIL, TPO, NKG2D-YA-G4Sx3-NKG2D-YA, etc.), and extracellular domains of receptors (e.g., NKp30, NKp44, NKp46, NKG2D, CD16A, etc.), adaptor binding domains (e.g., EZip, RZip, E4, R4, etc.) can be functionally linked to or near the amino terminus of the vL, vH, Vα, Vβ, Vγ, or Vδ chain of the SAR to confer additional antigen binding capability to the SAR.

[0342] In one embodiment, the two signaling adaptors of a double-chain SAR are of the same type (e.g., both chains are derived from CD3ε). An example of such a SAR is represented by SEQ ID NO: 4702. In one embodiment, the two signaling adaptors comprising the double-chain SAR are of different types (e.g., one signaling adaptor is derived from CD3ε and the second adaptor is derived from FcRγ, etc.). An example of such a SAR is represented by SEQ ID NO: 6733.

[0343] In one embodiment, a double-chain SAR can comprise one chain derived from a non-TCR receptor signaling chain (e.g., CD16A) and another chain derived from a signaling adaptor (e.g., CD3ζ or FcRγ). An example of such a SAR is represented by SEQ ID NO:4670.

[0344] In one embodiment, the double-chain SAR may comprise one chain comprising a signaling adaptor (eg, CD3ε) and another chain comprising a TCR constant chain (eg, TCRα-T48C).

[0345] In one embodiment, the optional linker is a long linker. In one embodiment, the optional linker between the vL / vH, Vα / Vβ, Vγ / Vδ chains of the heterologous antigen-binding domain and the non-TCR signaling chain is an Ig-like linker (SEQ ID NOs: (DNA): 1142-1175) and (PRT): 3536-3569) shown in Table 13.

[0346] In some embodiments, the present disclosure provides cells that are not T cells but have T cell target recognition properties and functions. In one embodiment, the present disclosure provides cells that are not T cells (i.e., non-T cells) that express a receptor that confers T cell receptor-like target recognition and / or signaling to the cell. In one embodiment, the present disclosure provides cells that are not T cells (i.e., non-T cells) that express a dual- or multi-chain receptor that confers T cell receptor-like target recognition and / or signaling to the cell. Transformation. In one embodiment, the dual- or multi-chain receptor comprises at least two membrane-associated domains (e.g., transmembrane domains or membrane anchor domains). In one embodiment, the dual- or multi-chain receptor comprises at least two transmembrane domains. In one embodiment, T cell receptor-like recognition comprises specific binding to a peptide target presented by an MHC molecule. In one embodiment, the cells that are not T cells (i.e., non-T cells) lack expression of T cell chains and / or lack expression of a functional TCR chain. In one embodiment, the cells that are not T cells (i.e., non-T cells) lack expression of a functional TCR / CD3 complex. In one embodiment, the cell that is not a T cell (i.e., the non-T cell) lacks expression of one or more of CD3ε, CD3γ, and CD3δ, or variants or fragments thereof. In one embodiment, the cell that is not a T cell (i.e., the non-T cell) has not been engineered to exogenously express one or more CD3ε, CD3γ, and CD3δ chains, or variants or fragments thereof. In one embodiment, the cell that is not a T cell (i.e., the non-T cell) has not been engineered to exogenously express one or more TCR chains, or variants or fragments thereof. In one embodiment, the cell that is not a T cell (i.e., the non-T cell) is not activated by a CD3 agonist antibody. In one embodiment, the cell that is not a T cell (i.e., the non-T cell) is not activated by an OKT3 antibody.

[0347] In one embodiment, the present disclosure provides non-T cells with T cell receptor-like target recognition generated from NK cells, g-NK cells, memory-like NK cells, cytokine-induced killer cells (CIKs), iPSCs, modified HLA-deficient iPSCs, iPSC-derived NK cells, iPSC-derived T cells, B cells, macrophages / monocytes, granulocytes, dendritic cells, immortalized cell lines, immortalized NK cell lines, NK92 cell lines, NK92MI cell lines, or derivatives thereof. In one embodiment, the present disclosure provides non-T cells with T cell receptor-like target recognition generated after introduction of a single receptor into cells that are not functional T cells. In one embodiment, the present disclosure provides non-T cells with T cell receptor-like target recognition generated without genetic modification involving ectopic expression of the four CD3 chains, namely, CD3ε, CD3γ, CD3δ, and CD3ε. Functional T cells.

[0348] For example, non-T cells expressing a SAR (e.g., uTCR-SAR) result in the recruitment of at least one signaling adaptor upon specific binding to a target antigen. In one embodiment, non-T cells expressing a dual-chain receptor result in the activation of at least one signaling pathway upon specific binding to a target antigen. In an exemplary embodiment, the signaling pathway is selected from the group of, but not limited to, the NFAT, NF-κB, PI3K, or ERK pathways. In one embodiment, non-T cells expressing a dual-chain receptor result in the activation of at least one biological activity upon binding to a target antigen. In one embodiment, the biological activity is selected from the group of, but not limited to, cell activation, proliferation, differentiation, cytokine secretion, phagocytosis, migration, or cytotoxicity.

