Methods and compositions for gene transfer and synthesis and for controlling the activity of immune receptors

Abstract

This application provides novel viral envelope glycoproteins for pseudotyping viral vectors, novel designs of synthetic antigen receptors (SARs), novel signaling chains for constructing SARs, novel antigen-binding domains, and novel methods for producing SAR-expressing cells. These novel methods and compositions are widely used in cell therapy.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 478,612, filed January 5, 2023, the disclosure of which is incorporated herein by reference in its entirety. [Technical Field]

[0002] TECHNICAL FIELD This disclosure relates to the field of biotechnology, and more particularly to single-chain and multi-chain synthetic antigen receptors. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application contains a sequence listing that has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML file was created on January 5, 2024, is named "NKSAR-13.xlm", and is 99.899 megabytes in size. Background

[0004] Chimeric antigen receptors (CARs) are synthetic receptors that can reprogram T cells to selectively kill tumor cells. To overcome the design limitations of conventional second-generation CARs, several alternative designs, collectively referred to as next-generation CARs, have been proposed. These include Ab-TCRs (WO 2017 / 070608 A1, incorporated herein by reference in its entirety), TCR receptor fusion proteins or TFPs (WO 2016 / 187349 A1, incorporated herein by reference in its entirety), synthetic immunoreceptors (SIRs) (WO 2018 / 102795 A1, incorporated herein by reference in its entirety), trifunctional T cell antigen binding factors (Tri-TACs) (WO 2015 / 117229 A1, incorporated herein by reference in its entirety), and synthetic antigen receptors (SARs), including universal TCR-SARs (uTCR-SARs) (PCT / US22 / 17177, incorporated herein by reference in its entirety). STAR (WO2020029774, incorporated herein by reference in its entirety) and HLA-independent TCR (HIT) (WO201915745 A1, incorporated herein by reference in its entirety) have designs similar to the SIR platform. These alternative CAR designs generally lack costimulatory domains. This disclosure describes novel synthetic antigen receptors (SARs), novel antigen-binding domains, novel viral envelopes, and in vivo methods for producing SARs. Overview

[0005] The present disclosure provides monospecific, bispecific, multispecific, and universal synthetic antigen receptor (SAR) designs. The term SAR refers to any non-natural antigen-binding receptor expressed on the surface of a cell (such as an immune cell). In embodiments, the SAR comprises a single polypeptide chain. In embodiments, the SAR comprises multiple polypeptide chains. In embodiments, the SAR comprises two polypeptide chains. The present disclosure also provides novel accessory modules containing co-receptors (e.g., CD8a, CD8b, and CD4) that can be co-expressed with the disclosed SARs (e.g., uTCR-SAR, HC-SAR, zSIR, zCD16-SAR, etc.). Examples of co-stimulatory co-receptors are set forth in (SEQ ID NOs: (DNA): 644-646 and SEQ ID NOs: (PRT): 9024-9026).

[0006] The present disclosure provides methods for generating cells that express any one or more accessory modules having any one or more SARs of the present disclosure.

[0007] The present disclosure provides novel designs of monospecific, bispecific, and multispecific SARs (e.g., SIRs, Ab-TCRs, etc.) comprising one or more hybrid TCR constant chains or functional variants thereof, including variants from non-human species (e.g., mouse, cat, dog, monkey, etc.). Examples of hybrid TCR constant chains are provided in SEQ ID NOs: (DNA): 529-544, 21350-21814, 22794-23019, 23021-23070, 23072-23231, 23901-27920, and SEQ ID NOs: (PRT): 8909-8924, 22112-22576, 23420-23677, 27936-31955. The present disclosure also provides that SIRs can be constructed using TCR constant chains that encode polypeptides having at least 70%, 80%, 90%, 95%, 98%, 99% or 100% amino acid identity to the hybrid TCR constant chains provided in SEQ ID NOs: (PRT): 8909-8924, 22112-22576, 23420-23677, 27936-31955. The present disclosure also provides hybrid TCR constant chains in which N-terminal deletions of the TCR constant chains of SEQ ID NOs: (PRT): 8909-8924, 22112-22576, 23420-23677, 27936-31955 encode polypeptides of 1 to 100 amino acids. The present disclosure also provides that SIRs can be constructed using functional variant TCR constant chains and functional fragments (including deletion variants) of the hybrid TCR constant chains provided in SEQ ID NOs: (PRT): 8909-8924, 22112-22576, 23420-23677, 27936-31955. In embodiments, the hybrid TCR chains are codon-optimized and optionally human codon-optimized. In some embodiments, the hybrid TCR chains contain cysteine ​​mutations at specific residues that result in the formation of an additional (second) disulfide bond between complementary TCR chains. In some embodiments, the hybrid TCR chains contain mutations in which human amino acid residues are replaced with the corresponding amino acid from a non-human species, such as a mouse TCR chain.In embodiments, the SAR comprises a hybrid T cell receptor alpha (Cα) chain constant region and a hybrid T cell receptor beta (Cβ) chain constant region, wherein the Cα region comprises mutations at amino acid positions 10C, 15C, 21F, 32I, 45C, 48C, 61R, 72T, 91S, 92D, 93V, 94P, 95R, 95S, 116L, 119V, 120L, and any combination thereof, corresponding to the reference Cα chain represented by SEQ ID NO: 8833, and the Cβ region comprises mutations at amino acid positions 15C, 17C, 18K or R, 22A, 23R, 39P, 54D, 57C, 59C, 77C, 79G, 131G, 131S, 133I, 136A, 139H, and any combination thereof, corresponding to the reference Cβ chain represented by SEQ ID NOs: 8847 and 8848. In embodiments, both chains comprising a double-chain SAR (e.g., SIR, Ab-TCR, HIT, STAR, zSIR, zSAR, zCD16 SAR, etc.) or a single-chain / half-chain SAR (e.g., SIR, Ab-TCR, HIT, STAR, etc.) comprise a hybrid TCR chain. In embodiments, one of the two chains of a double-chain SAR or a single-chain / half-chain (OHC) SAR comprises a hybrid TCR constant chain, and the other chain comprises a non-hybrid TCR constant chain. In embodiments, at least one chain of a double-chain SAR or OHC comprises a hybrid TCR constant chain. In embodiments, at least one chain of a double-chain SAR or OHC comprises a hybrid TCR constant chain with additional modifications, which may be selected from deletion, substitution, and / or mutation of one or more amino acids. Examples of double-chain SARs and single-chain / half-chain SARs (e.g., SIRs) with hybrid TCR constant chains are shown in Tables 16-20 of the provisional application. The human genome is composed of two highly homologous TCR β constant chains, Cβ1 and Cβ2. However, while the majority of the Cβ chain sequences in the current application (e.g., SARs, hybrid TCR chains and their fragments) are based on the sequence of the Cβ2 chain, those skilled in the art will recognize that these sequences can be replaced with corresponding sequences based on the Cβ1 chain.

[0008] Second-generation CAR-T cells currently in clinical use are associated with several toxicities, including cytokine release syndrome (CRS), immune effector cell-associated neurological complications (ICAN), non-ICAN neurological complications, and secondary cancers. Many of these complications are related to tonic signaling via the CAR construct, resulting in antigen-independent CAR-T cell proliferation. In some embodiments, the present disclosure provides that SARs with hybrid TCR chains exhibit less tonic signaling and antigen-independent proliferation compared to second-generation CARs. CAR-T cells have also been shown to recognize low levels of antigens expressed in normal, healthy tissues. This results in on-target, off-tumor toxicity. To overcome this issue, affinity-tuned CAR-T constructs have been described that utilize antigen-binding domains (e.g., scFvs, vHHs) with lower affinity. However, affinity tuning of antigen-binding domains is an expensive and time-consuming process and can lead to loss of affinity of the antigen-binding domain or the acquisition of new, unexpected binding properties. To overcome this limitation, the present disclosure provides a simple method for generating a diverse panel of SARs with different affinities by using different signaling chains. In one embodiment, the present disclosure provides that the hybrid and / or mutant TCR chains of the present disclosure can be used to rapidly generate a diverse panel of SARs with various affinities (e.g., SIRs, Ab-TCRs, HITs, STARs, zSIRs, zSARs, zCD16 SARs, etc.) containing the same antigen-binding domain. Thus, the present disclosure provides a simple method for generating a diverse panel of affinity-tuned and affinity-enhanced SAR constructs with different affinities based on the same antigen-binding domain (e.g., vL, vH, scFv, Fv, vHH, etc.). Thus, the disclosed method overcomes the limitations and risks associated with mutagenesis of antigen-binding domains to generate a diverse panel of affinity-tuned and / or affinity-enhanced SAR and CAR constructs.The diversity of the SAR pool can be further increased by the use of different junctions within the hybrid chain and different linkers that may be present between different domains of the SAR (e.g., antigen binding domain and TCR constant domain). The diversity of the T cell expressing pool can be further increased by the use of different accessory modules and therapeutic controls as described in the disclosure.

[0009] This diverse pool of SARs can be used to generate diverse immune responses against disease-causing or disease-associated cells expressing the antigen. Alternatively, the diverse pool of SARs can be optionally DNA barcoded using techniques known in the art and then used to select single or subgroups of SARs with optimal biological and clinical properties. These properties include, but are not limited to, performance in in vitro biological assays (e.g., cytotoxicity, cytokine secretion, binding affinity, cell surface expression, off-target effects, T cell proliferation, expression of exhaustion markers, and terminal differentiation), performance in in vivo assays (e.g., survival, tumor regression, T cell persistence, T cell proliferation), and clinical experience (e.g., disease remission, relapse rate, toxicity). The disclosed SARs can be used alone or in combination to generate diverse pools of immune effector cells for the prevention and treatment of various disease states caused by or associated with other SIRs, CARs, cTCRs, zSIRs, zCD16SARs, and cells expressing their target antigens, as well as other SIRs known in the art.

[0010] In any of the embodiments described herein, effector cells expressing one type of SAR exhibit diverse properties compared to effector cells expressing a different type of SIR (e.g., effector cells displaying on their surface an SIR comprising the antigen-binding domain of a first SIR but with a different TCR chain, e.g., an SIR comprising an scFv, vL, and / or vH fragment comprising the antigen-binding domain of a first SIR but with a different TCR chain) when compared under similar conditions. Because SIRs are modular in design, one skilled in the art can generate additional SIR types by replacing one module with another. Exemplary properties that different types of SIRs may exhibit diversity when expressed in immune effector cells include, but are not limited to, binding affinity, cell-surface expression, cytotoxicity, cytokine production, cell proliferation, terminal differentiation, depletion, and in vivo biological activity. In an exemplary embodiment, effector cells expressing SAR1 (SEQ ID NO: 40140), which contains a hu-mROO5-1-based CD19-targeting domain, have higher binding to the CD19-ECD-GGSG-NLuc-AcV5 fusion protein after 60 minutes of incubation at 4°C compared to corresponding effector cells expressing SAR2 (SEQ ID NO: 40146) or SAR3 (SEQ ID NO: 40147), which target CD19, when examined under similar conditions and when both SAR types target the TRAC (TCR alpha constant chain) genomic locus, ruling out expression variability due to random sites of integration of different SIR constructs. In some embodiments, target antigen binding of effector cells expressing one type of SIR (e.g., SAR1) is at least 5, 10, 20, 30, 40, 50% or 100% greater than target antigen binding of effector cells expressing a different type of SAR (e.g., SAR2) when examined under similar conditions after 60 minutes of incubation at 4°C, and when both SAR types target the TRAC (Constant Chain) genomic locus.In some embodiments, after 60 minutes of incubation at 4°C, target antigen binding of effector cells expressing different types of SARs (e.g., SAR1, SAR2, SAR3, etc.) containing the same binding domain varies by 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more when examined under similar conditions and when both SIR types target the TRAC (TCRα constant chain) genomic locus. Techniques for targeting genomic inserts to specific genomic loci are known in the art. In some embodiments, the standard deviation of target antigen binding of effector cells expressing different types of SIRs (e.g., SIR1, SIR2, SIR3, etc.), containing the same binding domain after 60 minutes of incubation at 4°C, is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more compared to the standard deviation of target antigen binding of an independently isolated population of effector cells expressing the corresponding cTCR, when examined under similar conditions, where the different SIR types and cTCRs target the TRAC locus. In other embodiments of the disclosure, the standard deviation of cytotoxicity of effector cells expressing different types of SIRs (e.g., SAR1, SAR2, SAR3, etc.) is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more compared to the standard deviation of cytotoxicity of an independently isolated population of effector cells containing and expressing the same binding domain after 4 hours of incubation with target cells at 37°C. The standard deviation is the square root of the variance and can be measured by methods known in the art. In some embodiments, the SIR-expressing effector cells are SIR T cells. In some embodiments, the SIR-expressing effector cells are SIR-expressing Jurkat T cells.

[0011] The present disclosure also provides that SARs having one or more hybrid TCR chains are less likely to pair with endogenous TCR chains than cTCRs (chimeric T cell receptors) or SIRs. In some embodiments, the present disclosure provides that SARs having one or more hybrid TCR chains exhibit higher expression and functional activity (e.g., cytokine production, cytotoxicity, etc.) when expressed in immune cells (e.g., T cells, NK cells) than SARs having wild-type TCR constant chains (i.e., cTCRs). In some embodiments, the present disclosure provides that SARs having one or more hybrid TCR chains exhibit lower expression and functional activity (e.g., cytokine production, cytotoxicity, etc.) when expressed in immune cells (e.g., T cells, NK cells) than second-generation CAR constructs. In some embodiments, the present disclosure provides that SARs having one or more hybrid TCR chains exhibit lower expression, binding affinity, and functional activity (e.g., cytokine production, cytotoxicity, etc.) compared to SARs having non-hybrid TCR chains (e.g., SIR, STAR, HIT, etc.) or second-generation CARs when expressed in immune cells (e.g., T, NK cells). In some embodiments, the present disclosure provides that immune cells expressing SARs having one or more hybrid TCR chains produce less cytokines and have fewer side effects when administered to a subject. In some embodiments, immune cells expressing SARs having one or more hybrid TCR chains exhibit less on-target extratumoral toxicity when administered to a subject. In embodiments, immune cells expressing SARs having one or more hybrid TCR chains are safer when administered to a subject. In embodiments, immune cells expressing SARs having one or more hybrid TCR chains exhibit effective anti-tumor activity when administered to a subject. In embodiments, immune cells expressing SARs with one or more hybrid TCR chains exhibit superior anti-tumor activity against solid tumors compared to conventional second-generation CARs.

[0012] The present disclosure also provides SARs having one or more hybrid TCR chains expressed from an endogenous TCR locus. Furthermore, the present disclosure also provides SARs having one or more hybrid TCR chains that target the locus of an endogenous T cell gene (e.g., TCRα, TCRβ, TCRγ, TCRδ, or CD3ζ). Examples of targeting constructs for targeting SARs having hybrid TCR constant chains to the TRAC locus are also shown in SEQ ID NOs: 8396-8404. Furthermore, additional targeting constructs can be constructed by replacing one or more modules (e.g., vL, vH, vHH, FHVH, TCR chain, etc.) of the constructs shown in SEQ ID NOs: 8396-8404 with different modules described herein.

[0013] The present disclosure provides novel SIR, Ab-TCR, and MHC-SAR / HLA-SAR designs, including those with hybrid chains comprising an activation domain linked to one or both TCR constant chains. In embodiments, the activation domain is derived from the cytoplasmic domain of CD3z or FcRy, or a functional fragment or variant thereof. In embodiments, the activation domain is operatively linked to the C-terminus of one or both TCR constant chains comprising the SIR or Ab-TCR. In embodiments, the SIR comprises a costimulatory domain operatively linked to one or both TCR chains. In embodiments, the costimulatory domain is operatively linked to the C-terminal region of one or both TCR constant chains. In embodiments, the costimulatory domain is operatively linked to the C-terminal region of one or both activation domains that are linked to one or both TCR constant chains. In embodiments, the costimulatory domain is derived from the cytoplasmic domain of CD28, 4-1BB, OX40, CD40, CD27, CD2, etc. Examples of TCR chains with activation domains are set forth in SEQ ID NOs: (DNA): 23901-25240 and SEQ ID NOs: (PRT): 27936-29275. Examples of TCR chains with activation and costimulatory domains are set forth in SEQ ID NOs: (DNA): 25241-27920 and SEQ ID NOs: (PRT): 29276-31955. Examples of SIRs with TCR chains comprising activation and / or costimulatory domains are provided in SEQ ID NOs: (DNA): 27922-27927 and SEQ ID NOs: (PRT): 31957-31962. In embodiments, SIRs, Ab-TCRs, and MHC-SARs with activation and / or costimulatory domains in the TCR constant chain exhibit improved cytokine production, cytotoxicity, and proliferation (e.g., at least a 5% improvement) compared to SIRs, Ab-TCRs, and MHC-SARs lacking activation and / or costimulatory domains in the TCR chain.

[0014] Double-chain SAR constructs comprising an antigen-binding domain linked to one or two CD3z polypeptide chains via an optional linker have been described (see WO 2016 / 187349 A1, WO 2018 / 102795 A1, and PCT / US22 / 17177, which are incorporated herein by reference). The present disclosure provides SARs (e.g., uTCR-SAR, zSIR, zCD16-SAR, etc.) comprising a CD3z chain with a deletion of residue Q101 (dQ101) in the cytoplasmic domain or a functional variant thereof, or a homologue from a non-human species (e.g., mouse, dog, monkey, etc.). An example of a CD3z chain with a deletion of residue Q101 (CD3zECDTMCP-dQ101) is provided in SEQ ID NOs: (DNA): 943-966 and (PRT): 9323-9346. The present disclosure also provides that SARs (e.g., uTCR, zSIR, zCD16SAR, etc.) can be constructed using CD3z chains having at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 98%, or 99% amino acid identity to the CD3z chains provided in SEQ ID NOs: (PRT):9323-9346, 40592-40605. In embodiments, both chains of the dual-chain SAR comprise a CD3z chain with a deletion of the Q101 residue. In embodiments, the dual-chain SAR comprises at least one chain comprising a CD3z chain with a deletion of amino acid residue Q101.

[0015] In embodiments, the double-chain SAR comprises one CD3z chain with a deletion of the Q101 residue and a second chain comprising a transmembrane domain or a membrane-anchoring domain. In embodiments, the second chain may comprise a hinge domain and, optionally, a cytoplasmic domain. In embodiments, the double-chain SAR comprises one CD3z chain with a deletion of the Q101 residue and a second chain comprising a CD16 hinge and transmembrane domain. Such a SAR is referred to as z16SIR, zCD16SAR, or zCD16SIR. In embodiments, the second chain of the zCD16SAR may further comprise a CD16 cytoplasmic domain. In embodiments, the second chain of the zCD16SAR may comprise a costimulatory domain. In embodiments, the second chain of the zCD16SAR may comprise a costimulatory domain but may lack the CD16 cytoplasmic domain or may comprise a partial CD16 cytoplasmic domain. In embodiments, the costimulatory domain is derived from 4-1BB, CD28, OX40, 2B4, CD8a, CD8b, or CD4. In embodiments, the second chain of zCD16SAR may include a signaling domain. In embodiments, the signaling domain may be derived from a kinase. In embodiments, the signaling domain is derived from Lck, mutant Lck, LAT, ZAP-70, SLP-76, or a mutant or variant thereof.

[0016] In an embodiment, the dual-chain SAR comprises one CD3z chain with a deletion of the Q101 residue and a second chain comprising an FcRy hinge and transmembrane domain. Such a SAR is referred to as a zFcRy-SAR. In an embodiment, the second chain may further comprise a cytoplasmic domain. In an embodiment, the second chain of a zCD16SAR may comprise a costimulatory domain. In an embodiment, the first chain and / or the second cytoplasmic domain may comprise one or more ITAMs. In an embodiment, the first chain and / or the second cytoplasmic domain may comprise one or more costimulatory domains. In an embodiment, the first chain and / or the second cytoplasmic domain may comprise one or more co-receptor domains. For example, the co-receptor cytoplasmic domain comprises the cytoplasmic domain CD8a, CD8b, or CD4. In embodiments, SARs comprising a dQ101 mutation in the CD3z chain exhibit enhanced activity (e.g., NFAT activation, cytokine production, or cytotoxicity) compared to SARs comprising a CD3z chain lacking the dQ101 mutation. The present disclosure also provides that SARs (e.g., uTCRs, zSIRs, etc.) can be constructed using CD3z chains that are functional variants of the hybrid CD3z chains provided in SEQ ID NOS: (PRT): 9323-9346, including homologs from non-human species. Examples of SARs having a CD3z chain lacking residue Q101 are provided in SEQ ID NOS: (DNA): 3171-3243 and (PRT): 11551-11623.

[0017] The present disclosure also features novel SARs (e.g., "MHC-SARs") comprising portions of MHC molecules (e.g., class I, class II, non-classical MHC) and non-TCR signaling receptors or non-TCR signaling chains. Examples of such non-TCR signaling receptors and non-TCR signaling chains include CD3z, CD3z-dQ101, FcRγ, DAP10, DAP12, CD16A, CD16B, NKp30, NKp44, and NKp46, as well as functional variants and homologs thereof from non-human species. In some embodiments, the MHC-SAR comprises a portion of an antigenic peptide. In embodiments, the peptide comprises a disulfide trap to enhance the stability and expression of the MHC-SAR. The present invention also features cells expressing the MHC-SAR (cells expressing the MHC-SAR are referred to herein as "redirected cells"), such as T cells or NK cells, macrophages, B cells, dendritic cells, and granulocytes. Redirected cells expressing MHC-SAR (e.g., redirected T cells, NK cells, macrophages, etc.) mimic antigen-presenting cells (APCs), which are cells that normally express MHC molecules. In some cases, when the TCR of a target T cell binds to the MHC-SAR of the redirected cell, the target T cell can be destroyed. Therefore, in this case, the redirected cell may function as an "anti-T cell" T cell, NK cell, or macrophage. The present invention is not limited to redirected cells that function to destroy targets. For example, in some embodiments, the redirected cell is adapted to help reprogram the target cell, e.g., the redirected cell may deliver instructions to the target cell. In embodiments, the MHC-SAR can be used to eliminate autoreactive T cells for the prevention and / or treatment of autoimmune diseases (e.g., multiple sclerosis, diabetes, etc.). Examples of MHC-SAR are set forth in SEQ ID NOs: (DNA): 23780-23794 and (PRT): 23877-23891. These constructs contain the RQ13 peptide linked to the N-terminus of the HLA-DRB-B1 (or D1) domain.These MHC-SARs respond to T cells expressing the F24 CAR (SEQ ID NO: (DNA): 23236 and SEQ ID NO: (PRT): 23682), which recognizes the RQ13 peptide / HLA-DR complex. Because the MHC-SARs are modular in design, different MHC SARs can be constructed by replacing the signaling chain of the above MHC-SAR with a different signaling chain described in this disclosure. For example, the [hTCRb-S57C] and [hTCRa-T48C] chains of MHC-SAR CD8SP-Sph-R13Q-Kpn-G4S-R1-HLA-DRB-B1-[hTCRb-S57C]-F-P2A-SP-HLA-DRA-A1-[hTCRa-T48C] represented by SEQ ID NO: 23887 can be replaced with different TCR β and TCR α or TCR γ and TCR δ chains, including hybrid TCR β, TCR α, TCR γ and TCR δ chains described herein. Additionally, the [hTCRb-S57C] and [hTCRa-T48C] chains can be replaced with TCR chains comprising activation and / or costimulatory domains. Similarly, the RQ13 peptide RFYKTLRAEQ (SEQ ID NO: 23892) can be replaced with another peptide (e.g., MOG peptide, NY-ESO-1, or Glia-γ1 peptide) to target TCRs that recognize these peptide antigens in complex with HLA molecules. The peptides may further contain disulfide traps to enhance the stability of the peptide / MHC complex. Finally, the HLA-DRB-D1 and HLA-DRA-A1 modules can be replaced with different HLA modules, such as HLA-DQB1-D1 (SEQ ID NO: 23775) and HLA-DQA1-D1 (SEQ ID NO: 23771) or HLA-DPB1-D1 (SEQ ID NO: 23755) and HLA-DPA1-D1 (SEQ ID NO: 23752).For example, an MHC-SAR in which the RQ13 peptide is replaced with the Glia-γ1 peptide (SEQ ID NO: 20377), and HLA-DQB1-D1 (SEQ ID NO: 23775) and HLA-DQB1-D1 (SEQ ID NO: 23771) are replaced with the [hTCRb-S57C] and [hTCRa-T48C] modules, respectively, can target immune-reactive T cells that target the gluten-derived Glia-γ1 / HLA-DQ8.5 complex for the prevention and treatment of celiac disease.