[0349] In another aspect, the present disclosure provides modified cells, such as, but not limited to, natural killer (NK) cells, having major histocompatibility complex (MHC)-restricted antigen-specific cytotoxicity. The MHC can be any of MHC class I, MHC class II, and MHC-like molecules. A non-limiting example of an MHC-like molecule is HLA-E.

[0350]

[0003] A method is provided for producing modified cells, such as, but not limited to, natural killer (NK) cells or macrophages, that express a dual-chain receptor with two transmembrane / membrane-associated domains and TCR-like antigen recognition. The method involves providing cells (e.g., natural killer (NK) cells or macrophages) and modifying the cells to express an antigen-specific receptor with TCR-like binding properties. The modified cells can be any cell. Commonly used non-limiting examples are NK-92 cells, YTS cells, and primary human NK cells.

[0351] In another embodiment, the present disclosure provides a class of SARs with TCR-like binding properties that can be expressed in any cell type. Such SARs with TCR-like binding properties and universal expression are referred to as Universal TCR-SARs, uTCR-SARs, or uTCRs. Provided herein are single-chain and multi-chain (e.g., dual-chain) uTCR-SARs comprised of variable antigen-binding domains of TCRs (e.g., Va / Vα, Vb / Vβ, Vg / Vγ, Vd / Vδ, etc.) that can be expressed not only in T cells but also in any other cells, including, but not limited to, NK cells, monocytes, macrophages, dendritic cells, granulocytes, endothelial cells, epithelial cells, etc. In one embodiment, cells expressing uTCRs respond to target cells expressing that antigen by increasing cell proliferation, activation, cytokine secretion, and cytotoxicity. In one embodiment, the target antigen is a peptide presented as part of an MHC complex. In one embodiment, the target antigen is a lipid. The uTCR may also respond to non-MHC-restricted antigens if its TCR binding domain is derived from an HLA-independent TCR. In one embodiment, the present disclosure provides a cell that is not a T cell that functionally expresses a dual-chain receptor with TCR-like binding properties, including the property of binding to intracellular peptides when presented by an MHC complex.

[0352] In some embodiments, the present disclosure provides a single-chain uTCR-SAR comprising one or more heterologous antigen-binding domains comprising an scTCR, svd-TCR, or TCR-mimicking scFv, or fragments thereof, operably linked via an optional linker to all or a portion of the extracellular domain of a non-TCR signaling receptor. In some embodiments, the present disclosure provides that the single-chain SAR comprising the scTCR, svd-TCR, or TCR-mimicking scFv acquires TCR-like binding ability (e.g., the ability to bind to a peptide / MHC complex) and can be expressed in any cell, including cells that are not T cells or that express a TCR chain.

[0353] ...

Claims

1. A synthetic antigen receptor (SAR) that specifically binds to at least one target antigen, the SAR comprising a dimer of two polypeptide chains comprising: (i) a first module comprising one or more heterologous antigen-binding domains selected from the group consisting of: Rule: a) Antibodies; b) antibody fragments; c) the heavy chain variable region (vH domain) of an antibody or a fragment thereof; d) the light chain variable region (vL domain) of an antibody or a fragment thereof; e) a single chain variable fragment (scFv) or a fragment thereof; f) Single domain antibodies (SDABs) or fragments thereof; g) a vHH domain or a fragment thereof; h) a monomeric variable region of an antibody; i) a single vH domain (SVH) or a fragment thereof; j) a single vL domain (SVL) or a fragment thereof; k) non-immunoglobulin antigen-binding scaffolds; l) the ligand-binding domain of a receptor or a fragment thereof; m) the receptor binding domain of the ligand; n) Bispecific antibodies, antibody fragments, scFV, vHH, SDAB, non-immunoglobulin antigen binding Scaffolds, receptors or ligands; o) an autoantigen or a fragment thereof; p) an adaptor binding domain or a fragment thereof; q) an Fc binding domain or a fragment thereof; r) a TCR or an HLA-independent TCR or a fragment thereof; and s) Va, Vb, Vg or Vd fragments of TCR or fragments thereof; a first module, where, if present, a vH domain of the antibody binds to one polypeptide chain and a complementary vL domain of the antibody binds to another polypeptide chain, and the vH and vL domains form an Fv-like antigen-binding module that specifically binds to a target antigen, or a Va domain of the TCR binds to one polypeptide chain and a complementary Vb domain of the TCR binds to another polypeptide chain, and the Va and Vb domains form a TCR-Fv-like antigen-binding module that specifically binds to a target antigen, or a Vg domain of the TCR binds to one polypeptide chain and a complementary Vd domain of the TCR binds to another polypeptide chain, and the Vg and VdV domains form a TCR-Fv-like antigen-binding module that specifically binds to a target antigen; and (ii) a second module comprising a membrane-associated module (MAM), wherein the first module and the second module are operably linked, and the MAM of the first polypeptide chain (first MAM) and the MAM of the second polypeptide chain (second MAM) form a non-T cell receptor module (NTCRM) capable of activating at least one signaling pathway and / or recruiting at least one signaling adaptor.

2. The first and / or second MAM and / or NTCRM are comprised of a transmembrane / membrane anchoring domain, an optional cytosolic domain, an optional cytoplasmic domain, an optional hinge domain and / or an extracellular domain of any non-T cell receptor and / or any signaling adaptor, wherein optionally a. The non-T cell receptor is a naturally occurring receptor, optionally including CD16A, CD16B, CD64, CD32, NKp30, NKp44, NKp46, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL4, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2D, NKG2C, NKG2A, NKG2E, NKG2F, DNAM-1, 2B4, OX40, CD28, 4-1BB, selected from the group consisting of CD27, CD81, CD2, CD5, TNFR-I, TNFR-II, Fas, CD30, CD40, CRTAM, TIGIT, CD96, SLAMF6, SLAMF7, CD100, CD160, CEACAM, ILT2, KLRG1, LAIR1, CD161, variants of any of the foregoing, and fragments thereof; and b. The SAR of claim 1, wherein the signaling adaptor is optionally selected from the group consisting of CD3ζ, FcRγ, DAP10, variants of any of the foregoing, and fragments thereof.

3. The SAR according to claim 1 or 2, having one or more of the following characteristics: a) the first MAM and / or the second MAM does not contain the cytoplasmic domain and optionally the transmembrane domain of a CD3 chain selected from CD3ε, CD3γ, CD3δ or CD3ζ; b) the first MAM and / or the second MAM does not contain a transmembrane domain of a TCR chain and / or a CD3 chain; c) the first MAM and / or the second MAM does not contain the transmembrane domain of CD3ζ; d) at least one of the MAMs does not contain the transmembrane domain of CD3ζ; e) at least one of the MAMs does not contain the transmembrane domain of the CD3 chain; f) One of the MAMs comprises a transmembrane domain of a T cell receptor constant chain selected from the group consisting of the constant chains of TCR alpha, TCR beta, TCR gamma, TCR delta and preTCR alpha.

4. a. the first polypeptide chain further comprises a first peptide linker between the antigen binding domain and the first MAM and the second polypeptide chain further comprises a second peptide linker between the antigen binding domain and the second MAM, where optionally, the first and / or second peptide linker individually comprise one or more of the following: (i) a constant domain or fragment thereof from an immunoglobulin or T cell receptor subunit; (ii) a CH1, CH2, CH3, CH4 or CL antibody domain, or a fragment thereof; (iii) a Cα, Cβ, Cγ, or Cδ TCR domain, or a fragment thereof; (iv) mutations that increase the expression, affinity and / or pairing of the two polypeptide chains; (v) a sequence set forth in any one of SEQ ID NOs: 3536-3569 and 9627-9631, or a sequence having at least 70% identity thereto; (vi) a disulfide bond linking two polypeptide chains, and / or b. the first polypeptide further comprises a first hinge domain or fragment thereof located at the N-terminus of the first MAM, and / or the second polypeptide further comprises a second hinge domain or fragment thereof located at the N-terminus of the second MAM, optionally wherein a disulfide bond is located between a residue of the first MAM and a residue of the second MAM, and / or between a residue of the first hinge domain and a residue of the second hinge domain, and / or c. The SAR of claim 1 or 2, wherein the first polypeptide further comprises a first homologous antigen-binding domain or a fragment thereof located N-terminally to the first hinge domain, and / or the second polypeptide further comprises a second homologous antigen-binding domain or a fragment thereof located N-terminally to the second hinge domain, wherein the two homologous antigen-binding domains are derived from the same naturally occurring non-T cell receptor as the corresponding hinge domains.

5. The first polypeptide further comprises a cytoplasmic domain comprising an optional activation domain C-terminal to a first transmembrane / membrane anchoring domain of a first MAM, and / or the second polypeptide further comprises a cytoplasmic domain comprising an optional activation domain C-terminal to a second transmembrane / membrane anchoring domain of a second MAM, and optionally the cytoplasmic domain comprises a z-chain or homologue of the T cell receptor complex, a human CD3 chain selected from the group consisting of CD3ζ, CD3γ, CD3δ and CD3ε, a Syk family member optionally selected from the group consisting of Syk and ZAP70.

3. The SAR of claim 1 or 2, comprising a cytoplasmic domain or a functional fragment thereof of one or more molecules selected from the group consisting of leech tyrosine kinase, a src family tyrosine kinase optionally selected from the group consisting of Fyn and Lyn, a molecule involved in T cell transduction optionally selected from the group consisting of CD2, CD5 and CD28, common FcR gamma (FCER1G or FcRγ or FCRG), Fc gamma RIIIa, FcR beta (Fc epsilon R1b), CD79a, CD79b, DAP10, and DAP12.

6. The first polypeptide chain further comprises an accessory intracellular domain comprising one or more costimulatory domains or functional fragments thereof, optionally located C-terminal to a first transmembrane / membrane anchoring domain of the first MAM, and / or the second polypeptide chain further comprises an accessory intracellular domain comprising one or more costimulatory domains or functional fragments thereof, optionally located C-terminal to a second transmembrane / membrane anchoring domain of the second MAM, optionally wherein the costimulatory domain comprises a costimulatory domain of a costimulatory molecule or functional fragment thereof.