[0018] The present disclosure also provides novel accessory modules comprising costimulatory co-receptors (e.g., CD8a, CD8b, and CD4) that can be co-expressed with the disclosed SARs (e.g., uTCR-SARs, zSIRs, MHC-SARs, hybrid chain SARs, SIRs, HC-SARs, Ab-TCRs, etc.). Examples of costimulatory co-receptors are provided in SEQ ID NOs: (DNA): 644-646 and SEQ ID NOs: (PRT): 9024-9026).

[0019] The present invention also features engineered cells that express both a SAR (e.g., MHC-SAR, uTCR-SAR, hybrid chain SAR, SIR, zSIR, etc.) and a surrogate co-receptor (SCR). Engineered cells that co-express a SAR (e.g., MHC-SAR, uTCR-SAR, SIR, hybrid chain SAR, SIR, zSIR, etc.) and an SCR can exhibit enhanced effects (e.g., increased IL-2 expression) compared to engineered cells that express a SAR without co-expressing an SCR.

[0020] The present disclosure provides novel SARs comprising signaling chains comprising the cytoplasmic domains of CD4, CD8b, and CD8a or functional variants thereof. Examples of signaling chains having the CD4, CD8b, and CD8a cytoplasmic domains are set forth in (SEQ ID NOs: (DNA): 555-558 and (PRT): 8925-8934). The present disclosure also provides that SARs (e.g., uTCRs, zSIRs, etc.) can be constructed using signaling chains that have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid homology or identity to the signaling chains provided in SEQ ID NOs: (PRT): 8925-8934. The present disclosure also provides that SARs (e.g., uTCRs, zSIRs, etc.) can be constructed using signaling chains that are functional variants of the signaling chains provided in SEQ ID NOs: (PRT): 8925-8934, including homologs from non-human species. An example of a SAR having a signaling chain comprising a CD4 cytoplasmic domain is represented by SEQ ID NO: (DNA): 1029. An example of a SAR having a signaling chain comprising a CD8a cytoplasmic domain is represented by SEQ ID NO: (DNA): 1028. An example of a SAR having a signaling chain comprising a CD8a cytoplasmic domain and a second signaling chain comprising a CD8b cytoplasmic domain is represented by SEQ ID NO: (DNA): 1046. An example of a SAR having a signaling chain comprising a CD4 cytoplasmic domain and a second signaling chain comprising a CD8a cytoplasmic domain is represented by SEQ ID NO: (DNA): 1047.

[0021] The present disclosure provides novel SARs comprising signaling chains comprising costimulatory domains or functional variants thereof. Examples of signaling chains comprising the cytoplasmic domains of 41BB, CD28, and CD30 are provided in SEQ ID NOs: (DNA): 545-554 and (PRT): 8935-8938). The present disclosure also provides SARs comprising signaling chains comprising at least 70% (e.g., 70%, 75%, 80%, 90%, 95%, 98%, 99%, or 100%) amino acid identity to the signaling chains set forth in SEQ ID NOs: (PRT): 8935-8938, and functional variants thereof.

[0022] The present disclosure provides novel linkers (including novel Ig linkers) that can be used to generate the disclosed SARs (e.g., SIRs). In embodiments, the novel Ig linkers comprise deleted or mutated immunoglobulin-like linker domains derived from antibody and TCR constant chains. In embodiments, the novel Ig linkers comprise deletions or point mutations of any of the linkers set forth in SEQ ID NOS: (PRT): 8961-8994, or functional variants thereof. In embodiments, the novel Ig linkers comprise linkers having at least 70% (e.g., 70%, 75%, 80%, 90%, 95%, 98%, 99%, or 100%) amino acid identity to any one of the linkers set forth in SEQ ID NOS: 8961-8994. In embodiments, the disclosed novel Ig linkers comprise linkers having an N-terminal deletion of 1 to 100 amino acids of any of the linkers set forth in SEQ ID NOS: (PRT): 8961-8994, or functional variants thereof. Examples of TCR constant chains, including hybrid TCR constant chains containing N-terminal deletions of the immunoglobulin-like linker domain, are shown in SEQ ID NOs: (PRT): 18259-18914. The present disclosure also describes that SARs can be generated using functional variants or variants of the above TCR constant chains and / or linkers, including homologs from non-human species.

[0023] In embodiments, the linker comprises an Ig-like constant domain of a TCR chain and further comprises a TCR connecting peptide. For example, long Ig-like linkers are provided in SEQ ID NOs: 22827-22829, 22833-22835, 22839-22840, and 22843-22844, respectively, and functional variants and homologs encoding polypeptides having at least 75% sequence identity to the polypeptides encoded by any of the above sequences are also included. In embodiments, the long Ig-like linkers include N- or C-terminal deletion variants of SEQ ID NOs: 22827-22829, 22833-22835, 22839-22840, and 22843-22844, respectively, in which 1 to 40 (e.g., 1, 5, 10, 15, 20, 25, 30, or 40) N- or C-terminal amino acid residues are deleted.

[0024] The present disclosure provides novel double-chain SARs (e.g., SIRs) and novel single- and half-chain SARs (e.g., SIRs), which comprise one or more non-TCR antigen-binding domains, in which the immunoglobulin-like linker domain (e.g., SEQ ID NOs: (DNA): 597-614 and SEQ ID NOs: (PRT): 8977-8994) contained in one or both TCR constant chains of the double-chain SAR or single- and half-chain SAR is replaced with an Ig-like linker domain derived from a different TCR chain or a mutant or functional variant thereof. Furthermore, the present disclosure provides novel double-chain SARs (e.g., SIRs) and novel single-half-chain SARs (e.g., SIRs) comprising one or more non-TCR antigen-binding domains, in which the immunoglobulin-like linker domain (e.g., SEQ ID NOs: (DNA): 597-614 and SEQ ID NOs: (PRT): 8977-8994) and connecting peptide / hinge domain (e.g., SEQ ID NOs: (DNA): 615-622 and SEQ ID NOs: (PRT): 8995-9002) contained in one or both TCR constant chains of the double-chain SAR or single-half-chain SAR are replaced with an Ig-like linker domain and connecting peptide (or hinge domain) derived from a different TCR chain or a mutant or functional variant thereof. Examples of linking peptide / hinge domains of TCR alpha are represented by SEQ ID NOS: 8995-8996), TCR beta (SEQ ID NOS: 8997-8998), TCR gamma (SEQ ID NOS: 8999-9000), and TCR delta (SEQ ID NOS: 9001-9002) and are shown in Table 7 of the provisional patent application. The present disclosure also provides SARs comprising hybrid TCR constant chains comprising connecting peptides having at least 70% amino acid identity to the connecting peptides set forth in SEQ ID NOS: (PRT): 8909-8924, as well as SARs comprising homologues from non-human species, including functional variants and variants thereof.

[0025] The present disclosure provides novel double-chain SARs (e.g., SIRs) and novel single-half-chain SARs (e.g., SIRs) comprising one or more non-TCR antigen-binding domains, in which an immunoglobulin-like linker domain (e.g., SEQ ID NOs: (DNA): 597-614 and SEQ ID NOs: (PRT): 8977-8994) contained in one or both TCR constant chains of the double-chain SAR or single-half-chain SAR is replaced with a linker domain not derived from a TCR chain or a mutant or functional variant thereof.

[0026] The present disclosure also provides novel single-chain and half-chain SARs comprising one or more non-TCR antigen-binding domains in which the immunoglobulin-like linker domain (e.g., the immunoglobulin-like domain of one or both TCR constant chains of a double-chain SAR or single-and-half-chain SAR (e.g., SEQ ID NOs: (DNA(597-614) and SEQ ID NOs: (PRT)) 8977-8994) is replaced with an Ig linker domain derived from a different antibody / immunoglobulin or mutant or functional variant thereof. Examples of Ig linker domains derived from different antibodies / immunoglobulins are shown in Table 7 of the provisional application (SEQ ID NOs: (DNA): 581-596 and SEQ ID NOs: (PRT): 8961-8976)). The present disclosure also provides SARs comprising hybrid TCR constant chains comprising Ig linkers having at least 70 (e.g., 70%, 75%, 80%, 90%, 95%, 98%, 99% or 100%) amino acid identity to the Ig linkers set forth in SEQ ID NOs: (PRT):8909-8924, and homologs thereof from non-human species, including functional variants and variants thereof.

[0027] Examples of immunoglobulin-like linker domains of the TCR alpha constant chain are provided in SEQ ID NOs: (PRT):8977-78, 8986-88, including functional variants, mutants, and homologs thereof, including homologs from non-human species. In embodiments, the immunoglobulin-like linker domain of the TCR alpha constant chain comprises a polypeptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 85%, 90%, 95%, 98%, 99% of 100%) amino acid sequence identity to any of the sequences represented in SEQ ID NOs: (PRT):8977-78 and 8986-88, or functional variants, mutants, or homologs thereof. In embodiments, the immunoglobulin-like linker domain of the TCR alpha constant chain comprises a polypeptide having a deletion of up to 60 (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.) N-terminal amino acids of any of the sequences set forth in SEQ ID NOs: (PRT):8977-78 and 8986-88, or functional mutants, variants or homologs thereof.

[0028] Examples of immunoglobulin-like linker domains of the TCR β constant chain are provided in SEQ ID NOs: (PRT): 8979-80, 8985, 8989-90, including functional variants, mutants, and homologs thereof, including homologs from non-human species. In embodiments, the immunoglobulin-like linker domain of the TCR β constant chain comprises a polypeptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 85%, 90%, 90%, 95%, 98%, 99%) amino acid sequence identity to any of SEQ ID NOs: (PRT): 8979-80, 8985, 8989-90, and functional variants, mutants, and homologs thereof. In embodiments, the immunoglobulin-like linker domain of the TCR alpha constant chain comprises a polypeptide having a deletion of up to 60 (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.) N-terminal amino acids of any of the sequences represented by SEQ ID NOs: (PRT):8979-80, 8985, 8989-90, or functional variants, mutants or homologs thereof.

[0029] Examples of immunoglobulin-like linker domains of the TCR gamma constant chain are provided in SEQ ID NOs: (PRT):8981-82 and 8991-92, and include functional variants, mutants, and homologs thereof, including homologs from non-human species. In embodiments, the immunoglobulin-like linker domain of the TCR gamma constant chain comprises a polypeptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 85%, 90%, 90%, 95%, 98%, 99%) amino acid sequence identity to any of SEQ ID NOs: (PRT):8981-82 and 8991-92, or functional variants, mutants, or homologs thereof. In embodiments, the immunoglobulin-like linker domain of the TCR gamma constant chain comprises a polypeptide having up to 60 (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.) deletions. The N-terminal amino acid of any of the sequences represented by SEQ ID NOs: (PRT): 8981-82 and 8991-92, or a functional mutant, variant or homologue thereof.

[0030] Examples of immunoglobulin-like linker domains of the TCR delta constant chain are provided in SEQ ID NOs (PRT):8983-84 and 8993-94, and include functional variants, mutants, and homologs thereof, including homologs from non-human species. In embodiments, the immunoglobulin-like linker domain of the TCR delta constant chain comprises a polypeptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 90%, 95%, 98%, 99% of 100%) amino acid sequence identity to any of the sequences represented in SEQ ID NOs (PRT):8983-84 and 8993-94, or functional variants, mutants, or homologs thereof. In embodiments, the immunoglobulin-like linker domain of the TCR delta constant chain comprises a polypeptide having up to 60 (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.) deletions. The N-terminal amino acid of any of the sequences represented by SEQ ID NOs: (PRT): 8983-84 and 8993-94, or a functional mutant, variant or homologue thereof.

[0031] In various embodiments, the present disclosure provides novel double-chain (DC) SARs and novel single-half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked to one or more hybrid TCR constant chains via an optional linker. In embodiments, the hybrid TCR constant chains comprise chains in which the immunoglobulin-like linker domain of a TCR constant chain is replaced by the immunoglobulin-like linker domain of a different TCR constant chain or homolog or variant thereof having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 90%, 95%, 98%, 100% or 99%) amino acid sequence identity. In embodiments, the hybrid TCR alpha constant chain comprises a chain in which the immunoglobulin-like linker domain of the TCR alpha constant chain is replaced by the immunoglobulin-like linker domain of a TCR beta, TCR gamma, TCR delta or pre-TCR alpha constant chain, or a homolog or variant thereof, and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) to 99% amino acid sequence identity. In embodiments, the hybrid TCR beta constant chain comprises a chain in which the immunoglobulin-like linker domain of the TCR beta constant chain is replaced by the immunoglobulin-like linker domain of a TCR alpha, TCR gamma, TCR delta or pre-TCR alpha constant chain, or a homolog or variant thereof, and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) to 99% amino acid sequence identity. In embodiments, the hybrid TCR gamma constant chain comprises a chain in which the immunoglobulin-like linker domain of the TCR gamma constant chain is replaced by the immunoglobulin-like linker domain of a TCR alpha, TCR beta, TCR delta or pre-TCR alpha constant chain, or a homolog or variant thereof, and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) to 99%) amino acid sequence identity.In embodiments, the hybrid TCRδ constant chain comprises a chain in which the immunoglobulin-like linker domain of the TCRδ constant chain is replaced by an immunoglobulin-like linker domain that is a TCRα, TCRβ, TCRγ, or pre-TCRα constant chain, or a homolog or variant thereof, and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) >99% amino acid sequence identity. In embodiments, the hybrid pre-TCRa constant chain comprises a chain in which the immunoglobulin-like linker domain of the pre-TCRα constant chain is replaced by an immunoglobulin-like linker domain of a TCRα, TCRβ, TCRγ, TCRδ constant chain, or a homolog or variant thereof, and has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) >99% amino acid sequence identity.

[0032] In an embodiment of the invention, the disclosure provides novel double-chain (DC) SARs and novel single half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked via an optional linker to a hybrid TCR chain, wherein the immunoglobulin-like linker domain of the TCR alpha constant chain is replaced by the immunoglobulin-like linker domain of the TCR beta constant chain, or a mutant or variant thereof, and the immunoglobulin-like linker domain of the TCR beta constant chain is replaced by the immunoglobulin-like linker domain of the TCR alpha constant chain, or a mutant or variant thereof.

[0033] In an exemplary embodiment, the present disclosure provides novel single-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked to a hybrid TCR chain via an optional linker, wherein the immunoglobulin-like linker domain of the TCR gamma constant chain is replaced by the immunoglobulin-like linker domain of a TCR delta constant chain or a mutant or variant thereof, and the immunoglobulin-like linker domain of the TCR gamma constant chain, the immunoglobulin-like linker domain of the TCR gamma constant chain, the immunoglobulin-like linker domain of the TCR gamma constant chain, the immunoglobulin-like linker domain of the TCR gamma constant chain, the immunoglobulin-like linker domain of the TCR gamma constant chain, and the immunoglobulin-like linker domain of the TCR gamma constant chain. The TCR delta constant chain is replaced by the immunoglobulin-like linker domain of the TCR gamma constant chain, or a mutant or variant thereof.

[0034] In embodiments, the present disclosure provides novel dual-chain (DC) SARs and novel single-half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains linked via an optional linker to a hybrid TCR chain in which the immunoglobulin-like linker domain of a TCR alpha constant chain is linked via an optional linker to the immunoglobulin-like linker domain of a TCR gamma constant chain, or a mutant or variant thereof, and the immunoglobulin-like linker domain of a TCR gamma constant chain, or a mutant or variant thereof, and the TCR beta constant chain is replaced by the immunoglobulin-like linker domain of a TCR delta constant chain, or a mutant or variant thereof.

[0035] In exemplary embodiments, the present disclosure provides novel double-chain (DC) SARs and novel single half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked via any linker to a hybrid TCR chain, wherein the immunoglobulin-like linker domain of the TCR alpha constant chain is operatively linked to the immunoglobulin-like linker domain of the TCR delta constant chain, or a mutant or variant thereof, and the immunoglobulin-like linker domain of the TCR delta constant chain and the immunoglobulin-like linker domain of the TCR delta constant chain are less than 100 μm, and the TCR beta constant chain is replaced by the immunoglobulin-like linker domain of the TCR gamma constant chain, or a mutant or variant thereof.

[0036] In embodiments, the present disclosure provides novel double-chain (DC) SARs and novel single-half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked via an optional linker to a hybrid TCR chain, wherein the immunoglobulin-like linker domain of the TCR gamma constant chain is replaced by the immunoglobulin-like linker domain of a TCR alpha constant chain or a mutant or variant thereof, the immunoglobulin-like linker domain of the TCR gamma constant chain, the immunoglobulin-like linker domain of the TCR gamma constant chain, the immunoglobulin-like linker domain of the TCR gamma constant chain, and the immunoglobulin-like linker domain of the TCR gamma constant chain. The TCR delta constant chain is replaced by the immunoglobulin-like linker domain of a TCR beta constant chain, or a mutant or variant thereof.

[0037] In exemplary embodiments, the present disclosure provides novel double-chain (DC) SARs and novel single half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked via an optional linker to a hybrid TCR chain, wherein the immunoglobulin-like linker domain of the TCR gamma constant chain is replaced by the immunoglobulin-like linker domain of the TCR beta constant chain or a mutant or variant thereof, and the immunoglobulin-like linker domain of the TCR delta constant chain is replaced by the immunoglobulin-like linker domain of the TCR alpha constant chain or a mutant or variant thereof.

[0038] In embodiments, the present disclosure provides novel single-chain (OHC) SARs and novel single-half-chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked via an optional linker to a hybrid TCR chain, in which the immunoglobulin-like linker domains of a double-chain SAR or one TFR or one TCR constant chain (e.g., SEQ ID NOs(DNA):597-6714 and SEQ ID NOs(PRT):8977-8994) are operatively linked via an optional linker to the hybrid TCR chain, in which the half-chain SAR is replaced with an Ig linker domain derived from an immunoglobulin (Ig) light chain or an immunoglobulin (Ig) heavy chain, or a mutant or variant thereof.

[0039] An example of an immunoglobulin linker domain derived from an immunoglobulin light chain (IgCL) is provided in SEQ ID NO: (PRT):8961, and includes functional variants, mutants, and homologs thereof, including homologs from non-human species. In embodiments, an immunoglobulin linker domain of an immunoglobulin light chain (IgCL) comprises a polypeptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 85%, 90%, 90%, 95%, 98%, or 995%) amino acid sequence identity to the sequence set forth in SEQ ID NO: (PRT):8961 or a functional variant, mutant, or homolog thereof. In embodiments, an immunoglobulin linker domain of an immunoglobulin light chain (IgCL) comprises a polypeptide having up to 80 (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, etc.) deletions. The N-terminal amino acid of any of the sequences represented by SEQ ID NO: (PRT):8961 or a functional variant, mutant or homologue thereof.

[0040] For example, immunoglobulin linker domains derived from immunoglobulin heavy chains (e.g., IgG1-CH1, IgG2-OC-CH1, IgG4-CHI1, etc.) are provided in SEQ ID NOs: (PRT):8962-8976, including functional variants, variants, and homologs thereof, including homologs from non-human species. In embodiments, the immunoglobulin linker domain of an immunoglobulin heavy chain comprises a polypeptide having at least 70% (e.g., 70%, 75%, 80%, 80%, 85%, 90%, 95%, 98%, or 995%) amino acid sequence identity to the sequence represented in SEQ ID NOs: (PRT):8962-8976 or a functional variant, variant, or homolog thereof. In embodiments, the immunoglobulin linker domain of the immunoglobulin heavy chain comprises a polypeptide having a deletion of up to 80 (e.g., 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, etc.) N-terminal amino acids of any of the sequences represented by SEQ ID NOs: (PRT):8962-8976, or functional mutants, variants, or homologs thereof.

[0041] In embodiments, the present disclosure provides novel single and half chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked, via an optional linker, to a hybrid TCR chain in which the immunoglobulin-like linker domain of a TCR alpha or TCR gamma constant chain is replaced by an immunoglobulin linker domain of an immunoglobulin light chain (e.g., e.g., SEQ ID NO: (PRT): 8961) or a variant or variant thereof and the immunoglobulin-like linker domain of a TCR beta or TCR delta constant chain is replaced by an immunoglobulin linker domain of an immunoglobulin heavy chain (e.g., SEQ ID NO: (PRT): 8962-8976) or a variant or variant thereof.

[0042] In an exemplary embodiment, the present disclosure provides novel single and half chain (OHC) SARs comprising one or more non-TCR antigen-binding domains operatively linked, via an optional linker, to a hybrid TCR chain in which the immunoglobulin-like linker domain of a TCR β or TCR δ constant chain is replaced by an immunoglobulin linker domain of an immunoglobulin light chain (e.g., SEQ ID NO: (PRT):8961) or a variant or variant thereof, and the immunoglobulin-like linker domain of a TCR α or TCR γ constant chain is replaced by an immunoglobulin linker domain of an immunoglobulin heavy chain (e.g., SEQ ID NO: (PRT):8962-8976) or a variant or variant thereof.

[0043] In embodiments, the present disclosure provides novel single and half chain (OHC) SARs comprising one or more non-TCR antigen binding domains operatively linked, via an optional linker, to a hybrid TCR chain in which the immunoglobulin-like linker domain of a TCR gamma constant chain is replaced by an immunoglobulin linker domain of an immunoglobulin light chain (e.g., e.g., SEQ ID NO: (PRT): 8961) or a variant or variant thereof and the immunoglobulin-like linker domain of a TCR delta constant chain is replaced by an immunoglobulin linker domain of an immunoglobulin heavy chain (e.g., SEQ ID NO: (PRT): 8962-8976) or a variant or variant thereof.

[0044] The present disclosure provides novel single-chain and half-chain SARs in which the immunoglobulin-like linker domain (e.g., SEQ ID NOs: (DNA): 597-614 and (PRT): 8977-8994) of one or both TCR constant chains of the single-chain and half-chain SARs is replaced with an Ig linker domain derived from an antibody (e.g., IgCL, IgG1-CH1 domain) or a mutant or variant thereof. Ig linker domains derived from antibodies are provided in SEQ ID NOs: (DNA): 581-596 and (PRT): 8961-8975). Examples of such SAR constructs are set forth in SEQ ID NOs: (DNA): 18023-18258 and (PRT): 18915-19740.

[0045] The present disclosure also provides novel hybrid chain TCRs comprising two chains in which TCR variable domains (e.g., Va and Vb or Vg and Vd) are linked via an Ig linker (e.g., IgCL and IgG1-CH1 or IgCL and IgG4-CH1, etc.) to a TCR module comprising the transmembrane domains of the TCR chains. In embodiments, the TCR module further comprises a connecting peptide and a cytoplasmic domain of the TCR chain. Examples of such hybrid chain TCRs targeting the NY-ESO-1 peptide / HLA-A2 complex are set forth in SEQ ID NOs: (DNA): 19760 and 19761 and SEQ ID NOs: (PRT): 20380 and 20381, respectively. In NY-ESO-1-IG4-Vb-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-NY-ESO-1-IG4-Va-[IgG1-CH1-TCRa-wt-op2-6MD] (SEQ ID NO: 19760), the Vb domain of the TCR targeting the NY-ESO-1 TCR is joined via an IgCL linker to a TCRb-wt-opt2-6MD module containing the connecting peptide, transmembrane domain, and cytoplasmic domain of the human TCR β chain, while the Va domain of the TCR is joined via an IgG1-CH1 linker to a TCRa-wt-op2-6MD module containing the connecting peptide, transmembrane domain, and cytoplasmic domain of the human TCR α chain. The hybrid chain TCR constructs of SEQ ID NO: (DNA):19761 and SEQ ID NO: (PRT):20381 are similar, except that the TCRb-wt-opt2-6MD module is replaced with the module IgCL-TCRg-6MD, which contains the connecting peptide, transmembrane domain, and cytoplasmic domain of the human TCR gamma chain, and the TCRa-wt-op2-6MD module is replaced with the module TCR delta-6MD, which contains the connecting peptide, transmembrane domain, and cytoplasmic domain of the human TCR delta chain. The two TCR chains of a hybrid chain TCR are less likely to pair with endogenous TCR chains, and therefore T cells expressing such hybrid chain TCRs are less likely to cause autoimmunity or GVHD.In some embodiments, the IgCL and IgG1-CH1 linkers are replaced with other Ig linkers described in this application (e.g., IgCL and IgG4-CH1) to generate hybrid chain TCRs with unique structural and functional properties. Similarly, TCR modules can be exchanged with other TCR modules described in this application to generate unique hybrid chain TCRs. Finally, variable domains can be replaced with other variable domains described in this application to generate hybrid chain TCRs that target different peptide / HLA complexes. In some embodiments, one or more autonomous antigen-binding domains (AABDs) (e.g., vHH, FHVH, DARPIN, AFFIBODY, CENTYRIN, svd-TCR, etc.) are attached to or near the N-terminus of the TCR variable domains (e.g., Va, Vb, Vg, Vd) of the hybrid chain TCR to generate bispecific and multispecific hybrid chain TCRs that can target multiple antigens and / or antigen peptide / MHC complexes.