3. The SAR of claim 1 or 2, wherein, optionally, the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1BB), CD134 (OX40), 2B4, CD27, CD81, CD2, CD5, CD8, BAFF-R, CD30, CD40, HVEM, CD278 (ICOS), ICAM, TNFR-I, TNFR-II, Fas, Toll ligand receptor, LFA-1, MHC class I molecule, BTLA, NKp30, NKp44, NKp46, GITR, CD81, CD160, DAP10, B7-H3, TNFR superfamily, Lck, or a mutant or fragment thereof.

7. The first and / or second MAM and / or NTCRM are comprised of a transmembrane / membrane anchoring domain, an optional cytosolic domain, an optional hinge domain and / or an optional extracellular domain, having one or more of the following properties: (a) the transmembrane / membrane anchoring domain, any cytoplasmic domain, any hinge domain, and / or any extracellular domain are derived from a single non-T cell receptor and / or signaling adaptor or variant thereof; or (b) the transmembrane / membrane anchoring domain, any cytoplasmic domain, any hinge domain and / or any extracellular domain are all derived from a single non-T cell receptor and / or signaling adaptor or variant thereof or are derived from different non-T cell receptors and / or signaling adaptors or variants thereof; or (c) one MAM comprises a transmembrane domain of a non-T cell receptor or variant thereof, and the other MAM comprises a transmembrane domain of a signaling adaptor or variant thereof; or (d) one MAM comprises the transmembrane domain of CD3ζ or a variant thereof, and the other MAM comprises the transmembrane domain of FcRγ or a variant thereof; or (e) one MAM comprises the transmembrane domain of CD3ζ or a variant thereof, and the other MAM comprises the transmembrane domain of CD16 or a variant thereof; or (f) one MAM comprises the transmembrane domain of FcRγ or a variant thereof, and the other MAM comprises the transmembrane domain of CD16 or a variant thereof; or (g) one MAM comprises the transmembrane domain of CD3ζ or a variant thereof, and the other MAM comprises the transmembrane domain of DAP10 or a variant thereof; or (h) one MAM comprises the transmembrane domain of FcRγ or a variant thereof, and the other MAM comprises the transmembrane domain of DAP10 or a variant thereof; or (i) one MAM comprises the transmembrane domain of CD3ζ or a variant thereof, and the other MAM comprises the transmembrane domain of a TCR chain or a variant thereof, or (j) one MAM comprises the transmembrane domain of FcRγ or a variant thereof and the other MAM comprises the transmembrane domain of a TCR chain or a variant thereof; or (k) one MAM comprises a transmembrane domain of CD16 or a variant thereof, and the other MAM comprises a transmembrane domain of a TCR chain or a variant thereof; or (l) one MAM comprises the transmembrane domain of CD3ζ or a variant thereof and the other MAM comprises the transmembrane domain of CD64 or a variant thereof; or (m) one MAM comprises the transmembrane domain of FcRγ or a variant thereof, and the other MAM comprises the transmembrane domain of CD64 or a variant thereof; or (n) one MAM comprises a transmembrane domain of CD3ζ or a variant thereof, and the other MAM comprises a transmembrane domain of NKp30, NKp40 or NKp46 or a variant thereof; or (o) one MAM comprises a transmembrane domain of FcRγ or a variant thereof, and the other MAM comprises a transmembrane domain of NKp30, NKp40 or NKp46 or a variant thereof; or (p) the two transmembrane / membrane anchoring domains, any cytoplasmic domain, any costimulatory domain, any hinge domain, and / or any extracellular domain are identical in sequence and all derived from a) a single protein or b) different proteins.

8. a) the first polypeptide chain comprises a first antigen-binding domain comprising a vL, a Vα or a Vγ domain and a first membrane-associated module (MAM); and b) the second polypeptide chain comprises a second antigen-binding domain comprising a vH, Vβ or Vδ domain and a second membrane-associated module (MAM), (i) the vL domain of the first antigen-binding domain and the complementary vH domain of the second antigen-binding domain form an Fv-like antigen-binding module that specifically binds to a target antigen; or (ii) the Vα domain of the first antigen-binding domain and the complementary Vβ domain of the second antigen-binding domain form a TCR-Fv-like antigen-binding module that specifically binds to a target antigen; or (iii) The synthetic antigen receptor (SAR) of claim 1, wherein the Vγ domain of the first antigen-binding domain and the complementary Vδ domain of the second antigen-binding domain form a TCR-Fv-like antigen-binding module that specifically binds to a target antigen.

9. 9. The SAR of any one of claims 1, 2 and 8, wherein the first and / or second polypeptide chain further comprises one or more autonomous antigen-binding domains (AABDs) attached at or near the N-terminus of the first and / or second antigen-binding domain, optionally the AABDs are selected from one or more of a single vH domain (SVH), a single vL domain (SVL), a vHH domain, a single domain antibody, a single variable domain of a TCR (svd-TCR), a non-immunoglobulin antigen-binding scaffold, a ligand-binding domain of a receptor, a receptor-binding domain of a ligand, an autoantigen, an adaptor-binding domain, an Fc-binding domain, fragments thereof and / or variants thereof.

10. The target antigen is CD19;CD123;CD22;CD30;CD171;CS-1;C-type lectin-like molecule-1;CD33;epidermal growth factor receptor variant III (EGFRviii);ganglioside G2 (GD2);ganglioside GD3;TNF receptor family member B cell maturation (BCMA);Tn antigen (Tn Ag);prostate-specific membrane antigen (PSMA);receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not hematopoietic progenitor cells, glycosylated CD43 epitope expressed in nonhematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); Kit (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11 Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1; cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostases; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta, 9 (LMP2); glycoprotein 100 (GPL00); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5);high molecular weight melanoma-associated antigen (HMWMAA);o-acetyl-GD2 ganglioside (OAcGD2);folate receptor beta;tumor endothelial marker 1 (TEM1 / CD248);tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6);thyroid-stimulating hormone receptor (TSHR);G protein-coupled receptor class C group 5;member D (GPRC5D);X chromosome open reading frame 61 (CXORF61);CD97;CD179a;anaplastic lymphoma kinase (ALK);polysialic acid;placenta-specific 1 (PLAC1);hexasaccharide portion of globoH glycoceramide (GloboH);mammary differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cell receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3);pannexin 3 (PANX3);G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex;locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma associated antigen 1 (MAGE-A1); MAGE-A2, MAGE-A3, MAGE-A4, PRAME, PSA, ETS translocation mutant gene located on chromosome 12p 6 (ETV6-AML); Sperm protein 17 (SPA17); X antigen family, member lA (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein P53 (p53); p53 mutant; Prostein; Survival; Telomerase; Prostate cancer tumor antigen-1 (PCT A-1 or galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor;cyclin BL;v-MYC avian myeloma viral oncogene neuroblastoma-derived homolog (MYCN);ras homolog family member C (RhoC);tyrosinase-related protein 2 (TRP-2);cytochrome P450 lB 1 (CYP1B 1);CCCTC-binding factor (zinc finger protein)-like (sibling of BORIS or regulator of imprinted sites);squamous cell carcinoma antigen recognized by T cells 3 (SART3);paired box protein Pax-5 (PAX5);proacrosin-binding protein sp32 (OY-TES1);lymphocyte-specific protein tyrosine kinase (LCK);kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2);receptor for advanced glycation end products (RAGE-1);renal ubiquitous 1 (RU1);renal ubiquitous 2 (RU2);legumain;human papillomavirus E6 (HPV E6);human papillomavirus E7 (HPV E7);intestinal carboxylesterase;mutated heat shock protein 70-2 (MUT HSP70-2);CD79a;CD79b;CD72;leukocyte-associated immunoglobulin-like receptor 1 (LAIR1);Fc fragment of IgA receptor (FCAR or CD89);leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2);CD300 molecule-like family member f (CD300LF);C-type lectin domain family 12 member A (CLEC12A);bone marrow stromal cell antigen 2 (BST2);EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2);lymphocyte antigen 75 (LY75);glypican-3 (GPC3);Fc receptor-like 5 (FCRL5);immunoglobulin lambda-like polypeptide 1 (IGLL1) and MPL;biotin;c-MYC epitope tag;CD34;LAMP1 TROP2;GFR alpha 4;CDH17;CDH6;NYBR1;CDH19;CD200R;Three (CA19.9; Sialyl Lewis Antigen) Fucosyl-GM1;PTK7;gpNMB;CDH1-CD324;DLL3;CD276 / B7H3;IL11Ra;IL13Ra2;CD179b-IGLl1;Alc;TCR gamma delta;NKG2D;CD32(FCGR2A);TN ag;CSPG4-HMW-MAA;Tim1- / HVCR1;CSF2RA (GM-CSFR-alpha);TGF beta R2;VEGFR2 / KDR;TCR-β1 chain;TCR-β2 chain;TCR-γ chain;TCR-delta chain;Fit;Luteinizing hormone receptor (LHR);Follicle-stimulating hormone receptor (FSHR);Chorionic gonadotropin hormone receptor (CGHR);CCR4;GD3;SLAMF6;SLAMF4;HIV1 envelope glycoprotein;HTLV1-Tax;CMV pp65;EBV-EBNA3c;Influenza A hemagglutinin (HA);GAD;PDL1;Guanylyl cyclase C (GCC);Autoantibody to desmoglein 3 (Dsg3);Autoantibody to desmoglein 1 (Dsg1);HLA;HLA-A;HLA-A2;HLA-B;HLA-C;HLA-DP;HLA-DM;HLA-DOA;HLA-DOB;HLA-DQ;HLA-DR;HLA-G;IGE;CD99;RAS The SAR of any one of claims 1, 2 and 8, selected from the group consisting of G12V; tissue factor 1 (TF1); AFP; GPRC5D; claudin 18.2 (CLD18A2 or CLDN18A.2); P-glycoprotein; STEAP1; LIV1; nectin-4; Crypto; GPA33; BST1 / CD157; low-conductance chloride channel and SARS-CoV2 spike protein.