[0046] In some embodiments, immunoresponsive cells (e.g., T, NK, NKT, etc.) or hematopoietic stem cells expressing SAR are obtained from an individual's circulating blood by apheresis. In one aspect, the cells are collected from a subject in which T cells, NK cells, or stem cells have been mobilized by administration of a drug. In some embodiments, the immune cells are obtained from a donor who has been administered a CXCR4 antagonist (e.g., plerixafor, BL-8040, BPRCX714, BPRCX807), a cytokine (e.g., G-CSF, GM-CSF, or sargramostim, neurasta, or pegfilgastrim, IL2, IL15), a beta 2 agonist (e.g., epinephrine), a tyrosine kinase inhibitor (e.g., dasatinib), or a chemotherapeutic agent (e.g., cyclophosphamide, doxorubicin, etc.) prior to collection of the immune cells. In some embodiments, the donor is an autologous donor, and in other embodiments, the donor is an allogeneic donor.

[0047] The present disclosure also provides proliferation-free methods for generating immunoresponsive cells expressing the disclosed SARs (e.g., SIRs, zSIRs, Ab-TCRs, uTCR-SARs, CD16-SARs, etc.). In embodiments, the SARs are double-chain SARs. In embodiments, the SARs are single-half-chain SARs. In embodiments, the SARs lack a cytoplasmic signaling domain. In embodiments, the SARs lack a cytoplasmic activation domain containing an ITAM. In embodiments, the SARs lack a costimulatory domain. In embodiments, the SARs do not contain a CD28 or 41BB costimulatory domain. In embodiments, the SARs provide physiological T cell receptor or NK receptor signaling. In embodiments, the SARs contain a T cell receptor module. In embodiments, the SARs are capable of recruiting a signaling adaptor. In embodiments, the signaling adaptor is selected from the group consisting of CD3z, FcRy, DAP10, and DAP12. In embodiments, the SARs are not second-generation chimeric antigen receptors. In some embodiments, the SAR does not include a CD3z or FcRy activation domain. In some embodiments, the SAR includes a TCR signaling chain selected from TCRα, TCRβ, TCRγ, TCRδ, preTCRα, CD16, NKp30, NKp44, NKp46, or a functional fragment, variant, or homolog thereof. In some embodiments, the SAR is selected from the group consisting of a double-chain SIR, a one-chain SIR, a one-and-a-half-chain SIR, a double-chain Ab-TCR, and a double-chain SIR with a hybrid TCR chain, and a one-and-a-half-chain SIR with a hybrid TCR chain, zSIR, UTCR-SAR, TFP, CD16-SAR, FceRγ-SIR, and vFLIP-CAR.

[0048] In embodiments, one or more steps of the expansion-free method for manufacturing a cell therapy product are performed in a closed system. In embodiments, one or more steps of the expansion-free method for manufacturing a cell therapy product are performed in an automated manner. In embodiments, one or more steps of the expansion-free method for manufacturing a cell therapy product are performed using a Prodigy (Miltenyi), Cocoon (Lonza), or Cellares cell shuttle. In embodiments, one or more steps of the expansion-free method for manufacturing a cell therapy product are performed using a gas permeable device. In embodiments, one or more steps of the expansion-free method for manufacturing a cell therapy product are performed in a G-Rex apparatus (Wilson-Wolf Manufacturing). In embodiments, one or more steps of the expansion-free method for manufacturing a cell therapy product are performed using a Wave bioreactor.

[0049] In embodiments, an expansion-free method for generating a SAR expressing a SAR comprises a) obtaining a population of immune effector cells (e.g., PBMCs) from a subject, wherein the subject optionally administers to the subject one or more mobilizing agents (e.g., a CXCR4 antagonist, e.g., Plerixafor, BL-8040, BPRCX714, BPRCX807, etc.), cytokines (e.g., G-CSF, GM-CSF, or sargramostim, Neulasta, or Pegfilgastrim, IL2, IL15);b) an optional step of isolating a subpopulation of immune effector cells (e.g., T cells, CD4, CD8, Treg, αβT cells, γδT cells, NK, NKT cells, Pgp-expressing stem cells such as T cells or NK cells); c) an optional step of eliminating the expression of one or more genes (e.g., β2M, TRAC, TRBC, etc.) in the immune effector cells; d) an optional step of activating the immune effector cells with one or more cytokines, optionally wherein the cytokines are selected from the group of IL2, IL7, IL15, or combinations thereof; e) an optional step of activating the immune effector cells with an agent that activates the TCR, where the agent is an antibody against CD3 or CD3 antibody-coated beads; and f) an optional step of adding a co-stimulatory agent during the activation step of (e), optionally wherein the co-stimulatory agent activates the CD28 receptor. g) a step of introducing a SAR expression construct into immune effector cells, optionally wherein the SAR expression construct is introduced by contacting the immune effector cells with a viral vector encoding the SAR for a sufficient period of time, optionally wherein the viral vector is a lentiviral vector or a gamma retroviral vector, and optionally wherein the contacting is performed in the presence of an agent that enhances gene transfer by the viral vector, optionally wherein the agent is selected from the group consisting of polybrene, protamine, retronectin, vectofusin, or a combination thereof; h) an optional step of removing the activator or activator-coated beads (i.e., debeading); i) an optional washing step; j) an optional step of concentrating the cell therapy product; k) an optional step of cryopreserving the cell therapy product; and l) optional procedures for safety and efficacy testing.

[0050] In embodiments, the SAR expression construct (e.g., RNA, viral vector) is introduced into immune effector cells or stem cells capable of giving rise to immune effector cells, which can be produced in less than 12 hours (e.g., 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 1 minute, etc.) of removal of the cells from the subject. In embodiments, the SAR expression construct (e.g., RNA, viral vector) is introduced into immune effector cells or stem cells, which can give rise to immune effector cells during a collection step (e.g., apheresis). In embodiments, the SAR expression construct (e.g., RNA, viral vector) is introduced into immune effector cells or stem cells capable of giving rise to immune effector cells in the same container (e.g., bag) in which the apheresis cell product is collected from the subject. In embodiments, the SAR expression construct (e.g., RNA, viral vector) is introduced into immune effector cells or stem cells capable of giving rise to immune effector cells at the same location (e.g., collection facility, room) where the apheresis cell product is collected from the subject. In embodiments, the SAR expression construct (e.g., RNA, viral vector) is introduced into immune effector cells or stem cells capable of giving rise to immune effector cells using a unit operatively attached to the apheresis machine. In embodiments, the SAR expression construct (e.g., RNA, viral vector) is introduced into immune effector cells or stem cells capable of giving rise to immune effector cells using a closed unit operatively attached to the apheresis machine.

[0051] In embodiments, the cells expressing the SAR expression construct are administered to the subject less than 36 hours (e.g., less than 36 hours, less than 24 hours, less than 20 hours), less than 12 hours, less than 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, 5 minutes, less than 1 minute) after collection from the subject (i.e., vein-to-vein time). In embodiments, the cells expressing the SAR expression construct are administered to the subject from the same container (e.g., bag) from which the cells are collected at the time of apheresis. In embodiments, the cells expressing the SAR expression construct are administered to the subject at the same location (e.g., collection facility, collection room) from which the cells are collected from the subject at the time of apheresis. In embodiments, the cells expressing the SAR expression construct are administered to the subject using a unit operatively attached to, part of, or integrated with the apheresis machine. In embodiments, cells expressing a SAR expression construct are administered to a subject using a closed unit that is operatively attached to an apheresis machine.

[0052] In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into the immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to the subject are performed in less than 36 hours (e.g., less than 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, less than 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 1 minute, etc.). In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into the immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to the subject are performed in a single visit. In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into the immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to the subject are performed at a single location (e.g., a collection facility, collection room). In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to a subject are performed using a single machine. In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to a subject are performed using machines (units) that are operatively linked to each other. In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to a subject are performed using a single machine or machines (units) that share a single power source (e.g., electricity). In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to a subject are performed using a single machine or machine (unit) controlled by a single computer or software. In embodiments, the steps of collecting immune cells or stem cells (i.e., apheresis), introducing SAR into the immune cells or stem cells, and administering the SAR-expressing immune cells or stem cells to a subject are performed using a single closed system.In embodiments, the SAR is introduced into stem cells capable of giving rise to immune cells via any method of gene transduction known in the art, including but not limited to, viral vectors (e.g., lentivirus, gamma retrovirus), virus-like particles, lipid nanoparticles, electroporation, lipofection, or by causing a transient perturbation in the cell membrane.

[0053] In embodiments, the proliferation-free cell method includes incubating a population of immune cells (e.g., T cells, e.g., freshly isolated T cells, e.g., freshly isolated resting T cells) in medium without serum or containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, or 2% serum, e.g., for at least about 1-10 hours, e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours, e.g., at least about 2-6 hours; (ii) transducing the population of immune cells with a nucleic acid molecule on a lentiviral vector encoding a SAR in medium containing serum (e.g., at least about 4, 5, or 6% serum) and optionally deoxynucleosides (e.g., at least about 40 μM to 1.5 mM deoxynucleosides, e.g., at least about 40 μM); 45 μM, 50 μM, 55 μM, 60 μM, 70 μM, 80 μM, 90 μM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM deoxynucleoside), e.g., for about 14 to 30 hours, e.g., about 14, 16, 18, 20, 22, 24, 26, or 28 hours. Optionally, step (ii) is performed at a cell concentration of at least about 0.7 x 10, 0.8 x 10, 0.9 x 10, 1 x 10, 2 x 10, 4 x 10, 6 x 10, 8 x 10, or 1 x 10 cells / mL, e.g., step (ii) is performed at a cell concentration of about 1 x 10 cells / mL. In embodiments, optionally, the population of immune cells is not contacted in vitro with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule, e.g., an anti-CD3 antibody and / or an anti-CD28 antibody.In embodiments, the expansion-free method includes (iii) recovering or administering the population of immune cells for storage (e.g., reformulating the population of immune cells in cryopreservation medium), wherein (a) step (iii) is performed within 48 hours, e.g., within 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 hours after the initiation of step (i); (b) the population of immune cells from step (iii) is not expanded or is expanded by no more than 10, 20, 30, 40, or 50%, e.g., no more than 10%, compared to the population of immune cells at the initiation of step (i); (c) the proportion of naive cells, e.g., naive T cells, in the population of immune cells from step (iii) is not reduced; Alternatively, the proportion of naive cells, e.g., naive T cells, in the population of immune cells at the start of step (i) is reduced by no more than 10%, 20%, or 30%; and / or (d) the proportion of differentiated cells, e.g., differentiated T cells, e.g., terminally differentiated T cells, e.g., CCR7low T cells, in the population of immune cells from step (iii) is not increased; or the proportion of differentiated cells, e.g., differentiated T cells, e.g., terminally differentiated T cells, e.g., CCR7low T cells, in the population of immune cells at the start of step (i) is increased by no more than 10%, 20%, or 30%. In an embodiment, step (i) comprises incubating a population of immune cells (e.g., T cells, e.g., freshly isolated T cells, e.g., freshly isolated resting T cells) in serum-free medium for about 2 to 6 hours.

[0054] In embodiments, the cell therapy product is expanded for 1 to 10 days after introduction of the SAR expression construct.

[0055] In embodiments, a viral vector (e.g., a lentivirus or retrovirus gamma) encoding a SAR is pseudotyped with a modified baboon envelope glycoprotein, a modified HERV-W1 envelope glycoprotein, or a VSVG envelope glycoprotein. In embodiments, a viral vector (e.g., a lentivirus or retrovirus gamma) encoding a SAR is pseudotyped with a modified baboon envelope glycoprotein described herein. In embodiments, the modified baboon envelope glycoprotein has a sequence having SEQ ID NOs: (DNA): 70-96, 98, 115-122, 264-273 or SEQ ID NOs: (PRT): 8450-8476, 8478, 8495-8502, 8644-8655, 50005-50010, or SEQ ID NOs: (PRT): 8450-8476, 8478, 8495-8502, 8644-8655, 50005-50010 extracellular domain. In embodiments, the modified baboon envelope glycoprotein encodes a protein comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 95%, 98% sequence identity within the transmembrane domain to SEQ ID NOs: (PRT): 8450-8476, 8478, 8495-8502, 8644-8655, 50005-50010. In embodiments, the modified baboon envelope glycoprotein encodes a protein that binds to hASCT1 or hASCT2.

[0056] In embodiments, viral vectors (e.g., lentiviruses or retrovirus gamma) encoding SARs are pseudotyped with modified HERV-W1 envelope glycoproteins described herein. In embodiments, the modified HERV-W1 envelope glycoproteins are encoded by a recombinant polynucleotide encoding an exogenous signal peptide sequence. In embodiments, viral vectors (e.g., lentiviruses or retrovirus gamma) encoding SARs are pseudotyped with modified HERV-W1 envelope glycoproteins having SEQ ID NOs: (DNA): 149-151, 159-164 or SEQ ID NOs: (PRT): 8529-8530, 8539-8544, 8581, or encoded envelope glycoproteins having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to SEQ ID NOs: (PRT): 8529-8530, 8539-8544, 8581 extracellular domain. In embodiments, the modified HERV-W1 envelope glycoprotein encodes a protein comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to the transmembrane domain of SEQ ID NOs: (PRT): 8529-8530, 8539-8544, 8581. In embodiments, the modified HERV-W1 envelope glycoprotein encodes a protein that binds to hASCT1 or hASCT2.

[0057] In embodiments, immune effector cells are transduced with a SAR-encoding viral vector pseudotyped with a combination of two or more different envelope glycoproteins (e.g., baboon and VSVG or HERV-W1 or VSVG, etc.). In embodiments, the SAR expression construct is introduced into immune effector cells using a non-viral delivery method. In embodiments, the non-viral method includes virus-like particles (VLPs) or lipid nanoparticles (LNPs). In embodiments, the SAR expression construct is introduced into immune effector cells using electroporation, lipofection, or by causing a transient perturbation in the cell membrane. In embodiments, the SAR is expressed from an endogenous locus. In embodiments, the SAR expression cassette is inserted into the genomic locus of an endogenous gene. In embodiments, the SAR expression cassette is inserted into the genomic locus of an endogenous gene. In embodiments, insertion of the SAR at the endogenous locus results in disruption of expression of the endogenous gene. In embodiments, the SAR is expressed under the promoter and regulatory elements of the endogenous gene. In embodiments, the endogenous locus is selected from the TRAC, TRBC, TRDC, TRGC, CD3z, CD16, and FceRy1 loci.

[0058] In embodiments, SAR-encoding cell therapy products produced using expansion-free methods are superior to products produced using methods involving expansion of the cell therapy product in one or more of the following: a) are produced in a shorter period of time (e.g., between 1 and 12 days); b) result in cost savings (e.g., 5-95% cost reduction compared to expansion methods); c) have fewer production failures (e.g., 5-95% fewer production failures compared to expansion methods); d) exhibit superior in vitro properties (e.g., at least a 5% improvement in one or more of the following parameters compared to expansion methods: cell death, cytokine production, lack of exhaustion markers, maintenance of CD4:CD8 ratio; lack of terminal differentiation, maintenance of stem-like / naive / memory phenotype; e) exhibit superior in vivo activity (e.g., at least a 5% improvement in one or more of the following parameters compared to expansion methods: long-term persistence, lack of fatigue, anti-tumor activity, etc.). f) when administered to a subject in need thereof, demonstrates at least 5% superior disease control (e.g., tumor control); g) demonstrates superior safety (e.g., cytokine release syndrome or neurotoxicity is reduced by at least 5% compared to products manufactured using expansion methods, etc.); and h) requires the administration of a lower cell dose (e.g., at least a 5% reduction in the administered cell dose compared to cell therapy products manufactured using expansion methods).

[0059] The present disclosure provides single- and double-stranded next-generation SAR designs that provide physiological signaling.

[0060] The present disclosure provides next-generation single-chain and double-chain SARs comprising one or more heterologous antigen-binding domains (e.g., vL, vH, scFv, vHH, FHVH, non-immunoglobulin antigen-binding scaffolds, epitopes, etc.) operatively linked to or near the N-terminus of the extracellular domain of a CD16 chain comprising a deleted and / or mutated CD16 cytoplasmic domain. Examples of CD16 chains with deleted and mutated cytoplasmic domains that can be used to construct SARs are set forth in SEQ ID NOs: (PRT): 8945-8948. In embodiments, SARs can be constructed using CD16 chains with at least 70% amino acid sequence homology to SEQ ID NOs: (PRT): 8945-8948 or functional variants thereof. Examples of SARs with deleted and mutated cytoplasmic domains are set forth in SEQ ID NOs: (DNA): 1111-2234 and SEQ ID NOs: (PRT): 9491-10614. In embodiments, the SAR can be configured to have one or more antigen-binding domains attached at or near the N-terminus of the entire or partial extracellular domain of a CD16 chain represented by SEQ ID NO: (PRT):8945-8948 or a functional variant thereof.

[0061] The present disclosure provides next-generation single-chain and double-chain SARs comprising one or more heterologous antigen-binding domains operatively linked to or near the N-terminus of the extracellular domain of a CD16 chain (e.g., FCGR1A, FCGR1B, FCGR1C). Examples of CD64 chains with deleted and mutated cytoplasmic domains that can be used to construct SARs are set forth in SEQ ID NO: (PRT):20383. In embodiments, SARs can be constructed using CD64 chains with at least 70% amino acid sequence homology to SEQ ID NO: (PRT):20382-83 or functional variants thereof. In embodiments, SARs can be configured such that one or more antigen-binding domains are attached to or near the N-terminus of the entire or partial extracellular domain of a CD64 chain represented by SEQ ID NO: (PRT):20382-83 or functional variants thereof.

[0062] The present disclosure provides novel antigen-binding domains (e.g., scFv, vL, vH, vHH, etc.) that target different antigens. The novel vL fragments are represented by SEQ ID NOs: 339-354, 19766-19776, and 32006-32068, and the complementary vH fragments are represented by SEQ ID NOs: 363-378, 19785-19795, and 32069-32131 (Tables 3 and 5). The present disclosure also provides novel vHH domains, including humanized vHHs, that target different antigens. These vHH domains are represented by SEQ ID NOs: 412-426 and 32195-32213. These novel antigen-binding domains (e.g., scFv, vL, vH, vHH, etc.) can be used to construct SARs (SIR, zSIR, Ab-TCR, CAR, etc.), antibodies, bispecific antibodies, antibody-drug conjugates, etc. The present disclosure also provides novel antigen-binding domains having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 99%) amino acid sequence identity in the framework regions to the novel antigen-binding domains (e.g., scFv, vL, vH, vHH, etc.) provided in Tables 3 and 5. The present disclosure also provides novel antigen-binding domains having up to two amino acid differences (i.e., 1 or 2) in each CDR of the novel antigen-binding domains (e.g., scFv, vL, vH, vHH, etc.) defined in Tables 3 and 5. The light chain complementarity-determining regions 1 to 3 (LC-CDR1-3) of these novel vL domains are as set forth in any of SEQ ID NOs: 20989-21015, 41591-41861, 21024-21050, 41862-42132, 21059-21085, and 42133-42403. Heavy chain complementarity determining regions 1-3 (HC-CDR1-3) are as set forth in any of SEQ ID NOS: 21094-21120 and 42404-42674; 21129-21155, 42675-42945; and 21164-21190, 42946-43216. CDR1-3 of the novel vHH domain are provided in SEQ ID NOS: 43304-43318. Examples of SARs containing these novel antigen-binding domains are provided in SEQ ID NOS (DNA): 3303-3929, 19823-21960, 33860-40139, and 40173-40427.

[0063] The present disclosure provides novel TCR variable domains (Table 4) that can be used in constructing the disclosed SARs (e.g., uTCR-SARs). Examples of uTCR-SARs comprising these TCR variable domains are shown in Table 12 of the provisional patent application. In embodiments, the TCR variable domains are capable of binding to peptide / MHC complexes independent of a co-receptor. In embodiments, the TCR variable domains are capable of binding to peptide / MHC complexes independent of CD8a, CD8b, or CD4. In embodiments, the TCR variable domains are capable of binding to peptide / MHC complexes with higher affinity than naturally occurring TCRs. In embodiments, the TCR variable region comprises an exogenous disulfide bond. In embodiments, the present disclosure provides SARs (e.g., uTCR-SARs) that are capable of binding to peptide / MHC complexes independent of a co-receptor. In embodiments, the SARs (e.g., uTCR-SARs) are capable of binding to peptide / MHC complexes independent of CD8a, CD8b, or CD4. In embodiments, the SAR (e.g., uTCR-SAR) comprises a TCR variable region comprising an exogenous disulfide bond. In embodiments, the SAR (e.g., uTCR-SAR) comprises a TCR constant region comprising an exogenous disulfide bond. In embodiments, the SAR (e.g., uTCR-SAR) comprises a mutation that creates a cysteine ​​residue in the TCR variable domain, resulting in the formation of an exogenous disulfide bond. In embodiments, the exogenous disulfide bond is an intrachain disulfide bond. In embodiments, the SAR (e.g., uTCR-SAR) comprises a TCR constant region comprising an exogenous disulfide bond. In embodiments, the exogenous disulfide bond is an interchain disulfide bond between two TCR constant chains.

[0064] The present disclosure provides a γδ T cell expressing a double-chain SAR or a single-half-chain SAR (e.g., a double-chain SIR or a single-half-chain SIR) comprising a non-TCR antigen-binding domain (e.g., vL, vH, scFv, vHH, etc.) operatively linked to a TCR α and a TCR β constant chain. In embodiments, the SAR (SIR) comprises a TCR α / β constant chain having a wild-type nucleic acid sequence. In embodiments, the SAR (SIR) comprises a TCR α / β constant chain having a wild-type amino acid sequence. In embodiments, the TCR α and TCR β constant chains comprise mutations (e.g., T48C and S57C) that result in the formation of a second disulfide bond. In embodiments, the TCR α and TCR β constant chains comprise mutations that result in better matchups between them and reduced reconciliation with endogenous TCR α and / or TCR β constant chains. In embodiments, the TCR α and TCR β constant chains (e.g., SIR) of the SAR comprise murine amino acid residues that result in better expression. In embodiments, the TCR α and TCR β constant chains of the SAR are of murine origin. In embodiments, the TCR α and TCR β constant chains of the SAR are deleted (e.g., an N-terminal deletion of 1 to 60 amino acids).

[0065] The present disclosure provides a γδ T cell expressing a double-chain SAR or a single-half-chain SAR (e.g., a double-chain SIR or a single-half-chain SIR) comprising a non-TCR antigen-binding domain (e.g., vL, vH, scFv, vHH, etc.) operatively linked to a hybrid TCR constant chain (e.g., a hybrid TCR α and TCR β constant chain or a hybrid TCR γ and TCR δ chain). In embodiments, the SAR (SIR) comprises a hybrid TCR α / β or TCR γ / δ constant chain having the wild-type nucleic acid sequence of the TCR α / β or TCR γ / δ constant chain. In embodiments, the SAR (SIR) comprises a hybrid TCR α / β constant chain or a hybrid TCR γ / δ constant chain having the wild-type amino acid sequence. In embodiments, the hybrid TCR α and TCR β constant chains comprise mutations (e.g., T48C and S57C) that result in the formation of a second disulfide bond. In embodiments, the hybrid TCR α and TCR β constant chains contain mutations that result in better matchups between them and reduced matchups with endogenous TCR α and / or TCR β constant chains. In embodiments, the hybrid TCR α and TCR β constant chains (e.g., SIRs) of the SAR contain mouse amino acid residues that result in better expression. In embodiments, the hybrid TCR α, β, γ, or δ constant chains of the SAR are of mouse origin. In embodiments, the hybrid TCR α, β, γ, or δ constant chains of the SAR are derived from a species other than human (i.e., mouse, cat, dog, monkey, etc.). In embodiments, the hybrid TCR α, β, γ, or δ constant chains of the SAR have a deletion (e.g., an N-terminal deletion of 1 to 60 amino acids).

[0066] The present disclosure provides γδ T cells expressing a double-chain SAR or a single-half-chain SAR (e.g., a double-chain SIR or a single-half-chain SIR) comprising a non-TCR antigen-binding domain (e.g., vL, vH, scFv, vHH, etc.) operatively linked to TCRγ and TCRδ constant chains. In embodiments, the SAR (SIR) comprises a TCRγ / δ constant chain having a wild-type nucleic acid sequence. In embodiments, the SAR (e.g., SIR) comprises a TCRγ / δ constant chain having a wild-type amino acid sequence. In embodiments, the TCRγ and TCRδ constant chains of the SAR have a deletion (e.g., an N-terminal deletion of 1 to 60 amino acids). In embodiments, the T cells lack or have reduced expression of endogenous TCRγ chains and / or TCRδ chains.

[0067] The present disclosure provides γδ T cells expressing a double-chain SAR, single-chain SAR, or single-half-chain SAR (e.g., a double-chain SIR or a single-half-chain SIR) comprising a non-TCR antigen-binding domain (e.g., vL, vH, scFv, vHH, etc.) operatively linked to pre-TCRα and TCRβ constant chains. In embodiments, the SAR (SIR) comprises pre-TCRα and TCRβ constant chains with wild-type nucleic acid sequences. In embodiments, the SAR (SIR) comprises pre-TCRα and TCRβ constant chains with wild-type amino acid sequences. In embodiments, the pre-TCRα and TCRβ constant chains of the SAR have a deletion (e.g., an N-terminal deletion of 1 to 60 amino acids). In embodiments, the T cells lack or have reduced expression of endogenous TCRα and / or TCRβ chains.

[0068] In embodiments, the SAR-expressing γδ T cells (e.g., SIR or NK-SAR) are derived from embryonic stem cells. In embodiments, the SAR-expressing γδ T cells (e.g., SIR or NK-SAR) are derived from induced pluripotent embryonic stem cells (iPSCs). In embodiments, the SAR-expressing γδ T cells (e.g., SIR or NK-SAR) are derived from umbilical cord blood. In embodiments, the SAR-expressing γδ T cells (e.g., SIR or NK-SAR) are derived from a donor. In embodiments, the donor is an autologous donor. In embodiments, the donor is an allogeneic donor.

[0069] The present disclosure provides αβ T cells expressing a double-chain SAR or single-half-chain SAR (e.g., a double-chain SIR or a single-half-chain SIR) comprising a non-TCR antigen-binding domain (e.g., vL, vH, scFv, vHH, etc.) operatively linked to TCRγ and TCRδ constant chains. In embodiments, the SAR (SIR) comprises a TCRγ / δ constant chain having a wild-type nucleic acid sequence. In embodiments, the SAR (SIR) comprises a TCRγ / δ constant chain having a wild-type amino acid sequence. In embodiments, the TCRγ and TCRδ constant chains of the SAR have a deletion (e.g., an N-terminal deletion of 1 to 60 amino acids). In embodiments, the SAR (SIR) comprises a hybrid TCRα / β or TCRγ / δ constant chain. In embodiments, the αβ T cells lack or have reduced expression of endogenous TCR α and / or TCR β chains.

[0070] In embodiments, the SAR-expressing αβ T cells (e.g., SIR or NK-SAR) are derived from embryonic stem cells. In embodiments, the SAR-expressing αβ T cells (e.g., SIR or NK-SAR) are derived from induced pluripotent embryonic stem cells (iPSCs). In embodiments, the SAR-expressing αβ T cells (e.g., SIR or NK-SAR) are derived from umbilical cord blood. In embodiments, the SAR-expressing αβ T cells (e.g., SIR or NK-SAR) are derived from a donor. In embodiments, the donor is an autologous donor. In embodiments, the donor is an allogeneic donor.

[0071] The present disclosure also provides embryonic stem cells (e.g., iPS cells) expressing one or more disclosed SARs (e.g., SIRs, NK-SARs, uTCR-SARs). The present disclosure also provides αβ and γδ T cells derived from embryonic stem cells (e.g., iPSCs) expressing one or more disclosed SARs (e.g., SIRs, NK-SARs, uTCR-SARs). In embodiments, the embryonic stem cells (e.g., iPSCs) lack or have reduced expression of one or more endogenous TCR chains. In embodiments, the embryonic stem cells (e.g., iPSCs) have reduced or decreased expression of β2M and / or HLA molecules.

[0072] The present disclosure also provides NK cells, NKT cells, umbilical cord-derived NK cells, umbilical cord-derived T cells, umbilical cord-derived T cells, and umbilical cord-derived stem cells expressing any of the SARs described in this disclosure (e.g., SIR, HC-SAR, zSIR, zCD16-SIR, uTCR-SAR, etc.).

[0073] In embodiments, SAR-expressing cells (e.g., iPSCs, NK, T, NKT, CD34+ cells, etc.) lack or have reduced expression of TNFα, IL1α, IL1β, and / or IFNγ. In embodiments, SAR-expressing cells (e.g., iPSCs, NK, T, NKT, CD34+ cells, etc.) lack or have genetic disruption of TNFα, IL1α, IL1β, and / or IFNγ genes. In embodiments, SAR-expressing cells (e.g., iPSCs, NK, T, NKT, CD34+ cells, etc.) overexpress CD47 and / or Fc receptors (e.g., CD64, CD16). In an embodiment, SAR-expressing cells (e.g., iPSCs, NK, T, NKT, CD34+ cells, etc.) express a CD16- or CD64-based SAR (i.e., a SAR comprising an exogenous antigen-binding domain linked to a CD64 or CD16 chain, comprising the extracellular domain, hinge domain, transmembrane domain, and optionally the cytoplasmic domain of CD64 or CD16). An example CD16-SAR is provided in Tables 9 and 10 of the provisional patent application. An example CD64-based SAR is described in WO2022178367, which is incorporated herein by reference. Alternatively, a CD64-based SAR can be generated by replacing the CD16 signaling chain in the SARs described in Tables 9 and 10 of the provisional patent application with the CD64 signaling chain (SEQ ID NOs: (DNA): 900-901 or SEQ ID NOs: (PRT): 9280-81).

[0074] The present disclosure also provides regulatory T cells (Tregs) expressing one or more of the disclosed SARs (e.g., SIRs, NK-SARs, uTCR-SARs). In embodiments, the Tregs overexpress Fox3P. The disclosure provides that regulatory T cells expressing the disclosed SARs can be used to regulate immune responses. In embodiments, Tregs expressing the disclosed SARs (e.g., SARs targeting HLA molecules) can be used to enhance transplant tolerance in subjects receiving HLA-mismatched transplants. In embodiments, Tregs expressing the disclosed SARs can be used to regulate immune responses in subjects with autoimmune diseases (e.g., inflammatory bowel disease or multiple sclerosis).

[0075] The present disclosure provides novel viral envelope proteins for pseudotyping lentiviral vectors. Examples of viral envelope proteins include modified baboon envelope (mBaEV) and modified HERV-W1 envelope proteins. These viral envelope proteins can be used to transduce cells that are difficult to infect, such as NK cells and CD34+ stem cells. In embodiments, the viral envelope proteins can be used for in vivo delivery of nucleic acids without eliciting a significant immune response. The present disclosure provides that a combination of two different pseudotyped viruses can be used to enhance gene transfer into cells. The present disclosure also provides novel envelope glycoproteins (e.g., SEQ ID NOs: 8539 and 8450-8476) that can be used to transduce cells without prior activation or stimulation. Examples of novel viral envelope glycoproteins are provided in SEQ ID NOs: (DNA): 70-96, 98, 115-122, 145, 159 and SEQ ID NOs: (PRT): 8539 and 8450-8476, 8525, 8539.

[0076] The present disclosure provides cell lines stably expressing reporter genes (e.g., marine luciferase and thermostable beetle luciferase), which are rendered replication-incompetent by treatment but can be used to measure cytotoxicity using the Matador assay or the Matador-Glo cytotoxicity assay. In embodiments, the cell lines are rendered replication-competent by treatment with mitomycin-C. In embodiments, the cell lines are rendered replication-competent by treatment with irradiation. In embodiments, the cell lines are rendered replication-enabled by treatment with ionizing irradiation.

[0077] The disclosed SAR can be expressed and functionally active in any mammalian cell. In an embodiment, the mammalian cell is a T cell, a NK cell, a macrophage, a granulocyte, or the like. In an embodiment, the cell is an umbilical cord-derived cell. In an embodiment, the cell is an umbilical cord-derived T cell, a NK cell, a NKT cell, or a stem cell. In some embodiments of any of the mammalian cells described herein, the mammalian cells are selected from the group consisting of CD8+ T cells, CD4+ T cells, memory T cells, naive T cells, T stem cells, Treg cells, natural killer T (NKT) cells, iNKT (innate innate killer cells), NK cells, g-NK cells, memory-like NK cells, cytokine-induced killer cells (CIK), iPS cell-derived NK cells, α / β T cells, γ / δ T cells, iPS cell-derived T cells, B cells, macrophages / monocytes, iPS cells. In some embodiments of any of the mammalian cells described herein, the mammalian cells are selected from the group consisting of iPSCs (induced pluripotent stem cells), embryonic stem cells, and hematopoietic stem cells capable of giving 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, YTS, or derivatives thereof. In some embodiments of any of the mammalian cells described herein, the mammalian cells are mammalian cells obtained from a subject. In some embodiments of any of the mammalian cells described herein, the subject is diagnosed or identified as having cancer or an autoimmune disease (e.g., lupus, multiple sclerosis, etc.). In some embodiments of any of the mammalian cells described herein, the subject is human. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. Examples of diseases that can be targeted by the antigen-targeting SAR of the present invention are provided in PCT / US19 / 035096, which is incorporated herein by reference.

[0078] Also provided herein are SARs (e.g., uTCR-SAR, HC-SAR, zCD16-SAR, etc.) that can be functionally expressed in cells other than T cells, i.e., cells including, but not limited to, NK cells, monocytes, macrophages, dendritic cells, granulocytes, stem cells, embryonic stem cells, and / or iPS cells. In embodiments, cells expressing SARs (e.g., uTCR-SAR, HC-SAR, zCD16-SAR, etc.) are engineered to express a co-receptor. In embodiments, the co-receptor is CD8a, CD8b, or CD4. In embodiments, the cells overexpress a chimeric CD8 molecule. In embodiments, the cells overexpress a chimeric CD8a / CD8b molecule. In embodiments, the cells overexpress a chimeric CD8 / CD4 molecule. In embodiments, SAR-expressing cells (e.g., uTCR-SAR, HC-SAR, zCD16-SAR, etc.) are engineered to express IL12 or an IL12 fusion protein (e.g., IL12f or membrane-anchored IL12f). In embodiments, the cells lack expression of β2M, MHC class I and class II, and HLA-E. In embodiments, the cells overexpress CD47 and an Fc receptor (e.g., CD64). In embodiments, the cells have a mutation in the calreticulin gene. In embodiments, the cells have genetic disruption or reduced expression of TNFα, IL1α, IL1β, IL6, IFNα, IFNβ, and / or IFNγ genes. In embodiments, the cells express IL2, IL7, or IL15, optionally in a membrane-bound form.

[0079] Also provided herein are polypeptides encoding any of the SARs described herein.

[0080] 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.

[0081] Also provided are pharmaceutical compositions 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 are kits comprising any of the pharmaceutical compositions described herein.

[0082] In some embodiments, a method for killing a target cell presenting one or more target antigens is provided, which includes contacting the target cell with an effector cell expressing 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.

[0083] 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 is in vitro.

[0084] In some embodiments, methods are provided for the detection, isolation, purification, expansion, enrichment, and removal of cells that express any of the SARs described herein.

[0085] Also provided herein are methods for producing cells that express single-, double-, and multi-chain SARs and / or accessory modules, including introducing into a mammalian cell any of the nucleic acids encoding any of the nucleic acids described herein, or any of the sets of nucleic acids encoding any of the multi-chain SARs described herein.

[0086] Also provided herein are methods for treating or preventing a disease in a subject (e.g., cancer, an infectious disease, an allergy, an autoimmune disease, etc.), comprising administering to the subject a therapeutically effective amount of any of the mammalian cells described herein. The present disclosure also provides methods comprising administering to the subject a SAR molecule, a cell expressing a SAR molecule, or a cell comprising a nucleic acid encoding a SAR molecule. In one embodiment, the subject has a disorder described herein, e.g., the subject has a cancer, an infectious disease, an allergic disease, a degenerative disease, or an autoimmune disease that expresses a target antigen described herein. In yet one embodiment, the subject is at increased risk for a disorder described herein, e.g., the subject is at increased risk for a cancer, an infectious disease, an allergic disease, a degenerative disease, or an autoimmune disease (e.g., lupus, multiple sclerosis, diabetes, inflammatory bowel disease, etc.), which expresses a target antigen described herein.

[0087] In some embodiments of any of the SARs described herein, the heterologous antigen-binding domain is an antibody, an antibody fragment (vL, vH, Fab, etc.), scFv, (scFv)2, a VHH domain, a FHVH (fully human vH domain), a single domain antibody, a non-immunoglobulin antigen-binding scaffold (e.g., centyrin, affibody, ZIP domain, adaptor, etc.), a VNAR domain, a ligand, a TCR, a variable domain of a TCR (Va, Vb, Vg, Vd), and a receptor.

[0088] In some embodiments of any of the 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 SARs described herein, the tumor antigen is selected from the antigens listed in Table B.

[0089] Also provided herein are mammalian cells containing any of the vectors described herein.

[0090] Also provided herein are methods for generating SAR-expressing cells, methods comprising introducing any of the nucleic acids described herein or any of the vectors described herein into a mammalian cell.

[0091] In another or further embodiment of any of the foregoing, any immune cell or stem cell comprises multiple SAR polypeptides. In embodiments, the multiple SAR polypeptides are based on different SAR architectures (e.g., backbones), such as SIR, CAR, zSIR, uTCR-SAR, TFP, etc. In another or further embodiment of any of the foregoing, at least one SIR polypeptide (e.g., SIR) of the plurality of SARs targets a different antigen than at least one other SAR polypeptide. In some embodiments, one SAR targets an antigen expressed on blood cells (e.g., CD19, CD20, CD22, BCMA, etc.), and a second SAR targets an antigen preferentially expressed on solid tumor cells (e.g., PSMA, Her2, MSLN, etc.). In another or further embodiment of any of the foregoing, at least one SAR polypeptide of the plurality of SAR polypeptides targets the same antigen. In another or further embodiment of any of the foregoing, at least one SAR polypeptide of the plurality of SAR polypeptides comprises a binding affinity for an antigen that differs from at least one other SAR polypeptide. In another or further embodiment of any of the foregoing, the immune cell further comprises at least one SAR encoding an SIR or zSIR or Ab-TCR and a second SAR encoding a chimeric antigen receptor (CAR) polypeptide. In another or further embodiment of any of the foregoing, the antigen-binding domain of the first SAR polypeptide targets a different antigen than the antigen-binding domain of the CAR polypeptide. In another or further embodiment of any of the foregoing, the CAR polypeptide comprises an intracellular signaling domain that includes a costimulatory signaling domain but not a primary signaling domain, or an intracellular signaling domain that includes a primary signaling domain but not a costimulatory signaling domain.In another or further embodiment of any of the foregoing, the CAR polypeptide comprises a costimulatory signaling domain comprising a functional signaling domain of a protein selected from the group consisting of 4-1BB, CD28, CD27, or OX-40, or the CAR molecule comprises a primary signaling domain comprising a functional signaling domain of CD3 zeta. In another or further embodiment of any of the foregoing, the CAR polypeptide is an inhibitory CAR polypeptide, the inhibitory CAR polypeptide comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule, wherein the inhibitory molecule is PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5. In alternative or further embodiments of any of the foregoing, the CAR polypeptide further comprises an intracellular signaling domain comprising a primary signaling domain and / or an intracellular signaling domain, wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional domain of CD3 zeta and a costimulatory signaling domain comprising a functional domain of 4-lBB or CD28 or both.

[0092] Also provided herein are methods of treating cancer or an autoimmune disease 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 prior to the administering step, 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 culture medium between the introducing and administering steps. In some embodiments, the SAR-T-expressing cells are generated in vivo. In some embodiments of any of the methods described herein, the subject is human.

[0093] In some embodiments, the cells are exposed to an agent that improves the efficacy and / or safety of the cells. In embodiments, the agent is selected from a tyrosine kinase inhibitor, e.g., a Src kinase inhibitor, e.g., an Lck inhibitor, e.g., dasatinib, ponatinib, a JAK / STAT inhibitor (e.g., ruxolitinib), an mTOR inhibitor, or a bispecific or multispecific T or NK cell activating antibody (e.g., BiTE, BiKE, TriKE, etc.), or a CSF1R antibody. In some embodiments, the cells are exposed to the agent in vitro. In some embodiments, the cells are exposed to the agent in vivo. In some embodiments, the subject is administered the agent before, simultaneously with, or after administration of cells expressing a SAR. In some embodiments, the subject is administered the agent before, simultaneously, or after administration of a vector expressing a SAR. In some aspects, the subject receives a single dose of the agent, while in other embodiments, the subject receives multiple doses of the agent.

[0094] In some embodiments of any of the multi-stranded SARs described herein, the SAR lacks an ITAM but recruits a signaling protein that comprises an ITAM-containing primary stimulatory domain, hi some embodiments, the SAR recruits a signaling protein selected from the group consisting of CD3z, FcRγ, DAP10, and DAP12.

[0095] In some embodiments, a method for producing a T cell expressing a hybrid chain synthetic antigen receptor (HC-SAR or HC-SAR) or hybrid-SAR is provided. The method can include contacting a T cell with a first nucleic acid encoding a first non-TCR antigen-binding domain (e.g., a vL, vH, vHH, FHVH, scFv, a non-immunoglobulin antigen-binding scaffold, an adapter, an epitope, a receptor, or a ligand, etc.), that is, operatively linked via an optional linker to a first hybrid TCR constant chain, wherein the first hybrid TCR chain comprises a transmembrane domain and a second TCR chain constant domain, but does not comprise the first TCR chain constant domain. The method may include contacting a T cell with a second nucleic acid encoding a second non-TCR antigen-binding domain (e.g., vL, vH, vHH, FHVH, scFv, non-immunoglobulin antigen-binding scaffold, adapter, epitope, receptor, or ligand, etc.) operatively linked to a second hybrid TCR chain via an optional linker, wherein the second hybrid TCR chain comprises a second chain transmembrane domain and a first TCR chain constant domain, but not a second TCR chain constant domain. In embodiments, the non-TCR antigen-binding domain operatively linked to the first hybrid TCR constant chain is a vL fragment of an antibody, and the non-TCR antigen-binding domain operatively linked to the second hybrid TCR constant chain is a complementary vH fragment of that antibody. In embodiments, the vL and vH fragments can form an Fv-like antigen-binding module that specifically binds to a target antigen.

[0096] In some embodiments, a method for generating a T cell expressing a hybrid chain synthetic antigen receptor (HC-SAR) or hybrid-SAR is provided. The method can include contacting the T cell with a first nucleic acid encoding a first hybrid TCR constant chain, where the first hybrid TCR chain comprises a transmembrane domain, a variable domain of an antibody light chain (vL), and a second TCR chain constant domain, but does not comprise the first TCR chain constant domain. The method can include contacting the T cell with a second nucleic acid encoding a second hybrid TCR chain, where the second hybrid TCR chain comprises a second chain transmembrane domain, a variable domain of an antibody heavy chain (vH), and the first TCR chain constant domain, but does not comprise the second TCR chain constant domain. The first chain variable domain can comprise a vL variable domain, the first chain constant domain can comprise an alpha or gamma chain constant domain, the first chain transmembrane domain can comprise an alpha or gamma chain transmembrane domain, the second chain variable domain can comprise a vH variable domain, the second chain constant domain can comprise a beta or delta chain constant domain and a second chain, the transmembrane domain can comprise a beta or delta chain transmembrane domain; or the first chain variable domain can comprise a vH variable domain, the first chain constant domain can comprise an alpha or delta chain constant domain, the first chain transmembrane domain can comprise an alpha or delta chain transmembrane domain, the second chain variable domain can comprise a vL variable domain, the second chain constant domain can comprise a beta or gamma chain constant domain and a second chain, the transmembrane domain can comprise a beta or gamma chain transmembrane domain. Thus, the method can include configuring a T cell to express an HC-SAR comprising the first hybrid chain and the second hybrid chain. In some embodiments, the first hybrid chain further comprises a second chain-connecting peptide, and the second hybrid chain further comprises a first chain-connecting peptide, where the first chain-connecting peptide comprises an alpha- or gamma-chain connecting peptide and the second chain-connecting peptide comprises a beta- or delta-chain connecting peptide; or where the first chain-connecting peptide comprises an alpha- or delta-chain connecting peptide and the second chain-connecting peptide comprises a beta- or gamma-chain connecting peptide.In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain constant domain comprises an alpha chain constant domain, the first chain transmembrane domain comprises a beta chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain constant domain comprises a beta chain constant domain, and the second chain transmembrane domain comprises an alpha chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain constant domain comprises a gamma chain constant domain, the first chain transmembrane domain comprises a gamma chain transmembrane domain, the second chain variable domain comprises a vH domain, the second chain constant domain comprises a delta chain constant domain, and the second chain transmembrane domain comprises a delta chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain constant domain comprises an alpha chain constant domain, the first chain transmembrane domain comprises a gamma chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain constant domain comprises a beta chain constant domain, and the second chain transmembrane domain comprises a delta chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL domain, the first chain constant domain comprises an α chain constant domain, the first chain transmembrane domain comprises a delta chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain constant domain comprises a beta chain constant domain, and the second chain transmembrane domain comprises a gamma chain transmembrane domain. It should be understood that the vL domain and vH domain in the above constructs can be substituted such that the first chain comprises a vH domain and the second chain comprises a complementary vL domain. Furthermore, one or more autonomous antigen-binding domains (AABDs), such as vHH, FHVH, DARPIN, Centyrin, adaptor, receptor, or ligand, may be attached to or near the N-terminus of the vL and / or vH domains of the two-chain hybrid SIR. Some example monospecific, bispecific, and universal SIRs targeting different antigens are provided in Tables 16-18 of the provisional patent application.

[0097] In some embodiments, a method for generating a T cell expressing a hybrid chain synthetic antigen receptor or hybrid SAR is provided. The method may include contacting the T cell with a first nucleic acid encoding a non-TCR antigen-binding domain (e.g., a vL, vH, vHH, FHVH, scFv, a non-immunoglobulin antigen-binding scaffold, an adapter, an epitope, a receptor, or a ligand, etc.) operatively linked via an optional linker to a first hybrid TCR constant chain, the first hybrid TCR chain comprising a first TCR chain transmembrane domain that is a hybrid of two TCR chains (e.g., TCRα and TCRδ or TCRβ and TCRγ) and a second TCR chain constant domain. The method may include contacting the T cell with a second nucleic acid encoding a second hybrid TCR chain, where the second hybrid TCR chain transmembrane domain is a hybrid of two TCR chains (e.g., TCRα and TCRδ or TCRβ and TCRγ).

[0098] The method can include contacting a T cell with a first nucleic acid encoding a non-TCR antigen-binding domain (e.g., vL, vH, vHH, FHVH, scFv, non-immunoglobulin antigen-binding scaffold, adaptor, epitope, receptor, or ligand, etc.) operatively linked via an optional linker to a first hybrid TCR constant chain, the first hybrid TCR chain comprising a first TCR chain transmembrane domain that is a hybrid of two TCR chains (e.g., TCRα and TCRδ or TCRβ and TCRγ) and a second TCR chain constant domain. The method can include contacting the T cell with a second nucleic acid encoding a second hybrid TCR chain, wherein the second hybrid TCR chain transmembrane domain is a hybrid of two TCR chains (e.g., TCRα and TCRδ or TCRβ and TCRγ).