11. 10. The SAR of any one of claims 1, 2, and 8, wherein the SAR has one or more of the following characteristics: a) the encoded SAR polypeptide comprises one or more heterologous antigen binding domains and / or autonomous antigen binding domains (AABDs) selected from the group consisting of: (i) an antibody heavy chain variable region (vH) comprising a sequence set forth in any of SEQ ID NOs: 2682-2918, or a sequence having at least 90% identity thereto, including the three complementarity determining regions (CDRs); (ii) a complementary light chain variable region (vL) of an antibody comprising a sequence set forth in any of SEQ ID NOs: 2440-2676, or a sequence having at least 90% identity thereto, including the three complementarity determining regions (CDRs) of said sequence; (iii) a single chain variable fragment (scFv) comprising a sequence set forth in any of SEQ ID NOs: 2924-3160, or a sequence having at least 90% identity thereto, comprising the six complementarity determining regions (CDRs) set forth above; (iv) a non-immunoglobulin scaffold encoded by a polynucleotide comprising a sequence set forth in any of SEQ ID NOs: 3366-3377, or a sequence having at least 90% identity thereto; (v) a ligand-binding domain of a receptor comprising a sequence set forth in any one of SEQ ID NOs: 3378-3395, 3880, 3882, 3886, 3893, 3896, 3897, or a sequence having at least 90% identity thereto; (vi) a receptor binding domain of a ligand comprising a sequence set forth in any one of SEQ ID NOs: 3396-3406, 10786-10787, or a sequence having at least 90% identity thereto and encoding a polypeptide that binds to its cognate; (vii) an adaptor binding domain encoding a polypeptide that binds to the adaptor, comprising a sequence set forth in any one of SEQ ID NOs: 3407-3435, 10771-10780, or a sequence having at least 90% identity thereto; (viii) an autoantigen comprising a sequence set forth in any one of SEQ ID NOs: 10788-10791, or a sequence having at least 90% identity thereto, and encoding a polypeptide that binds to an autoantibody or an autoantibody-producing cell; (ix) a TCR variable region (Va, Vb, Vg, or Vd) comprising a sequence set forth in any one of SEQ ID NOs: 3357-3364, 9606-9614, 10781-10782, or a sequence having at least 90% identity thereto, including the three complementarity determining regions (CDRs) described above; (x) a single variable TCR domain (svd-TCR) comprising a sequence set forth in any of SEQ ID NOs: 9613-9614, or a sequence having at least 90% identity thereto, including the three aforementioned complementarity determining regions (CDRs). b) the naturally occurring receptor and / or signaling adaptor or fragment thereof comprises a sequence selected from SEQ ID NOs: 3743-3966, 3385, 3394, 7818-7822, 9633-9859, or a sequence having at least 90% identity thereto; c) the polypeptide comprising the hinge domain, transmembrane domain, and cytoplasmic domain of a naturally occurring receptor and / or signaling adaptor comprises a sequence selected from SEQ ID NOs: 9669-9704, 3813, 8721, 8733, and 8746, or a sequence having at least 90% identity thereto; d) the membrane-associated domain of the naturally occurring receptor and / or signaling adaptor comprises a sequence selected from SEQ ID NOs: 3914-3928, 9741-9776, 9852-9855, or a sequence having at least 90% identity thereto; e) the cytoplasmic domain of the naturally occurring receptor and / or signaling adaptor comprises a sequence selected from SEQ ID NOs: 3944-3958, 9777-9812, 9856-9859, or a sequence having at least 90% identity thereto; f) the activation domain of a naturally occurring receptor and / or signaling adaptor comprises a sequence selected from SEQ ID NOs: 9856-9859 and 9777, or a sequence having at least 90% identity thereto; g) the costimulatory domain comprises a sequence selected from SEQ ID NOs: 9807-9810, or a sequence having at least 90% identity thereto; h) a leader sequence or signal peptide at the N-terminus of each chain, optionally comprising a sequence selected from the group consisting of SEQ ID NOs: 2425-2430.

12. The two polypeptide chains of SAR are represented by SEQ ID NOs: 6283-6294, 6315-6326, 6347-6358, 6379-6390, 6411-6422, 6443-6454, 6475-6486, 6507-6518, 6539-6550, 6571-6582, 6603-6614, 6635-6646, 6667-6678, 66 99-6710, 6731-6742, 6763-6774, 6795-6806, 6827-6839, 6859-6870, 6891-6902, 6923-6934, 6955-6966, 6987-6998, 7019-7030, 7051-7062, 7083-7094, 10338, 10345, 10347, 10350-1 The SAR according to any one of claims 1, 2 and 8, comprising a sequence of two polypeptide chains of SAR selected from the group consisting of 0353, 10355-10362, 10364, 10369-10375, 10378-10379, 10387-10392, 10394, 10396-7, 10421-10425, 10427-10428, 10466-10694, 10838-10841, or a sequence having 75% or more identity thereto.