[0099] In some embodiments, a method for producing an immune cell (e.g., a T cell) that expresses a hybrid chain synthetic antigen receptor (HC-SAR) or hybrid-SAR is provided. The method can include contacting a T cell with a first nucleic acid encoding a non-TCR antigen-binding domain (e.g., a vL, vH, vHH, FHVH, scFv, a non-immunoglobulin antigen-binding scaffold, an adapter, an epitope, a receptor, or a ligand, etc.), which is operatively linked via an optional linker to a first hybrid TCR constant chain, wherein the first hybrid TCR chain comprises a first TCR chain transmembrane domain and a second TCR chain Ig-like domain, but does not comprise the first TCR chain Ig-like domain. The method may include contacting a T cell with a second nucleic acid encoding a second hybrid (or hybrid) TCR chain, wherein the second hybrid TCR chain comprises a second chain transmembrane domain, a non-TCR antigen-binding domain (e.g., vL, vH, vHH, FHVH, scFv, non-immunoglobulin antigen-binding scaffold, adapter, epitope, receptor, or ligand, etc.), and a first TCR chain Ig-like domain, but does not comprise a second TCR chain constant region. In embodiments, the vL of the first hybrid TCR chain and the vH of the second hybrid TCR chain interact to form a fragment variable (Fv) that can specifically bind to a target antigen when expressed on the surface of an immune cell (e.g., a T cell). In embodiments, immune cells expressing the hybrid SAR can initiate a signaling pathway when exposed to a target antigen-expressing cell. In embodiments, immune cells expressing the hybrid SAR can initiate cell activation, differentiation, proliferation, cytokine secretion, and / or cytotoxicity when exposed to a target antigen-expressing cell.

[0100] In embodiments, the SAR having the hybrid TCR chain exhibits higher cell surface expression when expressed on immune cells (e.g., T cells) than a cTCR comprising the wild-type nucleic acid and amino acid sequence of the TCR chain but comprising the same antigen-binding domain. In embodiments, the SAR having the hybrid TCR chain exhibits at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) higher cell surface expression when expressed on immune cells (e.g., T cells) than a cTCR comprising the wild-type nucleic acid and amino acid sequence of the TCR chain but comprising the same antigen-binding domain. The expression of the hybrid chain SAR and the cTCR is measured using techniques known in the art, such as protein L staining and / or Topanga assay.

[0101] In embodiments, a SAR having a hybrid TCR chain exhibits reduced chain pairing with endogenous TCR chains when expressed on an immune cell (e.g., a T cell) compared to a cTCR or SIR comprising the same antigen-binding domain. In embodiments, a SAR having a hybrid TCR chain exhibits at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) subchain pairing with endogenous TCR chains when expressed on an immune cell (e.g., a T cell) compared to a cTCR and / or SIR comprising the same antigen-binding domain.

[0102] In embodiments, when expressed on immune cells (e.g., T cells), the SAR having a hybrid TCR chain exhibits higher cell activation, differentiation, proliferation, cytokine secretion, and / or cytotoxicity than a cTCR comprising a wild-type nucleic acid and the amino acid sequence of a TCR chain but the same antigen-binding domain. In embodiments, when expressed on immune cells (e.g., T cells), the SAR having a hybrid TCR chain exhibits at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) higher cell activation, differentiation, proliferation, cytokine secretion, and / or cytotoxicity than a cTCR comprising a wild-type nucleic acid and the amino acid sequence of a TCR chain but the same antigen-binding domain. Cell activation, differentiation, proliferation, cytokine secretion, and cytotoxicity are measured using techniques known in the art, such as flow cytometry, ELISA, and Matador cytotoxicity assay.

[0103] In some embodiments, a method for generating a T cell expressing a hybrid chain synthetic antigen receptor (HC-SAR) or hybrid-SAR is provided. The method can include contacting the T cell with a first nucleic acid encoding a first hybrid (or hybrid) TCR constant chain, where the first hybrid TCR chain comprises a transmembrane domain, a variable domain of an antibody light chain (vL), and a second TCR chain Ig-like domain, but does not comprise the first TCR chain Ig-like domain. The method can include contacting the T cell with a second nucleic acid encoding a second hybrid (or hybrid) TCR chain, where the second hybrid TCR chain comprises a second chain transmembrane domain, a variable domain of an antibody heavy chain (vH), and the Ig-like domain of the first TCR, but does not comprise the Ig-like domain of the second TCR chain. The first chain variable domain can comprise a vL variable domain, the first chain Ig-like domain can comprise an α- or γ-chain Ig-like domain, the first chain transmembrane domain can comprise an α- or γ-chain transmembrane domain, the second chain variable domain can comprise a vH variable domain, and the second chain Ig-like domain can comprise a β- or delta-chain Ig-like domain, the transmembrane domain can comprise a beta- or delta-chain transmembrane domain; or the first chain variable domain can comprise a vH variable domain, the first chain Ig-like domain can comprise an α- or delta-chain constant domain, the first chain transmembrane domain can comprise an α- or delta-chain transmembrane domain, the second chain variable domain can comprise a vL variable domain, and the second chain Ig-like domain can comprise a β- or gamma-chain Ig-like domain and the second chain transmembrane domain can comprise a beta- or gamma-chain transmembrane domain. Thus, the method can include configuring a T cell to express an HC-SAR comprising the first hybrid chain and the second hybrid chain.In some embodiments, the first hybrid chain further comprises a second chain-connecting peptide, and the second hybrid chain further comprises a first chain-connecting peptide, where the first chain-connecting peptide comprises an α- or γ-chain connecting peptide and the second chain-connecting peptide comprises a beta- or delta-chain connecting peptide, or the first chain-connecting peptide comprises an alpha- or delta-chain connecting peptide and the second chain-connecting peptide comprises a beta- or gamma-chain connecting peptide. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain constant domain comprises an alpha chain constant domain, the first chain transmembrane domain comprises a beta chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain constant domain comprises a beta chain constant domain, and the second chain transmembrane domain comprises an alpha chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain Ig-like domain comprises a gamma chain Ig-like domain, the first chain transmembrane domain comprises a gamma chain transmembrane domain, the second chain variable domain comprises a vH domain, the second chain Ig-like domain comprises a delta chain Ig-like domain, and the second chain transmembrane domain comprises a delta chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain Ig-like domain comprises an alpha chain Ig-like domain, the first chain transmembrane domain comprises a gamma chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain Ig-like domain comprises a beta chain Ig-like domain, and the second chain transmembrane domain comprises a delta chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL domain, the first chain Ig-like domain comprises an α chain Ig-like domain, the first chain transmembrane domain comprises a delta chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain Ig-like domain comprises a β chain Ig-like domain, and the second chain transmembrane domain comprises a gamma chain transmembrane domain. It should be understood that the vL and vH domains in the above constructs can be interchanged such that the first chain comprises a vH domain and the second chain comprises a complementary vL domain.Additionally, one or more autonomous antigen-binding domains (AABDs), such as vHH, FHVH, DARPIN, Centyrin, adaptor, receptor, or ligand, may be attached to or near the N-terminus of the vL and / or vH domain of the two-chain hybrid SIR.

[0104] It is also understood that multi-chain SIRs with hybrid (or hybrid) TCR chains may include those in which an antigen-binding domain (e.g., scFV, vHH, FHVH, darpin, non-immunoglobulin antigen-binding domain, receptor, ligand, autoantigen, etc.) is attached to only one of the hybrid (or hybrid) TCR chains and co-expressed with a complementary hybrid (or hybrid) TCR chain lacking the antigen-binding domain. Such hybrid chain SIRs are referred to as one-and-half-chain hybrid SIRs. Furthermore, one or more autonomous antigen-binding domains (AABDs), such as vHH, FHVH, darpin, centyrin, adaptor, receptor, or ligand, may be attached to or near the N-terminus of the antigen-binding domain of the hybrid (or hybrid chain) SIR. Some example monospecific, bispecific, and universal single- and half-chain hybrid SIRs targeting different antigens are shown in Tables 19 and 20 of the provisional patent application.

[0105] In some embodiments, an expression vector is provided. The expression vector can include a first nucleic acid encoding a non-TCR antigen-binding domain (e.g., a vL, vH, vHH, FHVH, scFv, a non-immunoglobulin antigen-binding scaffold, an adapter, an epitope, a receptor, or a ligand, etc.) operatively linked via an optional linker to a first hybrid (or hybrid) TCR chain, wherein the first hybrid (or hybrid) TCR chain comprises a first TCR chain transmembrane domain and a second TCR chain constant domain, but does not comprise the first TCR chain constant domain. The method can include contacting the T cell with a second nucleic acid encoding a second hybrid (or hybrid) TCR chain, where the second hybrid TCR chain comprises a second chain transmembrane domain, a non-TCR antigen-binding domain (e.g., vL, vH, vHH, FHVH, scFv, non-immunoglobulin antigen-binding scaffold, adapter, epitope, receptor or ligand, etc.) and a first TCR chain constant domain, but does not comprise a second TCR chain constant region. In embodiments, the vL of the first hybrid TCR chain and the vH of the second hybrid TCR chain interact when expressed on the surface of an immune cell (e.g., a T cell) to form a fragment variable (Fv), which is capable of specifically binding to a target antigen.

[0106] In some embodiments, an expression vector is provided. The expression vector may include a first nucleic acid encoding a non-TCR antigen-binding domain (e.g., a vL, vH, vHH, FHVH, scFv, non-immunoglobulin antigen-binding scaffold, adapter, epitope, receptor, or ligand, etc.) operatively linked via an optional linker to a first hybrid (or hybrid) TCR chain, wherein the first hybrid (or hybrid) TCR chain does not comprise the first TCR chain transmembrane domain and the second TCR chain Ig-like domain, but does comprise the first TCR chain Ig-like domain. The method may include contacting a T cell with a second nucleic acid encoding a second hybrid (or hybrid) TCR chain, wherein the second hybrid TCR chain comprises the second chain transmembrane domain, the non-TCR antigen-binding domain, and the first TCR chain Ig-like domain, but does not comprise the second TCR chain Ig-like domain. In an embodiment, the vL of the first hybrid TCR chain and the vH of the second hybrid TCR chain interact when expressed on the surface of an immune cell (e.g., a T cell) to form a fragment variable (Fv), which can specifically bind to a target antigen.

[0107] The expression vector may comprise a first nucleic acid encoding a first hybrid (or hybrid) TCR constant chain, where the first hybrid (or hybrid) TCR chain comprises a first TCR chain transmembrane domain, a variable domain of an antibody light chain (vL), and a second TCR chain Ig-like domain, but not the first TCR chain Ig-like domain.The expression vector may comprise a second nucleic acid encoding a second hybrid (or hybrid) TCR chain, where the second hybrid TCR chain comprises a second chain transmembrane domain, a variable domain of an antibody heavy chain (vH), and the Ig-like domain of the first TCR, but not the Ig-like domain of the second TCR chain. The first chain variable domain may comprise a vL variable domain, the first chain Ig-like domain may comprise an α- or γ-chain Ig-like domain, the first chain transmembrane domain may comprise an α- or γ-chain transmembrane domain, the second chain variable domain may comprise a vH variable domain, and the second chain Ig-like domain may comprise a β- or delta-chain Ig-like domain, the transmembrane domain may comprise a beta- or delta-chain transmembrane domain; or the first chain variable domain may comprise a vH variable domain, the first chain Ig-like domain may comprise an α- or delta-chain constant domain, the first chain transmembrane domain may comprise an α- or delta-chain transmembrane domain, the second chain variable domain may comprise a vL variable domain, and the second chain Ig-like domain may comprise a β- or gamma-chain Ig-like domain and the second chain transmembrane domain may comprise a beta- or gamma-chain transmembrane domain. Thus, an expression vector may comprise a hybrid chain SAR comprising a first hybrid chain and a second hybrid chain. In some embodiments, the first hybrid chain further comprises a second chain-connecting peptide, and the second hybrid chain further comprises a first chain-connecting peptide, where the first chain-connecting peptide comprises an alpha- or gamma-chain connecting peptide and the second chain-connecting peptide comprises a beta- or delta-chain connecting peptide; or where the first chain-connecting peptide comprises an alpha- or delta-chain connecting peptide and the second chain-connecting peptide comprises a beta- or gamma-chain connecting peptide.In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain constant domain comprises an alpha chain constant domain, the first chain transmembrane domain comprises a beta chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain constant domain comprises a beta chain constant domain, and the second chain transmembrane domain comprises an alpha chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain Ig-like domain comprises a gamma chain Ig-like domain, the first chain transmembrane domain comprises a gamma chain transmembrane domain, the second chain variable domain comprises a vH domain, the second chain Ig-like domain comprises a delta chain Ig-like domain, and the second chain transmembrane domain comprises a delta chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL variable domain, the first chain Ig-like domain comprises an α chain Ig-like domain, the first chain transmembrane domain comprises a gamma chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain Ig-like domain comprises a β chain Ig-like domain, and the second chain transmembrane domain comprises a delta chain transmembrane domain. In some embodiments, the first chain variable domain comprises a vL domain, the first chain Ig-like domain comprises an α chain Ig-like domain, the first chain transmembrane domain comprises a delta chain transmembrane domain, the second chain variable domain comprises a vH variable domain, the second chain Ig-like domain comprises a β chain Ig-like domain, and the second chain transmembrane domain comprises a gamma chain transmembrane domain. It should be understood that the vL domain and vH domain in the above constructs can be substituted such that the first chain may comprise a vH domain and the second chain may comprise a complementary vL domain. Additionally, the expression vector may contain one or more autonomous antigen binding domains (AABDs), which may be attached at or near the N-terminus of the vL and / or vH domain of the two-chain hybrid SIR.

[0108] The expression vector can include a first nucleic acid encoding a first hybrid chain comprising a first chain transmembrane domain, a vL domain, and a second chain constant domain. The expression vector can include a second nucleic acid encoding a second hybrid chain comprising a second chain transmembrane domain, a vH domain, and a first chain constant domain. The first chain variable domain may comprise a vL domain, the first chain constant domain may comprise an alpha or gamma chain constant domain, the first chain transmembrane domain may comprise an alpha or gamma chain transmembrane domain, the second chain variable domain may comprise a vH domain, the second chain constant domain may comprise a beta or delta chain constant domain, and the second chain transmembrane domain may comprise a beta or delta chain transmembrane domain; or the first chain variable domain may comprise a vL domain, the first chain constant domain may comprise an alpha or delta chain constant domain, the first chain transmembrane domain may comprise an alpha or delta chain transmembrane domain, the second chain variable domain may comprise a vH, the second chain constant domain may comprise a beta or gamma chain constant domain, and the second chain transmembrane domain may comprise a beta or gamma chain transmembrane domain. In some embodiments, the first hybrid chain further comprises a second chain-linking peptide and does not comprise the first chain-linking peptide; the second hybrid chain further comprises a first chain-linking peptide and the first chain-linking peptide does not comprise the first chain-linking peptide; the first chain-linking peptide comprises an α-chain or γ-chain connecting peptide and the second chain-linking peptide comprises a beta-chain or delta-chain connecting peptide; or the first chain-linking peptide comprises an α-chain or delta-chain connecting peptide and the second chain-linking peptide comprises a beta-chain or gamma-chain connecting peptide.

[0109] In some embodiments, the first and second nucleic acids are part of the same expression vector, the expression vector further comprising a sequence encoding a 2A peptide flanked by the first and second nucleic acids, and the first and second nucleic acids are driven by a single promoter. In some embodiments, the first and second nucleic acids are part of separate expression vectors. Optionally, the first and second nucleic acids can each be driven by a different promoter. Optionally, the first and second nucleic acids can be part of a single expression vector and driven by their own separate promoters. In some embodiments, the expression vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the first and / or second and / or third nucleic acids comprising a SAR or accessory module / therapeutic control are driven by activation of a synthetic Notch (SynNotch) receptor or a variant thereof. SynNotch receptors are described in WO2022140159A1, the entire contents of which are incorporated by reference.

[0110] In some embodiments, genetically engineered cells are provided. In embodiments, the cells are T cells. In embodiments, the cells are NK cells, NKT cells, iNKT cells, G-NK cells, macrophages, monocytes, granulocytes, embryonic stem cells, iPS cells, or hematopoietic stem cells. The genetically engineered T cells can comprise a first nucleic acid encoding a first hybrid chain comprising a first chain transmembrane domain, a vL domain, and a second chain constant domain. The genetically engineered T cells can comprise a second nucleic acid encoding a second hybrid chain comprising a second chain transmembrane domain, a vH domain, and a first chain constant domain. The first chain variable domain may comprise a vL variable domain, the first chain constant domain may comprise an α-chain or γ-chain constant domain, the first chain transmembrane domain may comprise an α-chain or γ-chain transmembrane domain, the second chain variable domain may comprise a vH chain variable domain, and the second chain constant domain may comprise a beta-chain or delta-chain constant domain, the chain transmembrane domain may comprise a β-chain or delta-chain transmembrane domain; or the first chain variable domain may comprise a vH chain variable domain, the first chain constant domain may comprise an α-chain or delta-chain constant domain, the first chain transmembrane domain may comprise an α-chain or delta-chain transmembrane domain, the second chain variable domain may comprise a vL domain, the second chain constant domain may comprise a beta-chain or gamma-chain constant domain, the second chain variable domain may comprise a beta-chain or gamma-chain constant domain, the chain transmembrane domain may comprise a beta-chain or gamma-chain transmembrane domain. In this way, T cells can be configured to express an HC-SAR comprising a first hybrid chain and a second hybrid chain. In some embodiments, the first hybrid chain further comprises a second chain-connecting peptide, and the second hybrid chain further comprises a first chain-connecting peptide, where the first chain-connecting peptide comprises an alpha- or gamma-chain connecting peptide and the second chain-connecting peptide comprises a beta- or delta-chain connecting peptide; or the first chain-connecting peptide comprises an alpha- or delta-chain connecting peptide and the second chain-connecting peptide comprises a beta- or gamma-chain connecting peptide.In some embodiments, the engineered T cells are configured to express a first hybrid chain as a first polypeptide and a second hybrid chain as a second polypeptide, wherein the first hybrid chain and the second hybrid chain are separate molecules. In some embodiments, expression of an endogenous TCR is suppressed or eliminated in the engineered T cells.

[0111] In some embodiments, methods of inducing an immune response in a subject are provided. The method can include configuring isolated T cells to express a first hybrid chain comprising a first chain transmembrane domain, a vL domain, and a second chain constant domain, but not comprising a first chain variable domain and not comprising a first chain constant domain. The method can also include configuring isolated T cells to express a second hybrid chain comprising a second chain transmembrane domain, a vH domain, and the first chain constant domain, but not comprising a second chain constant domain. The first chain variable domain can comprise a vL domain, the first chain constant domain can comprise an alpha or gamma chain constant domain, the first chain transmembrane domain can comprise an alpha or gamma chain transmembrane domain, the second chain variable domain can comprise a vH domain, the second chain constant domain can comprise a beta or delta chain constant domain, and the second chain transmembrane domain can comprise a beta or delta chain transmembrane domain; or the first chain variable domain can comprise a vH domain, the first chain constant domain can comprise an alpha or delta chain constant domain, the first chain transmembrane domain can comprise an alpha or delta chain transmembrane domain, the second chain variable domain can comprise a vL domain, the second chain constant domain can comprise a beta or gamma chain constant domain, and the second chain domain can comprise a beta or gamma chain transmembrane domain. Thus, engineered T cells configured to express a hybrid synthetic immunoreceptor (HC-SAR) comprising a first hybrid chain and a second hybrid chain can be administered to a subject. In some embodiments, the first hybrid chain further comprises a second chain-linking peptide, and the second hybrid chain further comprises a first chain-linking peptide, where the first chain-linking peptide comprises an alpha or gamma chain-linking peptide and the second chain-linking peptide comprises a beta or delta chain-linking peptide; or the first chain-linking peptide comprises an alpha or delta chain-linking peptide and the second chain-linking peptide comprises a beta or gamma chain-linking peptide. In some embodiments, the isolated T cells are autologous to the subject. In some embodiments, the isolated T cells are allogeneic to the subject.In some embodiments, the T cells comprise CD4 T cells. In some embodiments, the T cells comprise CD8 T cells. In some embodiments, the T cells comprise regulatory T cells (Tregs). In some embodiments, the T cells are co-administered with a second genetically engineered T cell population. In some embodiments, the T cells are administered in a single dose. In some embodiments, the T cells are administered multiple times. In some embodiments, the subject has at least one of a tumor, cancer, an infectious disease, or an autoimmune disease and is in need of treatment. In some embodiments, the subject has reduced, ineffective, or exhausted T cells and is in need of treatment. In some embodiments, the T cells are induced to express multiple HC-SARs against an antigen sequence. In some embodiments, the T cells can be administered to the subject via at least one of intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, subcutaneous, intradermal, or intranasal administration. In some embodiments, the administered T cells are further monitored over time. In some embodiments, the method can be repeated as desired. [Brief explanation of the drawings]

[0112] Figure 1 shows bioluminescence imaging of NSG mice xenografted with JEKO-1 cells and administered either control T cells or T cells expressing the indicated CD79b SAR constructs.

[0113] Figure 2 shows bioluminescence imaging of NSG mice xenografted with LNCaP cells and administered either control T cells or T cells expressing the indicated STEAP2 SAR constructs.

[0114] Figure 2 shows bioluminescence imaging of NSG mice xenografted with NALM6 cells and administered either control NK cells or T cells expressing the indicated CD19 SAR constructs.

[0115] The present invention is further described below. In the following passages, various aspects of the invention are defined in more detail. Each aspect so defined may be combined with other aspects, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

[0116] Unless otherwise stated or implicit from context, the following terms and phrases have the meanings set forth below: Unless otherwise expressly stated or apparent from context, the following terms and phrases do not exclude the meaning that the term or phrase has acquired in the relevant art. Definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims.

[0117] 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.

[0118] As used herein, the terms "comprise" or "comprises" are used in reference to compositions, methods, and their respective components that are useful in embodiments but are open to the inclusion of unspecified elements, whether useful or not. Those skilled in the art will generally understand that the terms used herein are generally intended as "open" terms (e.g., the term "comprise" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including, but not limited to," etc.).

[0119] In general, the nomenclature used in connection with cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well 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, e.g., 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 immunology, molecular biology, analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the laboratory procedures and technical names thereof, are those 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.

[0120] The term "autonomous antigen-binding domain" or "AABD," as used herein, refers to an antigen-binding domain that can autonomously bind to an antigen, i.e., in the absence of another antigen-binding domain. An example of an AABD is a single vH domain or an 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 example of an AABD is a fully human vH domain (FHVH). As another example, an AABD is a single vL domain or an 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 autonomously bind to an antigen. In embodiments, the AABD is a non-scFv antigen-binding domain. For example, 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 DARPINs, affibodies, ZIP domains (e.g., RZIP, EZIP, E4, R4, etc.), affilins, adnectins, affitins, obodies, repebodies, finomers, alphabodies, avimers, atrimers, centrins, pronectins, anticalins, Kunitz domains, armadillo repeat proteins or fragments thereof. Further examples of non-scFV-based autonomous antigen-binding domains include the ligand-binding domain of a receptor (e.g., CD16-V158A, NKG2D) or a fragment thereof, a ligand (e.g., APRIL, thrombopoietin, etc.) or a fragment thereof, an adaptor (e.g., RZIP, EZIP, E4, K4, NKG2D-YA, NKG2D-AF, etc.) or a fragment thereof, an adaptor-binding protein (e.g., ULBP2R, ULBP2-S3, etc.) or a fragment thereof, an epitope or tag (e.g., Streptag, FLAG tag, etc.), an autoantigen or a fragment thereof, etc.

[0121] The present disclosure has described the use of AABDs, e.g., human VH (or vH) domains, e.g., multiple human VH domains, as building blocks for generating monospecific, bispecific, and multispecific SARs.

[0122] The term "about," when referring to a measurable value, such as an amount, temporal duration, or the like, is intended to encompass a variation of ±20%, or in some cases ±5%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value. 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 mutations" explicitly includes 1, 2, 3, 4, and / or 5 mutations.

[0123] The term "Ab-TCR" or "AbTCR" refers to the next generation CAR platform described in WO 2017 / 070608 A1, which is incorporated herein by reference.

[0124] "Accessory module" refers to one or more of PDL1, PDL2, CD80, CD86, crmA, p35, K13-opt, MC159, MyD88-L265P, TCL-1a, 41BBL, CD40L, vFLIP-K13, MC159, cFLIP-L / MRITα, IgSP-[hTRAC-opt2], IgSP-[hTRBC-opt2], multipurpose switch (e.g., IL2-tBCMA, IL15-tBCMA, IL2-RQR, IL15-RQR), NKG2C, CD94, DAP10, DAP12, CD3ε, CD3γ, CD3δ, CD3ζ, and FcRy. Their combinations expressed in immune cells (e.g., NK cells or T cells, e.g., SAR-NK cells, SAR-T cells, or TCR-T cells) reduce, regulate, or modify the activity of the immune cells. In embodiments, the accessory module is a therapeutic control (e.g., squapase 9). Nucleic acid and amino acid sequence numbers of some exemplary accessory modules and therapeutic controls are provided in Table 7 of the provisional patent application (e.g., SEQ ID NOs: 9038-9047, 9284-9308, 9348- 9349). In some embodiments, an accessory module is co-expressed with an immune receptor, such as a SAR or TCR, to increase, decrease, regulate, or modify the expression or activity of the SAR or TCR or a SAR-expressing or TCR-expressing cell.

[0125] 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," "chimerized," fully human, or non-human. Antibodies can have a single domain (e.g., a single vH domain).

[0126] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., bind, steric hindrance, stabilizing / destabilizing, spatial distribution) with an epitope of an antigen.

[0127] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformation, and usually determines the class to which the antibody belongs.

[0128] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in the naturally occurring conformation in antibody molecules. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0129] "Anticancer drugs" refer to drugs that inhibit abnormal cell division and proliferation, drugs that inhibit tumor cell migration, drugs that inhibit invasiveness, and drugs that prevent cancer proliferation and metastasis.

[0130] 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. An "anti-cancer effect" can also be manifested by the ability of SAR to prevent cancer from occurring in the first place.

[0131] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include antibody production, or activation of specific immunologically competent cells, or both. Non-limiting examples of antigens that can be specifically bound by either an antigen or an antigen-binding domain are listed in Table B.

[0132] 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. In exemplary embodiments, the target antigens and SEQ ID NOs of various antigen-binding domains are set forth in Tables 3-7 herein. In exemplary embodiments, the target antigens and SEQ ID NOs of vL, vH, scFVs, and their CDR regions are set forth herein in Tables 6A-C of patent application PCT / US18 / 53247 and Tables 3-4 of patent application PCT / US19 / 035096, which are incorporated herein by reference in their entireties.

[0133] 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, moglein 1, moglein 3, and fragments thereof.

[0134] "Avidity" refers to the strength of the interaction between an agent and its target.

[0135] As used herein, the term "backbone" or "architecture" refers to the organization of different components (e.g., antigen-binding domains, hinge domains, transmembrane domains, signaling domains), including different SARs and generally any accessory modules, if any.

[0136] Table 1: 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. Second-generation conventional CARs (conventional CAR2 or CAR II) have one costimulatory domain (e.g., 41BB or CD28) and one intracellular signaling domain (ISD) (e.g., CD3z). Third-generation conventional CARs (conventional CAR3 or CAR III) have two costimulatory domains (e.g., 41BB and CD28) and one intracellular signaling domain (ISD) (e.g., CD3z). Ab-TCRs are duel-chain receptors incorporating a vL-linker-TCR domain (TCRD) and a vH-linker-TCR domain (TCRD), as described in PCT / US2016 / 058305. A cTCR (chimeric T cell receptor) is a single-chain, single-and-a-half-chain, or double-chain receptor consisting of an antigen-binding domain derived from a vL and vH fragment, which is fused to one or more TCR constant chains (TCR-C), activating T cell signaling. The TCR constant chain of a cTCR is encoded by a wild-type nucleic acid sequence and a corresponding wild-type amino acid sequence. Various configurations of cTCRs are described in PCT / US2017 / 064379 or WO 2018 / 102795 A1. Synthetic immune receptors are next-generation CARs and are described in PCT / US2017 / 064379 or WO 2018 / 102795 A1. A SIR is a single-chain, single-and-a-half-chain, or double-chain receptor. In one embodiment, the antigen-binding domain of a SIR is derived from a vL and vH fragment fused to one or more TCR constant chains (TCR-C), activating T cell signaling. In some embodiments, the TCR constant chains of the SIRs are encoded by human codon-optimized nucleic acid sequences and contain one or more mutations that enhance their expression and chain pairing. zSIRs are dual-chain receptors that contain antigen-binding domains (e.g., vL, vH, etc.) operatively linked to two CD3z chains or fragments thereof using any linker, and are described in PCT / US2019 / 035096. JPEG2026501516000001.jpg57145

[0137] Tables 2A-2J show examples of monospecific, bispecific, and multispecific SAR architectures of the present disclosure. The 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 including optional linkers, where 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 derived from a TCR chain), ConP (connecting peptide), TM (transmembrane domain), CP (cytoplasmic domain), IC (intracellular domain), Ca (constant chain of TCR α), Cb (constant chain of TCR β), Cg (constant chain of TCR γ), Cd (constant chain of TCR δ), scFv (single-chain fragment variable), scTFv (single-chain fragment containing two variable fragments of TCR, e.g., Va and Vb), dCa / dCb / dCg / dCd (constant chains deleted at the N-terminus of TCR α, β, γ or δ lacking their Ig linker domains), connecting peptide of TCR α, β, γ or δ constant chain), Ca-ConP (connecting peptide of TCR α constant chain), IgCL (Ig linker derived from immunoglobulin light chain), IgCH1 (Ig linker derived from immunoglobulin heavy chain), CD3εγδ ECD (extracellular domain of CD3ε, γ, or δ chain), CSD (costimulatory domain), 4-1BB or BB (costimulatory domain of 4-1BB), CD28 or 28 (costimulatory domain of CD28), CD3z or zd or z (activation domain of 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, and δ), FCRG (FcRγ); hinge domain (Hn). JPEG2026501516000002.jpg179147JPEG2026501516000003.jpg70149The vL and vH in the constructs in Table 2D can be replaced with Va, Vb, Vg, or Vd fragments. The order of Va, Vb, Vg, Vd, vL, and vH can be reversed, and each can be linked to a different chain. Similarly, one or more CD3ζ fragments can be replaced with the corresponding FcRγ fragment. The Ig linker domain can be derived from an immunoglobulin or TCR constant chain. Additionally, the dQ101 mutation can be introduced into one or both of the cytoplasmic domains of CD3ζ. JPEG2026501516000004.jpg90148 The vL and vH in the constructs in Table 2E can be replaced with Va, Vb, Vg, or Vd fragments. The order of Va, Vb, Vg, Vd, vL, and vH can be interchanged, and each can be linked to a different chain. Similarly, one or more CD3ζ fragments can be replaced with the corresponding FcRγ fragment. The Ig linker domain can be derived from an immunoglobulin or TCR constant chain. In addition, the dQ101 mutation can be introduced into one or both of the cytoplasmic domains of CD3ζ. Table 2F. Examples of SARs (SIRs) with hybrid TCR chains JPEG2026501516000005.jpg204139JPEG2026501516000006.jpg210137JPEG2026501516000007.jpg28135 Table 2G. Examples of SARs (SIRs) with hybrid TCR chains JPEG2026501516000008.jpg200124JPEG2026501516000009.jpg211124JPEG2026501516000010.jpg208124JPEG2026501516000011.jpg31124 The vL and vH fragments in the following constructs can be replaced with Vα, Vβ, or Vγ and Vδ fragments. One or both of the Ig linkers (IgCL and IgG1-CH1) can be replaced with TCR constant domains. Table 2H. Examples of SARs (SIRs) with hybrid TCR chains JPEG2026501516000012.jpg205139JPEG2026501516000013.jpg210139JPEG2026501516000014.jpg12138 Table 2I. Examples of SARs with hybrid TCR chains JPEG2026501516000015.jpg207146JPEG2026501516000016.jpg207146JPEG2026501516000017.jpg211146Table 2J Examples of TCRs with hybrid chains containing antibody constant domains JPEG2026501516000018.jpg139115

[0138] "Binds to the same epitope" means that the antibody, scFv, or other antigen-binding domain is capable of binding to the target antigen and has the same epitope as the exemplified antibody, scFv, or other antigen-binding domain.

[0139] Unless explicitly stated otherwise, when the disclosure relates to a polypeptide, protein, polynucleotide, antibody, SAR, or fragment thereof, it should be inferred that equivalents or biologically equivalents thereof are contemplated within the scope of this disclosure. As used herein, the terms "biologically equivalent" or "variant" or "functional variant" are intended to be synonymous with "equivalent thereof" when referring to a reference protein or fragment thereof, antibody or fragment thereof, receptor or fragment thereof, ligand or fragment thereof, non-immunoglobulin antigen-binding domain or fragment thereof, SAR or fragment thereof, SIR or fragment thereof, or CAR or fragment thereof. A "biologically equivalent" or "variant" or "functional variant" polypeptide or nucleic acid is intended to have minimal homology while maintaining the desired structure or function. Unless otherwise specified herein, it is intended that any of the above also include their equivalents, including alternatively spliced ​​isoforms and equivalents from other animal species. For example, a variant refers to a polypeptide, antibody, or fragment thereof, or nucleic acid that has 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 98% homology or identity, and exhibits substantially the same biological activity as the reference protein. Alternatively, when referring to a polynucleotide, the variant is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement. Alternatively, when referring to a polypeptide or protein, the variant is a polypeptide or protein expressed from a polynucleotide that hybridizes under stringent conditions to the polynucleotide or its complement encoding the reference polypeptide or protein.

[0140] It is recognized that proteins can share identity or homology with one another and retain similar or identical functions. In embodiments, a polypeptide "variant" or "functional variant" as used herein is a polypeptide that differs from the described polypeptide 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. For immunoreactive polypeptides, variants can alternatively be identified by modifying the amino acid sequence of one 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 functional variants of SARs, SAR components, and SAR fragments (e.g., extracellular, hinge, transmembrane, and cytosolic regions of CD16, TCRα, TCRβ, TCRγ, TCRδ, and CD3z, etc.) that have 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 while retaining biological activity. The present disclosure also includes antigen-binding domains, extracellular domains, hinge domains, transmembrane domains, cytoplasmic domains, costimulatory domains, accessory modules, 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 and functional variants include homologs and alternative splicing isoforms from other species (e.g., mouse, dog, cat, monkey, etc.).

[0141]

[0013] Embodiments and aspects of the present invention include proteins, fusion proteins, and sequence variants and fragments of polynucleotides encoding proteins and fusion proteins, wherein the length of each peptide region or domain comprising the fusion protein varies independently at the amino terminus, carboxy terminus, or both termini, based on the native sequence of the polypeptide from which the peptide region or domain is derived. In some aspects, sequence variants and fragments have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, etc.) of the activity of the particular fusion protein on which they are based.

[0142] In one aspect of this embodiment, the sequence variant or fragment is a sequence variant or fragment in which the length of at least one peptide region or domain comprising the fusion protein varies independently by up to 10 amino acids at the amino terminus, the carboxy terminus, or both termini, based on the native sequence of the polypeptide from which the peptide region or domain is derived.

[0143] Each embodiment and aspect of the present invention also includes sequence variants and fragments of proteins, polypeptides, and fusion proteins, and the polynucleotides encoding them, that have at least 70% (e.g., 70%, 75%, 80%, 85%, 85%, 90%, 95%, 98%, 99%, etc.) sequence identity to a specific protein, polypeptide, or fusion protein defined herein over the entire length of that particular fusion protein. In certain aspects, these sequence variants will have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, etc.) of the activity of the protein, polypeptide, and specific fusion protein on which they are based.

[0144] In embodiments, the present invention provides sequence variants and fragments of antibodies, including antibody fragments (e.g., Fab, vL, vH, vHH, scFv, FHVH, etc.), TCRs, and TCR variable domains (e.g., Vα, Vβ, Vγ, Vδ, etc.), and polynucleotides encoding same, having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, etc.) sequence identity to a particular antibody, antibody fragments, TCRs, and TCR variable domains as defined herein over their entire length excluding the complementarity determining regions (CDRs).

[0145] As used herein, the term "CD3 complex" refers to a cell surface molecular assembly that contains multiple proteins for transmembrane signaling of TCR activation.

[0146] As used herein, the term "CDR" or "complementarity-determining region" is intended to refer to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are defined by Kabat et al., J. Bio. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Bio. 196:901-917 (1987); MacCallum et al., J. Mol. Bio. 25, 262:732-745 (1996). The definitions include overlapping or subsets of amino acid residues relative to each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. As used herein, different CDRs of an antibody can be defined by a combination of different definitions. For example, vHCDR1 can be defined based on Kabat, and VHCDR2 can be defined based on Chothia. The amino acid residues encompassing the CDRs defined by each of the above references are as follows: JPEG2026501516000019.jpg78154 (residue numbers correspond to the specified references).

[0147] The sequence IDs of the CDRs of exemplary vL and vH segments that can constitute the antigen-binding domains of the SARs, bispecific antibodies, and other immunotherapies of the present disclosure are set forth in SEQ ID NOs: 13204-14121 and 14122-15039 (Table 6A, B) of PCT / US2018 / 053247, Tables 5-6, and Table 39 of PCT / US2017 / 064379, respectively, and PCT / US2021 / 022641, which are incorporated herein by reference. The SEQ IDs of exemplary vL and vH segments that can constitute the antigen-binding domains of the SARs, antibodies, and other immunotherapies are also set forth in Table 3 of the present disclosure. Light chain CDR1, CDR2, and CDR3 of the vL fragments and scFvs provided in this disclosure are provided in SEQ ID NOs: 20989-21015, 41591-41861, 21024-21050, 41862-42132, and 21059-21085, 42133-42403, respectively. 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: 21094-21120 and 42404-42674; 21129-21155, 42675-42945; and 21164-21190 and 42946-43216, respectively. CDR1-3 of the selective novel binders are shown in Table 8.

[0148] In embodiments, the present disclosure provides a SAR comprising: (1) a heavy chain variable domain (vH) comprising a heavy chain CDR1-3 sequence and a complementary light chain variable domain (vL) comprising a light chain CDR1-3 sequence, which binds to the same epitope as a monoclonal antibody comprising variable domains (vL and vH) of the sequences depicted in Table 3, or which binds to the same epitope as an scFv having the sequences depicted in Table 3; or (2) a heavy chain variable domain and a light chain variable domain shown in Table 3; or (3) an scFv shown in Table 3; or (4) at least 85% amino acid sequence identity to an amino acid sequence shown in Table 3. (5) a heavy chain variable domain having at least 75% amino acid sequence identity in the framework regions to an amino acid sequence set forth in Table 3, and a light chain variable domain having at least 75% amino acid sequence identity in the framework regions to an amino acid sequence set forth in Table 3. In embodiments, the antibody or antibody fragment is a bispecific antibody, a multispecific antibody, an Fv, an scFv, or an Fab. In embodiments, the SAR is a CAR or next-generation CAR (e.g., SIR, Ab-TCR, zSIR, uTCR-SAR, CD16SAR, zCD16SAR, etc.).

[0149] In embodiments, the present disclosure provides a single domain antibody or antibody fragment, or a SAR comprising: (1) a vHH domain having a sequence depicted in Table 5; or (2) a vHH domain having a sequence with at least 85% amino acid sequence identity to the amino acid sequence depicted in Table 5; or (3) a vHH domain having a sequence with at least 75% amino acid sequence identity in the framework regions of the amino acid sequence depicted in Table 5, and comprising CDR1-3 of the sequences depicted in Table 5. In embodiments, the single domain antibody or antibody fragment is a bispecific or multispecific antibody. In embodiments, the SAR is a CAR or next-generation CAR (e.g., SIR, Ab-TCR, zSIR, uTCR-SAR, CD16SAR, zCD16SAR, etc.).

[0150] "Cancerous" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0151] "Cell therapy" or "cell-based therapy" or "immune cell therapy" or "immune effector cell therapy" refers to therapies that use cells to prevent or treat disease.

[0152] "Chimeric antigen receptors" (CARs) are artificial (non-naturally occurring) immune cell (e.g., T cell) receptors that are contemplated for use as cancer treatments using a technique called adoptive cell transfer. In various embodiments, CARs are recombinant polypeptides that include an antigen-specific domain (ASD), a hinge region (HR), a transmembrane domain (TMD), an optional costimulatory domain (CSD), and an intracellular signaling domain (ISD).

[0153] "Codon optimization" or "control of species codon bias" refers to the preferred codon usage of a particular host cell. In embodiments, the present invention describes proteins, polypeptides, and fragments thereof that are human codon optimized.

[0154] "Co-expression" refers to the expression of two or more polynucleotides or genes.

[0155] 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.

[0156] As used herein, "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. Each costimulatory domain may include, for example, one or more costimulatory domains from any of the following: a member of the TNFR superfamily, CD28, (4-1BB), CD134, BAFF-R, HVEM, CD27, CD2, CD2, CD5, Fas, CD30, CD40, or a combination thereof.

[0157] The term "costimulatory molecule" or "costimulatory receptor" refers to a cognate binding partner on an immune cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation, activation, or cytokine secretion.

[0158] The term "cTCR" or "chimeric T cell receptor" refers to the nucleic acid coding sequence of a wild-type TCR and the corresponding wild-type TCR protein to which an antigen-binding domain not derived from a TCR has been linked. cTCRs are described in Gross, Waks, and Eshhar (1989). cTCRs are used in some embodiments and also serve as reference controls.

[0159] The terms "cytoplasm" or "cytoplasmic" refer to an agent, for example, a protein, that is located in the cytoplasm of a cell in its mature form.

[0160] The term "degenerative disease" refers to diseases that are the result of a continuous process based on degenerative cellular changes and affect tissues and organs.

[0161] "Derived from," as that term is used herein, denotes a relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the process or source of the first molecule from which the second molecule is derived.

[0162] A "dimerization molecule," as that term is used herein, refers to a molecule that promotes the association of a first switch domain with a second switch domain.

[0163] "Disease targeted by genetically modified cells" encompasses the targeting of any cells involved in any way in any disease by the disclosed genetically modified cells.

[0164] As used herein, a "diverse set of non-naturally occurring immune receptors" or a "diverse set of SARs" refers to multiple non-naturally occurring 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 embodiments, the diverse set of SARs may have a diverse range of binding affinities for the target antigen. In embodiments, the diverse set of SARs may exhibit different expression levels.

[0165] 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.

[0166] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell (e.g., T cell, NK cell, NKT cell, B cell, macrophage, neutrophil) and a tumor cell that results in activation of the immune cell.

[0167] 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.

[0168] A "functional portion" ("biologically active portion") of a protein refers to a portion of the protein that retains one or more functions of the full-length or mature protein.

[0169] The term "FcRγ" or "FCER1G" or "FCRG" or "FcRy" as used herein refers to the gene represented by Gene ID:2207.

[0170] 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). Functional portions include, for example, portions of a SAR that retain the ability to recognize target cells or detect, treat, or prevent disease to the same extent, the same extent, or a greater extent 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.

[0171] As used herein, the term "flexible polypeptide linker" refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link polypeptide chains together (e.g., linking variable heavy and variable light chain regions together). In one embodiment, the flexible polypeptide linker is a Gly / Ser linker.

[0172] As used herein, "genetically modified cells," "redirected cells," "genetically engineered cells," or "modified cells" refer to cells that express the disclosed SARs.

[0173] As defined herein, an "HLA-independent TCR" or "MHC-independent TCR" is a TCR that is able to recognize antigens independently of MHC restriction.

[0174] As defined herein, an "HLA-independent TCR variable domain" is a variable domain of a TCR that is capable of binding to an antigen in an HLA-independent manner.

[0175] 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 "non-MHC-restricted" antigen recognition.

[0176] 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 that has been introduced from one species into another. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native regulatory sequence, etc.). As another example, a heterologous gene includes a gene that is expressed in a previous or future cell lineage or differentiation state of a cell. A heterologous gene is distinguished from an endogenous gene in that the heterologous gene sequence is typically linked to a DNA sequence not naturally found in the gene's sequence in a chromosome or linked to a DNA sequence that is associated with a part of the chromosome where it is not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed).

[0177] The term "heterologous," when used in the context of a protein domain, refers to a domain that is not in its natural environment. For example, a heterologous protein domain is not part of a single, naturally occurring polypeptide or protein. Stated differently, two domains are heterologous if they are derived from two different polypeptides or proteins found in nature. For example, the TCRα constant domain and the TCRβ transmembrane domain are heterologous to each other because they are derived from two different polypeptides or proteins.

[0178] Two domains derived from homologs of the same protein but from two different species are considered "interspecies heterologous domains" if they share less than 80% amino acid sequence identity. For example, a human TCR α constant domain and a mouse TCR α transmembrane domain are considered heterologous to each other if the human TCR α constant domain is less than 80% identical to the mouse TCR α constant domain at the amino acid level, or if the human TCR α transmembrane domain is less than 80% identical to the mouse TCR α. For purposes of this disclosure, two domains are not considered interspecies heterologous domains if less than 20% of the amino acid residues in a protein domain belonging to one species are replaced by corresponding residues found in a different species. For example, a variant of a human TCR transmembrane domain and a human TCR constant domain is not considered interspecies heterologous if less than 20% of the amino acid residues in the human TCR constant domain are replaced by corresponding residues found in the mouse TCR constant domain.

[0179] As used herein, "hinge region" (HR) refers to the hydrophilic region located between the antigen-binding domain and the transmembrane domain of the SAR. Some exemplary hinge regions are provided in Table 29 of the provisional patent application.

[0180] As used herein, the term "hybrid TCR chain" or "hybrid chain" refers to a chain containing at least one domain selected from the group consisting of a TCR constant domain (CD or C), a TCR connecting peptide (ConnP), a TCR transmembrane (TM) domain, and a heterologous TCR cytoplasmic domain (CP or IC). In an embodiment of the present invention, a hybrid TCR chain refers to a TCR chain in which the TM domain is derived from one TCR chain and at least one domain selected from CD, ConnP, and IC is derived from a different TCR chain. For example, if the TM is derived from human TCRα, at least one domain selected from CD, ConnP, and IC is derived from TCRβ1 / β2, TCRγ, TCRδ, or pre-TCRα. A hybrid chain may also contain domains derived from two different species. Thus, a hybrid chain may contain a human TCRα constant domain and a non-human TCRα transmembrane domain. A hybrid TCR chain can have one, two, three, or more heterologous domains.

[0181] "Hybrid chain SIR" or (HC-SIR) or hybrid chain SAR (HC-SAR), as the terms are used herein, refers to a heterodimeric synthetic immunoreceptor (SIR) or synthetic antigen receptor (SAR) in which at least one TCR constant chain is a hybrid chain. A hybrid chain SIR or hybrid chain SAR can have both TCR chains that are hybrids.

[0182] "Immune effector cells," as that term is used herein, refer to cells that participate in an immune response, e.g., cells that are involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, monocytes / macrophages, and bone marrow-derived phagocytes.

[0183] "Immune effector function" or "immune effector response" refers to a specialized function of a differentiated cell. For example, the effector function of a T cell or NK cell can be cytolytic activity or helper activity, including secretion of cytokines. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing or inhibition of proliferation or growth of a target cell.

[0184] "Intracellular signaling domain" (ISD) or "activation domain" as the term is used herein refers to the intracellular signaling portion of a molecule.

[0185] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free from other materials.

[0186] A "long linker" or "long linker domain" is a linker that is 25 to 500 amino acids in length. In embodiments, a long linker is about 25 to 500 amino acids in length and any number of lengths therebetween. In embodiments, a long linker is 25 to 125 amino acids in length. In embodiments, a long linker is between 50 and 150 amino acids in length.

[0187] In embodiments, the linker encodes or comprises an immunoglobulin (Ig) domain or Ig-like domain, or a fragment thereof. The terms "Ig domain," "Ig linker domain," "Ig-like domain," or "Ig-like linker domain" are used interchangeably in this disclosure. Examples of Ig linker domains include IgCL (SEQ ID NO: 8961) and IgG1-CH1 (SEQ ID NO: 8962). Additional examples of Ig linkers are set forth in SEQ ID NOs (PRT): 8962-8976.