13. A recombinant polynucleotide encoding the SAR of claim 1.

14. 10. An effector or stem cell expressing at least one SAR and optional accessory module according to any one of claims 1, 2 and 8, optionally wherein the cell has one or more of the following properties: (a) The cells include α / β T cells, γ / δ T cells, CD8 + T cells, CD4 + T cells, memory T cells, naive T cells, T stem cells, Treg cells, natural killer T (NKT) cells, iNKT (innate natural killer cells), NK cells, g-NK cells, memory-like NK cells, cytokine-induced killer cells (CIK), hematopoietic stem cells, iPSCs, modified HLA-deficient iPSCs, iPSC-derived NK cells, iPSC-derived T cells, B cells, macrophages / monocytes, granulocytes, dendritic cells, immortalized cell lines, immortalized NK cell lines, NK92 cell lines, NK92MI cell lines, YTS cells or derivatives thereof; and / or (b) the cell lacks or has underexpression of a functional TCR, a functional HLA, beta2 macroglobulin, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD38, CD5, CD52, CD33, CD123, CLL-1, CIS, CBL-b, SOCS2, PD1, CTLA4, LAG3, TIM3, TIGIT, or any gene within the chromosome 6p21 region; and / or (c) the cells exhibit induced or increased expression of at least one of HLA-E, 41BBL, CD3ε, CD3γ, CD3δ, CD3ζ, FcRγ, DAP10, DAP12, CD4, CD8, CD16, CD47, CD94, CD113, CD131, CD137, CD80, PDL1, A2AR, an Fc receptor, an engager, or a surface triggering receptor for coupling with a bispecific or multispecific or universal engager; and / or (d) the cells have been modified to inhibit or reduce expression of the first endogenous TCR subunit and / or the second endogenous TCR subunit; and / or (e) the cell does not express a T cell receptor (TCR) and / or does not express CD3ε, CD3γ or CD3δ, and the cell is modified by recombinant expression to express a recombinant dual chain TCR exogenous to the cell, wherein the recombinant dual chain TCR is a SAR comprising a TCR antigen recognition domain comprising a) Vα and Vβ domains, or b) Vγ and Vδ domains and a non-T cell receptor module (NTCRM); and / or (f) the cell is operably linked via an optional linker to a non-T cell receptor module (NTCRM) consisting of a first MAM and a second MAM derived from a non-T cell receptor and / or a signaling adaptor, and further comprises a TCR antigen recognition motif including an optional cytoplasmic costimulatory domain; and / or (g) the cell comprises a plurality of SAR polypeptides; and / or (h) the cell contains multiple SAR polypeptides that target the same antigen or that target an antigen that is different from at least one other SAR polypeptide; and / or (i) the cell comprises a plurality of SAR polypeptides that comprise a binding affinity for an antigen that differs from that of at least one other SAR polypeptide; and / or (j) the cell comprises a plurality of SAR polypeptides, at least one SAR polypeptide of the plurality comprising a naturally occurring receptor or signaling adaptor that is distinct from at least one other SAR polypeptide; and / or (k) the cell comprises a plurality of SAR polypeptides, at least one SAR polypeptide of the plurality having an extracellular domain, a transmembrane domain, or a cytosolic domain that is distinct from at least one other SAR polypeptide; and / or (l) the cell comprises a plurality of SAR polypeptides, at least one SAR polypeptide of the plurality being an activating receptor and at least one other SAR polypeptide being an inhibitory receptor; and / or (m) the cell comprises a plurality of SAR polypeptides, and two or more SAR polypeptides of the plurality of SAR polypeptides are activating receptors or two or more SAR polypeptides of the plurality of SAR polypeptides are inhibitory receptors; and / or (n) the cell comprises a plurality of SAR polypeptides, and two or more SAR polypeptides of the plurality of SAR polypeptides recruit different signaling adaptors and / or activate different signaling pathways; and / or (o) the cell is a non-T cell but has T cell like antigen recognition; and / or (p) the cells lack functional expression of the CD3γ, CD3δ and / or CD3ε chains but have T cell like antigen recognition; and / or (q) the cell is a non-T cell but is capable of inducing T cell-like signaling upon antigen recognition; and / or (r) the cells lack functional expression of the CD3γ, CD3δ and / or CD3ε chains but are capable of inducing T cell-like signaling upon antigen recognition; and / or (s) the cell is a non-T cell but is capable of binding a peptide in complex with an MHC (HLA) molecule; and / or (t) the cell is a non-T cell but is capable of initiating at least one signaling pathway upon binding to a peptide / MHC complex; and / or (u) the cells express one or more accessory modules encoding one or more polypeptides selected from the group consisting of: a) the cytokine or variant thereof optionally comprises a sequence of SEQ ID NO: 7833-7842 or a variant having up to 70% sequence homology thereto; b) a membrane-anchored cytokine or a membrane-anchored cytokine with an epitope tag, optionally comprising a membrane-anchored cytokine or a variant thereof, or a sequence having SEQ ID NO: 7825-7832 or a variant having up to 70% sequence homology thereto; c) a multi-purpose switch that functions as a suicide, survival and marker function, optionally having the sequence of SEQ ID NO: 7843-7850 or a variant having up to 70% sequence homology thereto; d) a signaling adaptor molecule selectable from the group of CD3ζ, FcRγ, DAP10 and DAP12; e) Kill switch; f) truncated epidermal growth factor receptor (tEGFR), truncated epidermal growth factor receptor viii (tEGFRviii), truncated CD30 (tCD30), truncated BCMA (tBCMA), truncated CD19 (tCD19), CD34, thymidine kinase, cytosine deaminase, nitroreductase, xanthine-guanine phosphoribosyltransferase, human caspase 8, human caspase 9, inducible caspase 9 (icaspase 9), purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), selection marker, multipurpose switch, vFLIP-K13, vFLIP-MC159, 4-1BBL-CD40L, dihydroxyfolate receptor (DHFR), mutant DHFR, methylated DNA-protein-cysteine ​​methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), puromycin acetyltransferase (PAC), blasticidin resistance gene, mutant calcineurin a / b (Can / b), CNa12, CNb30, PDL1, PDL2, CD80, CD86, crmA, p35, hNEMO-K277A (or NEMO-K277A), hNEMO-K277A-delta-V249-K555, mNE MO-K270A, K13-opt, IKK2-S177E-S181E (or IKK2-SS / EE), IKK1-S176E-S180E (or IKK1-SS / EE), MyD88-L 265P, TCL-1a, MTCP-1, CMV-141, vFLIP-K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS Mutants, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1scFV, PD1-5C4scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-ipilimumab-scFv, CTLA4-ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, shRNA targeting hTERT, Fx06, Brd4, IgSP-[hTRAC-opt2], IgSP-[hTRBC-opt2], IL2-tBCMA, IL15-tBCMA, IL2-RQR, IL15-RQR, NKG2C, CD94, DAP10, DAP12, CD3ε, CD3γ, CD3δ, CD3ζ, FcRγ, IL2, IL-7, IL-15, IL12f, IL21, membrane anchored form of IL2, membrane anchored form of IL15, or combinations thereof; g) an agent that provides costimulation to SAR-expressing cells; h) an agent that provides costimulation to SAR-expressing cells in an inducible manner; i) agents that modulate the activity of SAR-expressing cells; j) agents that enhance or modulate the activity of SAR-expressing cells; k) Agents that promote the proliferation and / or persistence of SAR-expressing cells.