[0188] In some embodiments, the peptide linker is derived from a TCR subunit constant region. In embodiments, the linker comprises an Ig-like constant domain of a TCR chain and further comprises a TCR connecting peptide. For example, long Ig-like linkers are provided in SEQ ID NOs: 22827-22829, 22833-22835, 22839-22840, and 22843-22844, respectively, and also include functional variants and homologs that encode polypeptides having at least 75% sequence identity to the polypeptides encoded by any of the above sequences. In embodiments, the long Ig-like linker comprises an N- or C-terminal deletion mutant of SEQ ID NOs: 22827-22829, 22833-22835, 22839-22840, 22843-22844, respectively, in which 1 to 40 (e.g., 1, 5, 10, 15, 20, 25, 30, 40) N- or C-terminal amino acid residues are deleted.

[0189] As used herein, the term "linker" (or "linker domain" or "linker region") refers to an oligo or polypeptide (or an oligo encoding a polypeptide) that connects two or more domains or regions of a SAR polynucleotide or polypeptide disclosed herein. Linkers can range from 1 to 500 amino acids in length, or from 3 to 1500 nucleotides in length. In some embodiments, the "linker" is cleavable or non-cleavable. Linker module also refers to the TCR and antibody linkers set forth in Table 7 of the provisional patent application.

[0190] The term "lentivirus" refers to a genus of the Retroviridae family. 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.

[0191] A "multipurpose switch" or "multipurpose gene" encodes a protein that provides suicide, survival, and marker functions. In embodiments, all of the above functions are provided by a single polypeptide chain. Examples of multipurpose switches include IL2-tBCMA, IL15-tBCMA, IL2-RQR8, and IL2-tHer2.

[0192] 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 can be a double-chain SAR. A double-chain SAR contains two membrane-associated domains (e.g., transmembrane domains or membrane-anchored domains).

[0193] 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 or naturally expressed in a cell. Thus, a native or endogenous TCR α chain polypeptide of a T cell consists of a variable domain (Vα) attached to a TCR α constant chain.

[0194] As used herein, "native receptor" or "naturally occurring receptor" or "endogenous receptor" or "natural receptor" refers to any receptor that occurs in nature and contains an antigen-binding or 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 a "naturally occurring signaling receptor" if it is capable of transducing a cellular signal upon binding to its target. A naturally occurring receptor or natural receptor is native to a cell or naturally expressed within a cell. Examples of naturally occurring signaling receptors or natural 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.

[0195] As used herein, "non-TCR naturally occurring receptor" or "non-TCR naturally occurring signaling receptor" or "non-TCR receptor" or "non-TCR signaling receptor" refers to a receptor that is not a T cell receptor (TCR). A non-TCR receptor can be expressed in a cell other than a T cell. A non-TCR receptor can be expressed in a cell that lacks expression of the CD3ζ, CD3ε, CD3δ and / or CD3γ chains.

[0196] As used herein, "non-T cell receptor module" or "non-TCR module" or "non-TCR signaling module" or "NTCRM" refers to a module that lacks sequences consisting of a T cell receptor transmembrane domain and may further lack all or a portion of a T cell receptor-linked peptide and / or an intracellular domain. An NTCRM lacks sequences consisting of a transmembrane domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. An NTCRM may further lack all or a portion of a binding peptide and / or an intracellular domain of TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. For example, a non-TCR module (NTCRM) is composed of two CD3z transmembrane domains. As another example, an NTCRM is composed of a CD3z transmembrane domain and a CD16 transmembrane domain.

[0197] 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 embodiments, 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.

[0198] As used herein, the term "near the N-terminus" refers to within the N-terminal 30 amino acids. For example, "an AABD operably linked to the N-terminus 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 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 the vL or vH domain. Similarly, an "AABD operably linked to at or near the N-terminus of a Va and / or Vb domain" means an AABD that is operably linked to the N-terminus of a Va or Vb fragment, or 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 of a Va or Vb domain. A disclosed AABD may also be operably linked to at or near the N-terminus of another domain, either directly or through an intervening linker sequence.

[0199] As used herein, "natural killer cell receptor" or "NK receptor" refers to a cell surface receptor expressed on natural killer (NK) cells.

[0200] As used herein, "natural killer cells" ("NK cells") refer to a type of cytotoxic lymphocyte of the immune system.

[0201] As used herein, "non-naturally occurring agent" or "non-natural" or "exogenous" refers to an agent that is not naturally expressed in a cell. In other words, a non-naturally occurring agent is "engineered" to be expressed in a cell. A non-naturally occurring agent may be a cloned version of a naturally occurring agent. Examples of non-naturally occurring agents include SARs (e.g., CARs, SIRs, AB-TCRs, TFPs, recombinant TCRs). A non-naturally occurring agent may be expressed in a cell using gene transfer techniques known in the art, such as lentivirus- or retrovirus-mediated gene transfer.

[0202] As used herein, "non-naturally occurring immunoreceptor" or "exogenous immunoreceptor" refers to an immunoreceptor that is not naturally expressed in immune cells. In other words, non-naturally occurring immunoreceptors are "engineered" to be expressed in immune cells. Examples of non-naturally occurring immunoreceptors include SAR (second-generation CAR, SIR, cTCR, STAR, zSIR, Ab-TCR, TFP, recombinant TCR, etc.).

[0203] As used herein, a "non-naturally occurring TCR antigen-binding domain" or "exogenous TCR antigen-binding domain" refers to a binding domain operably linked to a chimeric, non-naturally occurring TCR constant region with respect to a naturally occurring TCR. Stated differently, a non-naturally occurring TCR antigen-binding domain is "engineered" and operably linked to a TCR using recombinant molecular biology techniques, and further, the antigen-binding domain is obtained or derived from a molecule other than a TCR found in nature. Antigen-binding domains other than TCRs include antibodies, antibody fragments, vH and vL fragments, scFvs, humanized antibody fragments, chimeric antibody fragments, adapters, non-immunoglobulin antigen-binding scaffolds, receptors, ligands, and the like.

[0204] As used herein, a "non-naturally occurring antigen-binding domain" or "non-naturally occurring extracellular antigen-binding domain" or "heterologous antigen-binding domain" refers to an antigen-binding domain that is not part of a naturally occurring receptor. Examples of 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.

[0205] The term "non-TCR antigen-binding domain" refers to an antigen-binding domain that is not a TCR antigen-binding domain. A non-TCR antigen-binding domain is structurally distinct from the variable domains (i.e., Vα, Vβ, Vγ, Vδ) found in TCRs. A non-TCR antigen-binding domain does not include the variable domains (i.e., Vα, Vβ, Vγ, Vδ) present in natural TCRs. A non-TCR antigen-binding domain also does not include variable domain (i.e., Vα, Vβ, Vγ, Vδ) TCRs generated using recombinant molecular biology techniques. Examples of non-TCR 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.), chimeric antibody fragments, adaptors, non-immunoglobulin antigen-binding scaffolds (e.g., DARPIN, Centyrins, D domains, etc.), adaptors, extracellular Fc-binding domains of receptors (e.g., CD16, CD64, etc.), ligands, cytokines, etc.

[0206] The terms "operably linked" or "functionally linked" or "operably linked" refer to a functional linkage or association between a first component and a second component such that each component functions.

[0207] "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 they have a specified percentage of amino acid residues or nucleotides that are identical (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, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or a specified region, as measured using one of the sequence comparison algorithms below, or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or 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.

[0208] Two examples of algorithms that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1977) Nucleic 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.

[0209] Non-limiting examples of target antigens are shown in Table B. The disclosed SARs may be linked 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.

[0210] Table B JPEG2026501516000020.jpg204151JPEG2026501516000021.jpg42151

[0211] 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.

[0212] 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, e.g., the CD3ζ signaling region described herein.

[0213] The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome. Examples of retroviral vectors include MSCVneo or MSCVpac.

[0214] The term "SAR" or "synthetic antigen receptor" as used herein refers to any non-natural antigen-binding receptor expressed on the surface of a cell (e.g., an immune cell). A "synthetic antigen receptor" or "SAR" can be expressed on the surface of a cell and comprises at least one heterologous antigen-binding domain and at least one membrane-binding domain, the membrane-binding domain being a transmembrane domain or a membrane-anchored domain (i.e., a GPI-linked domain). The antigen-binding domain of a SAR is heterogeneous with respect 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 comprise a hinge domain, an extracellular ligand-binding domain, and / or an optional cytoplasmic domain. In embodiments, a SAR comprises a polypeptide or a set of polypeptides that, when expressed in an effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and is accompanied by intracellular signal generation. A SAR can be one chain, two chains, or more than two chains. A SAR can be monospecific, bispecific, or multispecific. A SAR can have one or more heterologous antigen-binding domains. The term SAR includes traditional CARs (e.g., second generation CARs containing 41BB or CD28 costimulatory domains and CD3z activation domains) and also encompasses newer approaches to confer antigen specificity to cells, such as antibody-TCR chimeric molecules or Ab-TCR (WO 2017 / 070608 A1, incorporated herein by reference), TCR receptor fusion proteins or TFP (WO 2016 / 187349 A1, incorporated herein by reference). Synthetic immunoreceptors (SIRs) (see WO 2018 / 102795 A1, incorporated herein by reference), STARs (see WO 2020 / 029774), HLA-independent TCRs (see WO2019157454A1), Tri-functional T cell antigen binders (Tri-TACs or TACs) (see WO 2015 / 117229 A1, incorporated herein by reference), and zSIRs (see PCT / US2019 / 035096, incorporated herein by reference).Bispecific and multispecific SARs are described in PCT / US2021 / 022641. The term "SAR" encompasses not only CARs but also other antigen-binding receptors, including, but not limited to, recombinant TCRs. SARs also include compositions containing one or more regions derived from CD16A, CD16B, CD3ζ, DAP10, DAP12, FcRγ, TCRαβ, and TCRγδ, as well as variants and fragments thereof. The present disclosure encompasses SARs containing functional variants of the above genes and / or proteins, as well as alternative splicing isoforms, hybrid chains, and homologs from other species. Exemplary regions or fragments of the above genes and proteins that can be used to construct the disclosed SARs are provided in Tables 7 and 22 of the provisional patent application. SARs can also be constructed using polypeptides or fragments with 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to any of the fragments set forth in Tables 7 and 22 of the provisional patent 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 3-6. SARs can also be constructed using polypeptides or fragments containing light and heavy chain CDR regions with 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to the framework regions of any of the fragments defined in Tables 3-6, and with no more than one amino acid substitution in the CDR regions of the antigen-binding fragments (e.g., vL, vH, scFv, vHH, and FHVH, etc.) of the fragments listed in Tables 3-6. Examples of the disclosed SARs are provided in Tables 8-20 and 23-25 ​​of the provisional patent application. SARs are modular in design, and additional SARs can be constructed by exchanging one module of a SAR for another. The expression and activity of these novel SARs can be tested using the methods described in the disclosure to select SARs with optimal functional activity.

[0215] The term "single-chain synthetic antigen receptor" or "single-chain SAR" refers to a synthetic antigen receptor comprising a single polypeptide chain. Examples of such SARs are provided in SEQ ID NOs: 1392-2234 and Table 10 of the provisional patent application.

[0216] The term "double-chain synthetic antigen receptor" or "double-chain SAR" refers to a synthetic antigen receptor that comprises two polypeptide chains, each chain comprising at least one antigen binding domain and a signal transduction chain. Examples of double-chain SAR include synthetic immunoreceptors (SIR) (see WO 2018 / 102795 A1, which is incorporated herein by reference) and zSIR (see PCT / US2019 / 035096, which is incorporated herein by reference). Bispecific and multispecific SARs are described in PCT / US2021 / 022641.

[0217] The term "half-chain synthetic antigen receptor" refers to a synthetic antigen receptor composed of two polypeptide chains, one of which contains at least one antigen-binding domain and a signaling chain (e.g., a TCR alpha constant chain), and the other chain contains a signaling chain (e.g., a TCR beta constant chain) but lacks an antigen-binding domain. Examples of single-half-chain SARs include synthetic immunoreceptors (SIRs) (WO 2018 / 102795 A1, incorporated herein by reference). Examples of single-half-chain SARs are shown in SEQ ID NOs: 50061 and 50062.

[0218] The term "SAR-T" is typically used to refer to T cells engineered to express a synthetic antigen receptor. "SAR-NK" refers to NK cells modified to express a SAR.

[0219] 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, and involves intracellular signal generation. SIRs represent the next-generation CAR platform described in WO 2018 / 102795 A1, which is incorporated herein by reference. In typical embodiments, an SIR comprises one or more antigen-binding domains (e.g., antibodies or antibody fragments, ligands, or receptors) that bind to an antigen as described herein and are linked to one or more T cell receptor constant chains or regions via an optional linker. In some embodiments, the set of polypeptides are contiguous with each other. In some embodiments, an SIR comprises two or more sets of two or more polypeptides. The polypeptides of each set of an SIR are adjacent to each other (functional polypeptide unit 1) but not contiguous with the polypeptides of the other set (functional polypeptide unit 2). In some embodiments, the T cell receptor constant chain (or region) of the SIR is selected from the constant chains of human T cell receptor-α (TCRα or TCRα or TCRa or hTCRα or hTCRα or hTCRa ​​or Cα), human T cell receptor-β1 (TCR-β1 or TCRβ1 or TCRb1 or hTCRβ1 or hTCRβ1 or Cβ1) (also referred to as TCRβ2 or TCRβ2 or TCRb2 or hTCRβ2 or hTCRb2 or Cβ2). TCRβ, TCRβ or TCRb or Cβ), human pre-T cell receptor alpha ((preTCRα or preTCRα or preTCRa or preCα), human T cell receptor gamma (TCRγ or TCRg or hTCRγ or hTCRγ or hTCRγ1 or hTCRγ1 or Cγ), or human T cell receptor delta (TCRδ or TCRδ or hTCRδ 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 a nucleotide sequence that is not wild-type.In some embodiments, the TCR constant chain of the SIR is encoded by their human codon-optimized sequence. In some embodiments, the TCR constant chain of the SIR encodes a wild-type polypeptide sequence, and in other embodiments, the TCR constant chain of the SIR encodes a polypeptide having one or more mutations. In some embodiments, the TCR constant chain of the SIR is encoded by their codon-optimized sequence having one or more mutations.

[0220] 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. Examples of TCR constant chains are listed in Table 9B of the current application and Table 12 of the provisional patent application. TCR constant chains include an Ig-like C1 domain (e.g., SEQ ID NOs: 1168-1175; Table 13 of the provisional patent application), a connecting peptide (e.g., SEQ ID NOs: 1177-1184; Tables 9B and 14 of the provisional patent application), a transmembrane domain (SEQ ID NOs: 1187-1190; Table 9B of the current application and Table 15 of the provisional patent application), and a cytoplasmic domain (e.g., SEQ ID NOs: 1193-1196; Provisional Table 16). The cytoplasmic 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.

[0221] The term "single-chain variable region" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain.

[0222] 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, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules.

[0223] The term "signaling domain" refers to a functional region of a protein that transmits information within a cell and regulates cellular activity through a defined signaling pathway by generating second messengers or functioning as effectors in response to such messengers.

[0224] The term "signaling module" refers to a molecule or molecular complex that contains one or more signaling mediators or signaling adaptors that can initiate a cellular signal.

[0225] The term "signaling mediator" or "signaling adaptor" refers to a molecule that can initiate or inhibit cell signals when recruited by natural or non-natural signaling receptors. In contrast to signaling receptors, signaling adaptors lack their own antigen- or ligand-binding domains. Examples of signaling adaptors include CD3ζ (CD3z), FcRγ, DAP10, DAP12, CD3ε, CD3γ, and CD3δ.

[0226] 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. Examples of signaling chains include the constant chains of TCRα, TCRβ, TCRγ, and TCRδ. Additional exemplary signaling chains include those comprising the transmembrane and / or intracellular regions of CD16, NKp30, NKp44, NKp46, DAP10, DAP12, DNAM-1, NKG2D, CD32, CD64, KIR3DL1, KIR2DS4, FcRγ, and CD3z.

[0227] The term SVH domain as used herein refers to a single human VH domain antibody (VH sdAb). Therefore, these terms are used interchangeably. The term SVH is used interchangeably with an independent vH domain. An example of an SVH is the fully human vH domain (FHVH) shown in SEQ ID NO: (DNA): 425-426 and SEQ ID NO: (PRT): 8805-8806.

[0228] The term "subject" is intended to include any organism in which an immune response can be elicited (eg, any domesticated mammal or a human).

[0229] As used herein, the term "TCR" or "T cell receptor" refers to dimeric heterologous cell surface signaling proteins that form α-β or γ-δ receptors that are typically involved in the recognition of antigens presented by MHC molecules (i.e., antigen recognition in the context of MHC molecules).

[0230] As used herein, "TCR constant chain" refers to the constant chains TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, and pre-TCRα, as well as functional variants, mutants, alternative splicing isoforms, and homologs from non-human species. TCR constant chains lack a variable antigen-binding domain, but are composed of an Ig-like domain, a connecting peptide (or hinge domain), a transmembrane domain, and any intracellular or cytosolic domains.

[0231] As used herein, the term "T lymphocyte" or "T cell" refers to a cell that expresses CD3 (CD3+) and the T cell receptor (TCR+).

[0232] The term "non-T cell" refers to a cell that is not a T cell. In an embodiment, the non-T cell lacks cell surface expression of CD3 and a T cell receptor. In an embodiment, the non-T cell does not respond to a T cell activating antibody such as OKT3. In an embodiment, the non-T cell lacks surface expression of CD3. In an embodiment, the non-T cell lacks expression of one or more CD3 chains selected from the group of CD3ε, CD3γ, and CD3δ. Examples of non-T cells include NK cells, B cells, macrophages, granulocytes, dendritic cells, epithelial cells, etc. The non-T cell may be an immortalized cell line.

[0233] The term "T cell receptor module" or "TCRM" refers to a heterodimer comprising sequences derived from a T cell receptor. A TCRM comprises a T cell receptor transmembrane domain and may further comprise a T cell receptor-linked peptide and / or all or part of the intracellular domain.

[0234] The term "canonical TCRs" refers to TCRs formed by heterodimerization between canonical TCR chains, i.e., TCRα and TCRβ1 or TCRβ2, TCRγ and TCRδ, and pre-TCRα and TCRβ1 or TCRβ2. Furthermore, the term "canonical TCRs" refers to TCRs formed between two TCR chains belonging to the same species (e.g., human, mouse, etc.). For example, a canonical TCR is formed by heterodimerization of a first polypeptide chain containing the connecting peptide, transmembrane domain, and intracellular domain of human TCRα with a second polypeptide chain containing the connecting peptide, transmembrane domain, and intracellular domain of human TCRβ1 or TCRβ2. Similarly, a canonical TCR is formed by heterodimerization of a first polypeptide chain containing the connecting peptide, transmembrane domain, and intracellular domain of human TCRγ with a second polypeptide chain containing the connecting peptide, transmembrane domain, and intracellular domain of human TCRδ.

[0235] The term "non-canonical TCRs" refers to TCRs that are not formed by heterodimerization of canonical TCR chains, i.e., TCRα with TCRβ1 or TCRβ2, TCRγ with TCRδ, and pre-TCRα with TCRβ1 or TCRβ2. Non-canonical TCRs can also be formed by heterodimerization of TCRα with TCRγ, and TCRβ with TCRδ. Non-canonical TCRs can also be formed between variants of the TCRα, β, γ, and δ chains, including their deletion mutants and hybrid chains. For example, a non-canonical TCR is formed by HC-SAR (SEQ ID NO: 32272), which consists of a first chain containing the constant domain and connecting peptide of TCRβ fused in-frame with the transmembrane and intracellular domains of TCRγ, and a second chain that is the TCRα chain (SEQ ID NO: 8838).

[0236] The term "interspecies non-canonical TCRs" refers to TCRs formed by heterodimerization of two TCR chains belonging to different species (e.g., between a human TCRα and a mouse TCRβ chain). Interspecies non-canonical TCRs can also be formed when the components of the hybrid chain are derived from different species. For example, an interspecies non-canonical TCR is formed by heterodimerization of a human TCRα chain with a hybrid TCRβ chain that includes the constant domain of human TCRβ and the connecting peptide, transmembrane domain, and intracellular domain of mouse TCRβ.

[0237] As used herein, "Fragment variable TCR," "TCR-Fv," or "Fv-TCR" refers to the antigen-binding module formed by the variable domains of the TCR chains. The TCR-Fv can be formed by the Vα and Vβ domains, or the Vγ and Vδ domains.

[0238] As used herein, "Fv" or "fragment variable" refers to the antigen-binding module formed by the variable domains of an antibody. Fv can be formed by the vL domain and the vH domain.

[0239] As used herein, "transmembrane module" or "TMM" refers to a molecule or molecular complex that comprises a transmembrane protein (e.g., TCRα, TCRβ, TCRγ, TCRδ, CD16A, or CD3z).

[0240] The term "membrane-associated module" or "MAM" refers to a molecule or molecular complex comprising a transmembrane protein (e.g., CD16A, CD3ζ) or a membrane-anchored protein (e.g., CD16B). This term includes transmembrane proteins such as CD16A, CD3z (or CD3ζ), and GPI-linked proteins such as CD16B. A MAM may further comprise all or part of a hinge domain and / or a cytoplasmic domain.

[0241] As used herein, "therapeutic agent" refers to an agent used to, for example, treat, suppress, prevent, alleviate the effects of, reduce the severity of, reduce the likelihood of onset, slow the progression of, and / or cure a disease.

[0242] As used herein, "therapeutic control" refers to an element used to control the activity of SAR-expressing cells. Examples of therapeutic controls are shown in Table 24 of the provisional application.

[0243] The term "therapeutic 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, and the like.

[0244] As used herein, the phrase "therapeutically effective amount" means a sufficient amount of the composition to treat disorders, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0245] 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 to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic compounds, plasmids, and viruses.

[0246] As used herein, "transmembrane domain" (or TM domain) refers to the region of a receptor (eg, SAR) that crosses the cell membrane.

[0247] As used herein, "vector," "cloning vector," and "expression vector" refer to a vehicle for introducing a polynucleotide sequence (e.g., a foreign gene) into a host cell, transforming the host, and promoting the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

[0248] The term "viral vector" refers to a vector obtained or derived from a virus.

[0249] The term "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta," "CD3ζ" is 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 a "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, that are sufficient to functionally transduce the initial signal required for T cell activation.

[0250] Tables 9-20 and 23-25 ​​of the provisional patent application summarize the target antigens, clone IDs, SEQ ID(DNA), SEQ ID(PRT), and names of several exemplary SARs described in this disclosure. These constructs were generally created by combining the antigen-binding fragments described in Tables 3-6 and PCT / US22 / 17177 with the exemplary signaling chains described herein (including variants as defined in Table 7 of the provisional patent application). SARs are classified into various types based on their architecture or backbone, i.e., the type of signaling chain present in them (e.g., TCR constant chain, CD16 chain, CD3z chain, etc.). However, SARs are modular by design, and it should be understood that the scope of the present disclosure is not limited to the SARs described in Tables 9-20 and 23-25 ​​of the provisional patent application, and that different SARs can be generated by switching out different modules. Therefore, it is possible to combine the antigen-binding domain with other variants of the TCR constant chain, which are not included in the SARs described in Table 7 of the provisional patent application. It is also possible to design SARs using antigen-binding domains other than those listed in Tables 3-6. It is also possible to add, replace, or remove different therapeutic or accessory modules from SARs. Thus, while Tables 9-20 and Tables 23-25 ​​of the provisional patent application include some SARs with antibiotic resistance genes (e.g., PAC), this module can be removed or replaced with other therapeutic and accessory modules (e.g., IL12f, K13, MC159, icaspase, etc.). Finally, other SARs (e.g., CAR, TFP) can be expressed in combination with the SARs described herein.