15. 15. A composition comprising the cells of claim 14 and a pharma- ceutically acceptable excipient for treating cancer, an infectious disease, an autoimmune disease or a degenerative disease in a subject, optionally further comprising one or more agents that bind to one or more antigens expressed on the SAR-expressing effector cells, wherein the agents are optionally selected from the group consisting of an antibody, an antibody fragment, an antigen-binding domain, a non-immunoglobulin antigen-binding domain fragment, an autonomous antigen-binding domain, a bispecific engager, a bispecific T cell engager (BiTE), a bispecific killer engager (BiKE), a trispecific engager, a trispecific T cell engager, a trispecific killer engager (TRiKE), or a combination thereof.

16. A composition comprising a SAR according to any one of claims 1, 2 and 8 for use in treating a disease, wherein the disease is selected from the group consisting of cancer, a viral disease, an autoimmune disease, a degenerative disease, and an infectious disease.

17. At least one recombinant polynucleotide encoding the SAR of claim 1, wherein the sequences of the two polypeptide chains encoded by the SAR are selected from the group consisting of SEQ ID NOs: 6283-6294, 6315-6326, 6347-6358, 6379-6390, 6411-6422, 6443-6454, 6475-6486, 6507-6518, 6539-6550, 6571-6582, 6575-6586 ... 582, 6603-6614, 6635-6646, 6667-6678, 6699-6710, 6731-6742, 6763-6774, 6795-6806, 6827-6839, 6859-6870, 6891-6902, 6923-6934, 6955-6966, 6987-6998, 7019-7030, 7051-7062, 7083-7094, 10338 , 10345, 10347, 10350-10353, 10355-10362, 10364, 10369-10375, 10378-10379, 10387-10392, 10394, 10396-7, 10421-10425, 10427-10428, 10466-10694, 10838-10841, or a sequence having at least 75% identity thereto.

18. 18. At least one vector comprising the recombinant polynucleotide of claim 13 or 17, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, and a Sleeping Beauty transposon vector.

19. 20. A method for producing a SAR-expressing effector cell, comprising introducing at least one vector comprising a recombinant polynucleotide according to claim 13 or 17, or at least one recombinant polynucleotide according to claim 13 or 17, into an effector cell, cell line, hematopoietic stem cell, progenitor cell, or IPSC (induced pluripotent stem cell) capable of giving rise to an effector cell, under conditions such that the SAR polypeptide and any accessory module are expressed.

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