[0251] The provisional patent application of this disclosure provides tables of sequence numbers for several SARs and their components. These tables can be used to identify SARs that contain specific antigen-binding domains and belong to specific architectures. Alternatively, the sequences of SARs containing specific antigen-binding domains of this disclosure can be determined by homology searches of the sequence listing files accompanying this disclosure. Finally, because SARs are modular in design, the DNA and amino acid sequences of SARs containing specific modules can be generated by replacing modules with new modules. Table 3 JPEG2026501516000022.jpg206148JPEG2026501516000023.jpg209147JPEG2026501516000024.jpg207147JPEG2026501516000025.jpg21314 8JPEG2026501516000026.jpg208148JPEG2026501516000027.jpg209147JPEG2026501516000028.jpg211148JPEG2026501516000029.jpg69147 JPEG2026501516000030.jpg80141 Table 5 JPEG2026501516000031.jpg119149 Table 6 JPEG2026501516000032.jpg48130 Table 7: Peptide / MHC complexes targeted by SAR (uTCR-SAR) JPEG2026501516000033.jpg192133 Table 8: Light and heavy chain CDR1-3 for some novel antigen-binding domains JPEG2026501516000034.jpg192144JPEG2026501516000035.jpg159144

[0252] Table 9: Examples of diseases targeted by SAR. JPEG2026501516000036.jpg204144JPEG2026501516000037.jpg212144JPEG2026501516000038.jpg211144Table 9B JPEG2026501516000039.jpg170141Examples of diseases targeted by SAR are described in PCT / 2020 / 014237.

[0253] Patent application PCT / US22 / 17177 describes a new SAR design called universal TCR-SAR (or uTCR-SAR) that confers T cell receptor-like antigen binding specificity to any cell. In embodiments, the uTCR-SAR comprises: a) a first polypeptide chain comprising a first antigen-binding domain and a first membrane-associated module (MAM); and a second polypeptide chain comprising a second antigen-binding domain and a second membrane-associated module (MAM), wherein the first antigen-binding domain and the second antigen-binding domain form a TCR-like (e.g., TCR-Fv) antigen-binding module that specifically binds to a target antigen, and the first MAM and the second MAM form a non-T cell receptor module (NTCRM). In some embodiments, the first and second MAMs of the uTCR-SAR comprise transmembrane or membrane-associated domains of a signaling adaptor. In embodiments, the signaling adaptor is selected from, but not limited to, one or more of CD3ζ, FcRγ, DAP10 and / or DAP12, or variants or fragments thereof. In embodiments, the MAM of the uTCR-SAR comprises a non-TCR receptor (e.g., CD16).

[0254] The present disclosure provides uTCR-SARs comprising a Vα (or Va or a) chain reference amino acid sequence of SEQ ID NOs: 8685-8693, 40531-32, or variants thereof, and a Vβ (or Vb or b) chain reference amino acid sequence of SEQ ID NOs: 8702-8710, 40533-34, and variants thereof, wherein the uTCR-SARs target antigens set forth in Tables 4 and 7. Sequence ID numbers for exemplary peptide antigens listed in Table 4 are provided in Table 7. Variants may have amino acid sequences with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the reference amino acid sequences (e.g., with respect to either the a chain reference sequence and / or the b chain reference sequence). The uTCR-SAR may be encoded by an alpha (or a) strand reference nucleotide sequence of SEQ ID NOs: 305-313 or a variant thereof, and a beta (or b) strand reference nucleotide sequence of SEQ ID NOs: 322-330 or a variant thereof. Variants may have nucleotide sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the reference nucleotide sequences (e.g., with respect to either the a strand reference sequence and / or the b strand reference sequence).

[0255] According to the present invention, the uTCR-SAR may be a NY-ESO-1 uTCR-SAR which may comprise the Vα (or Va or a) chain reference amino acid sequence of SEQ ID NO: 8685 or a variant thereof, and the Vβ (or Vb or b) chain reference amino acid sequence of SEQ ID NO: 8702 or a variant thereof. The NY-ESO-1 uTCR-SAR may be encoded by the α (or a) chain reference nucleotide sequence of SEQ ID NO: 305 or a variant thereof, and the b chain reference nucleotide sequence of SEQ ID NO: 322 or a variant thereof. In embodiments, the NY-ESO-1 uTCR-SAR binds to the peptide antigen (-SLLMWITQC- or -SLLMWITQCF-) represented by SEQ ID NOs: 22798-99, optionally in a complex with HLA-A*02:01. Examples of uTCR-SARs targeting the NY-ESO-1 peptide (SLLMWITQC or SLLMWITQCFL) and containing different signaling chains are shown in SEQ ID NOs: 10621-10697 (Tables 11 and 12, Provisional Patent Application). Additionally, the nucleic acid and amino acid sequences of uTCR-SARs targeting the NY-ESO-1 peptide, including one or both CD3z signaling chains (e.g., CD3zECDTMCP; e.g., SEQ ID NO: 8880) and a CD3z signaling chain containing a deletion of residue 101 (i.e., CD3zECDTMCP-dQ101; SEQ ID NO: 9327), are shown in SEQ ID NOs: 11567-11623 (Table 13). Because uTCR-SARs are modular, signaling chains can be replaced with different signaling chains (e.g., SEQ ID NOs: 9327-9340) or functional variants thereof to generate new uTCR-SARs with different properties. Similarly, for example, the antigen-binding domain (e.g., Vα, Vβ, Vγ, Vδ, vL, vH, vHH, svTCR, etc.) and linker domain of a uTCR-SAR construct may be replaced with an antigen-binding domain targeting a different antigen (e.g., MAGE-A3, MAGE-A4, etc.) or a different linker (e.g., Ig or Ig-like linker) or functional variant thereof to generate a new uTCR-SAR with different properties.

[0256] This disclosure provides uTCR-SARs targeting MAGE-A4, WT-1, gp100, KRas, HPV-E6, HPV-E7, and PRAME. Va and Vb fragments targeting these antigens are listed in Table 4. For example, the nucleic acid and amino acid sequences of uTCR-SARs targeting different peptide antigens and containing different signaling chains are listed in Tables 11, 12, and 13 of the provisional patent application.

[0257] Patent application PCT / US22 / 17177 (incorporated herein by reference in its entirety) describes single-chain and two-chain synthetic antigen receptors based on CD16 (CD16a and CD16b) isoforms. The present invention describes CD16-based SARs comprising mutant CD16 chains. In embodiments, the mutant CD16 chains of the CD16-SARs of the present disclosure comprise deletions in the CD16 cytoplasmic domain. In embodiments, the mutant CD16 chains of the CD16-SARs of the present disclosure lack the CD16 cytoplasmic domain. In embodiments, the mutant CD16 chains of the CD16-SARs of the present disclosure comprise mutations in the CD16 transmembrane domain. In embodiments, the mutant CD16 chains of the CD16-SARs of the present disclosure lack full-length CD16.

[0258] Nucleic acid and amino acid sequences of the binding peptide, hinge, transmembrane (TM), cytoplasmic (CP) domains of CD16, CD3z, and FcRy chains, as well as coreceptors and costimulatory receptors that can be used to construct SARs, are shown in Table 9B. SEQ ID numbers for different Ig linkers and TCR constant domains are provided in the provisional patent application. Examples of CD16 chains with deleted and mutated cytoplasmic domains that can be used to construct SARs are set forth in SEQ ID NOs: (PRT): 8945-8948. In embodiments, SARs can be constructed using CD16 chains with at least 70% amino acid sequence identity to SEQ ID NOs: (PRT): 8945-8948 or functional variants thereof. Examples of SARs with deleted and mutated cytoplasmic domains are shown in SEQ ID NOs: (DNA): 1111-2234 and SEQ ID NOs: (PRT): 9491-10614 (see also Tables 9-10 of the provisional patent application). In embodiments, the SAR can be configured to have one or more antigen-binding domains attached at or near the N-terminus of the entire or partial extracellular domain of a CD16 chain represented by SEQ ID NO: (PRT):8945-8948 or a functional variant thereof.

[0259] The present disclosure provides single-chain, double-chain, and duplex heterodimeric SARs comprising partial or entire regions of CD16 (FcγRIII). The present disclosure provides SARs comprising CD16 or fragments thereof having at least 70% identity to any of the CD16 sequences (SEQ ID NOs: 8945-8948) described herein while retaining biological activity. For example, mutant CD16 nucleic acid and amino acid sequences that can be used in the construction of the disclosed CD16-SARs are provided in SEQ ID NOs: (DNA): 565-568 and (PRT): 8945-8948, or equivalent residues (i.e., homologs) from non-human species, such as mice, rodents, monkeys, apes, etc.

[0260] In some embodiments, CD16 sequences that can be used to construct the disclosed CD16 SARs can include variants and mutations that increase the affinity of CD16 for the immunoglobulin Fc region (e.g., CD16A--F158V) and further prevent its cleavage from the cell surface (e.g., CD16A-F158V-S197P).

[0261] In certain embodiments, the nucleic acid sequence of the SAR molecule comprises the nucleic acid sequence of human CD16 as set forth in SEQ ID NOs: 565-568. In certain embodiments, the nucleotide sequence of the SAR comprises a sequence encoding an amino acid sequence of CD16 having at least one, five or ten modifications but not more than 20 modifications of the amino acid sequence of SEQ ID NOs: 8945-8948, or a sequence having 70-99% homology to the amino acid sequence of SEQ ID NOs: 8945-8948. In certain embodiments, the SAR molecule comprises the amino acid sequence of SEQ ID NOs: 8945-8948 or the equivalent residues from a non-human species.

[0262] In embodiments, the disclosure provides a single-chain CD16 SAR comprising a partial or entire region of CD16 or a variant thereof. In embodiments, the disclosure provides a single-chain CD16 SAR comprising a partial or entire region of the CD16 extracellular domain. Exemplary CD16 extracellular domain sequences that can be used in constructing the disclosed CD16-SAR are provided in SEQ ID NOs: (DNA): 759-761 and (PRT): 9139-9141, or equivalent residues (i.e., homologs) from non-human species. In embodiments, the disclosure provides a CD16 SAR comprising a partial or entire region of the CD16 hinge domain. Exemplary CD16 hinge domain sequences that can be used in constructing the disclosed CD16-SAR are provided in SEQ ID NOs: (DNA): 763 and (PRT): 9143, or equivalent residues (i.e., homologs) from non-human species. In embodiments, the disclosure provides a CD16 SAR comprising a partial or entire region of the CD16 transmembrane domain. Exemplary CD16 transmembrane sequences that can be used in constructing the disclosed CD16-SARs are provided in SEQ ID NO: (DNA):764 and SEQ ID NO: (PRT):9144, or equivalent residues (i.e., homologs) from non-human species. In embodiments, the present disclosure provides CD16 SARs that include partial or entire regions of the CD16 cytoplasmic domain. The present disclosure also provides SARs that include variants of CD16 or fragments thereof that retain at least one biological activity of wild-type CD16 to which they share identity or homology.

[0263] In embodiments, the CD16 SAR comprises a CD16 extracellular domain comprising both immunoglobulin-like domains (i.e., D1 and D2) connected to the CD16 transmembrane domain via the CD16 hinge domain. In embodiments, the CD16 transmembrane domain comprises a mutation. In embodiments, the CD16 transmembrane domain comprises a S213Y mutation. One example of such a CD16 SAR targeting CD19 is represented by CD8SP-CD19-hu-mROO5-1-(vL-vH)-CD16A-F158V-S197P-v3-L639-S213Y (SEQ ID NO(DNA):2057, SEQ ID NO(PRT):10437). Additional examples of SARs comprising scFvs, FHVHs, vHHs, and non-immunoglobulin antigen-binding scaffolds targeting different antigens are provided in SEQ ID NOs(DNA):1954-2234 and SEQ ID NOs(PRT):10334-10614. Such CD16 SARs also retain the ability to bind to the Fc region of antibodies, antibody fragments, or bispecific / trispecific engagers and mediate antibody-dependent cellular cytotoxicity. Therefore, immune cells (e.g., T cells, NK cells, monocytes / macrophages, neutrophils, etc.) expressing the SAR CD8SP-CD19-hu-mROO5-1-(vL-vH)-CD16A-F158V-S197P-v3-L639-S213Y can target CD19-expressing target cells via the CD19-hu-mROO5-1 scFv region. Furthermore, such immune cells can be redirected to target Her2-expressing target cells in the presence of Herceptin. Alternatively, such immune cells (e.g., T cells or NK cells) can be redirected to target CD20-expressing target cells in the presence of rituximab.

[0264] In one embodiment, the CD16 SAR comprises a CD16 extracellular domain containing both immunoglobulin-like domains (i.e., D1 and D2) linked to the CD16 transmembrane domain via the CD16 hinge domain but lacking the complete CD16 cytoplasmic domain. Examples of such SARs include CD8SP-CD19-hu-mROO5-1-(vL-vH)-CD16A-F158V-S197P-v3-L639 (SEQ ID NO: (DNA): 1495, SEQ ID NO: (PRT): 9875). Further examples of such CD16-based SARs targeting different antigens are provided in SEQ ID NOs: (DNA): 1111-1953 and (PRT): 9491-10333. Such CD16-based SARs also retain the ability to bind to the Fc region of antibodies, antibody fragments, or bispecific / trispecific engagers and mediate antibody-dependent cellular cytotoxicity. Therefore, immune cells expressing such SARs (e.g., T cells, NK cells, monocytes / macrophages, neutrophils, etc.) can target cells via their exogenous antigen-binding domains (e.g., scFv, vHH, FHVH, etc.). Furthermore, such immune cells can be redirected to target Her2-expressing target cells in the presence of Herceptin. Alternatively, such immune cells (e.g., T cells or NK cells) can be redirected to target CD20-expressing target cells in the presence of rituximab.

[0265] In some embodiments, the CD16 SAR comprises a partial or entire CD16 hinge domain attached to a CD16 transmembrane domain. The CD16 transmembrane domain may contain a mutation (e.g., S213Y). Such CD16-SAR lacks both the D1 and D2 domains and therefore lacks the ability to bind to antibodies. In some embodiments, the CD16-based SAR comprises a heterologous hinge (spacer) domain located between the antigen-binding domain (e.g., scFv or AABD) and the hinge domain of CD16.

[0266] In embodiments, a CD16 SAR comprises an AABD (e.g., vHH, FHVH, chVH, centyrin, affibody, etc.) inserted between the D2 domain and the hinge domain of CD16, and an optional intervening linker (e.g., a Gly4-Ser linker). In an embodiment, the different domains from the amino to carboxy terminus of such a CD16 SAR include an N-terminal signal peptide, a CD16-D1 domain, a CD16-D2 domain, an optional linker, an AABD (e.g., vHH, FHVH, centyrin, affibody, etc.), an optional linker, a CD16-hinge domain, and a CD16 transmembrane domain.

[0267] It should be understood that the different CD16 domains (i.e., extracellular, D1, D2, hinge, and transmembrane) that can be used to construct SARs can include their entire sequences or deletion mutants or variants, so long as the domain retains at least one of its functional properties. The CD16 domains can include one or more of their wild-type sequences or high-affinity (e.g., F158V) or high-affinity non-cleavable (e.g., F158V / S197P or F158V / S197R) variants.

[0268] In embodiments, the antigen-binding domain of the CD16SAR is an scFv, vL, vH, Fv, Va, Vb, Vg, Vd, TCR-Fv, vHH, FHVH, single-domain antibody, single-chain TCR (scTCR), single variable domain TCR (svd-TCR), non-immunoglobulin antigen-binding scaffold, ligand (e.g., APRIL), or extracellular domain of a receptor (e.g., PD1, NKG2D, NKp30, NKp44, NKp46, etc.). The chain of the single-chain CD16SAR may bind to one antigen or multiple antigens (e.g., two, three, four, etc.). The chain of the single-chain CD16SAR may further comprise one or more adaptors (e.g., RZIP, EZIP, NKG2D-YA, etc.).

[0269] In some embodiments, the disclosed CD16 SAR comprises a molecule of the general formula:

[0270] AABD(n) - optional CD16 D1 domain - optional CD16 linker domain - optional - CD16 D2 domain, CD16 hinge domain - CD16 transmembrane domain. In one embodiment, n is at least 2, for example 2, 3, 4 or 5. The AABD (Autonomous antigen binding domain) forms the antigen binding domain and is located on the extracellular side when expressed intracellularly.

[0271] In some embodiments, the disclosed CD16 SAR comprises a molecule of the general formula:

[0272] scFv(n)-any CD16 D1-any CD16 linker domain-any-CD16 D2 domain, CD16 hinge domain-CD16 transmembrane domain, where n is 1 or more.

[0273] The nucleic acid and amino acid sequences of SARs comprising the CD16A=F158V=S197P=v3-L642 variant fused to different antigen-binding domains targeting different antigens are shown in Table 9 of the provisional patent application. The nucleic acid and amino acid sequences of SARs comprising other CD16 variants fused to different antigen-binding domains targeting different antigens are shown in Table 10 of the provisional patent application. In embodiments, exemplary SARs comprise a CD16 variant chain sequence and a vHH fragment or FHVH fragment attached to an scFv targeting CD19. For example, SARs comprising a CD16 variant chain sequence and an adapter (SEQ ID NOs: 1381-87) or scTCR are also provided.

[0274] The nucleic acid and amino acid sequences of exemplary SARs comprising mutant CD16A fused to vHH and FHVH fragments targeting different antigens are represented by SEQ ID NOs: (DNA): 1300-1368 and SEQ ID NOs: (PRT): 9680-9748, respectively.

[0275] The nucleic acid and amino acid sequences of exemplary SARs comprising mutant CD16A fused to a non-immunoglobulin antigen-binding domain (e.g., DARPIN, Centyrin, affibody), receptor extracellular domain (e.g., NKG2D), ligand / cytokine (e.g., TPO), adaptor (e.g., EZIP, K4, ULBP2R, and ULBP2-S3, etc.) are shown in Table 9 of the provisional patent application as SEQ ID NOs: (DNA): 1369-1387 and SEQ ID NOs: (PRT): 9747-9767, respectively.

[0276] T cells expressing single-chain CD16-based SARs with mutant CD16 chains can activate NFAT signaling, induce IL2 production, promote T cell proliferation, promote T cell activation, and exert cytotoxicity when exposed to cells expressing the cognate target antigen. In another example embodiment, NK cells expressing single-chain CD16-SARs with mutant CD16 chains can induce IL2 production, promote NK cell proliferation, promote NK cell activation, or exert cytotoxicity when exposed to cells expressing the cognate target antigen. In another example embodiment, monocytes / macrophages expressing single-chain CD16-SARs with mutant CD16 chains can induce phagocytosis of target cells when exposed to cells expressing the cognate target antigen. In another example embodiment, granulocytes (e.g., neutrophils) expressing single-chain CD16-SARs with mutant CD16 chains can induce phagocytosis of target cells when exposed to cells expressing the cognate target antigen.

[0277] In one embodiment, the disclosure provides a novel synthetic antigen receptor platform designated CD16-SAR, comprising two chains, at least one of which comprises a partial or entire sequence of a mutant CD16 chain or a variant thereof. In one embodiment, the disclosure provides a novel synthetic antigen receptor platform designated CD16-SAR, comprising two chains, at least one of which comprises a CD16 hinge domain and a CD16 transmembrane domain but lacks a cytoplasmic domain. In one embodiment, the disclosure provides a novel synthetic antigen receptor platform designated CD16-SAR, comprising two chains, at least one of which comprises a CD16 transmembrane domain but lacks a cytoplasmic domain. In one embodiment, the disclosure provides a novel synthetic antigen receptor platform designated CD16-SAR, comprising two chains, at least one of which comprises a CD16 transmembrane domain with one or more mutations but lacks a cytoplasmic domain. In one embodiment, the present disclosure provides a novel synthetic antigen receptor platform designated CD16-SAR, which comprises two chains, at least one of which comprises a CD16 hinge and transmembrane domain but lacks a cytoplasmic domain. In one embodiment, the present disclosure provides a novel synthetic antigen receptor platform designated CD16-SAR, which comprises two chains, at least one of which comprises a CD16 hinge domain and a CD16 transmembrane domain with one or more mutations but lacks a cytoplasmic domain. Examples of double-chain CD16 SARs are shown in SEQ ID NOs: (DNA): 1388-1391 and (PRT): 9768-9771. The sequences of additional exemplary double-chain CD16 SARs are shown in Table 10 of the provisional patent application.

[0278] In embodiments, the present disclosure provides a double-chain CD16-based SAR in which at least one chain comprises a partial or entire region of the CD16 extracellular domain and a CD16 transmembrane domain with a mutation (e.g., S213Y mutation). In embodiments, the present disclosure provides a double-chain CD16-based SAR in which at least one chain comprises a partial or entire region of the CD16 extracellular domain and comprises the CD16 transmembrane domain but lacks the cytoplasmic domain. For example, the CD16 extracellular domain, hinge domain, and transmembrane domain sequences that can be used in constructing the disclosed double-chain CD16-SAR are provided in the previous section.

[0279] The present disclosure provides dual-chain CD16-based SARs in which an antibody vL or vH fragment is operably linked to a first chain containing a CD16 transmembrane domain, and a complementary vH or vL fragment can be linked to a second chain containing a transmembrane domain of a signaling adaptor (e.g., CD3z, FcRy, DAP10, DAP12, etc.). When such two chains (e.g., vL-CD16 and vH-CD3zECDTMCP, or vL-CD16 and vH-FceRy1) are coexpressed in the same cell, the vL and vH fragments can bind to their cognate antigens and transduce cellular signals. Note that the vL and vH fragments of such SARs cannot bind to antigens by themselves in the absence of complementary fragments. Note that the antigen binding of the described CD16-based SARs may involve variable domains derived from TCRs (e.g., Vα, Vβ, Vγ, Vδ) rather than the antibody vL and vH fragments.

[0280] In embodiments, the CD16 chain may be a mutant chain (e.g., consisting of an S213Y mutation). In embodiments, the CD16 chain may lack a cytoplasmic domain or may include a partial cytoplasmic domain. In embodiments, the CD16 chain may include a partial cytoplasmic domain comprising 1, 2, 3, 4, 5, 6, 7, 10, 15, or 20 or fewer amino acid residues. In one example embodiment, when exposed to cells expressing a cognate target antigen, T cells expressing such a CD16-SAR can activate NFAT signaling, induce IL2 production, promote T cell proliferation, promote T cell activation, and exert cytotoxicity. In another example embodiment, when exposed to cells expressing a cognate target antigen, NK cells expressing such a CD16-SAR can induce IL2 production, promote NK cell proliferation, promote NK cell activation, or exert cytotoxicity. In another exemplary embodiment, monocytes / macrophages expressing such CD16-SAR can induce phagocytosis of target cells when exposed to cells expressing the cognate target antigen. In another exemplary embodiment, monocytes / macrophages expressing dual-chain CD16-SAR can induce phagocytosis of target cells when exposed to cells expressing the cognate target antigen. In another exemplary embodiment, granulocytes expressing dual-chain CD16-SAR can induce phagocytosis of target cells when exposed to cells expressing the cognate target antigen.

[0281] The expression and activity of dual-chain SARs (such as CD16-SAR, zSIR, uTCR-CAR, zSAR, and FceRγ1-SAR) can be further increased by incorporating a linker between the vL / vH, Vα / Vβ, or Vγ / Vδ and the signaling adaptor (such as CD3z, FceRγ1) and / or signaling chain (such as CD16). In particular, IgCL (SEQ ID NO: 8961) and Ig-CH1 domains (SEQ ID NOs: 8962-8976) derived from antibodies serve as useful linkers between the vL / vH and the signaling adaptor or signaling chain (such as CD16). Additional Ig-like domains are known in the art (SEQ ID NOs: (DNA): 597-614, 21451-21456 and SEQ ID NOs: (PRT): 8977-8994, 21466-21471) and can serve as useful linkers in alternative embodiments of the present disclosure.

[0282] In embodiments, the linker comprises the Ig-like constant domain of a TCR chain and further comprises a TCR connecting peptide. Such long Ig-like linkers are provided in SEQ ID NOs: 22827-22829, 22833-22835, 22839-22840, and 22843-22844, respectively. Long Ig-like linkers also include functional variants and homologs that encode polypeptides having at least 75% sequence identity to the polypeptides encoded by the above sequences. In embodiments, long Ig-like linkers include N- or C-terminal deletion variants of SEQ ID NOs: 22827-22829, 22833-22835, 22839-22840, and 22843-22844, respectively, in which 1 to 40 (e.g., 1, 5, 10, 15, 20, 25, 30, or 40) amino acid residues encoded at the N or C terminus are deleted.

[0283] In embodiments, the double-chain CD16-SAR comprises an antigen-binding domain (e.g., vL / vH, Vα / Vβ or Vγ / Vδ, scFv, Fab, vHH, non-immunoglobulin antigen-binding scaffold, DARPIN, receptor, cytokine / ligand, adaptor, etc.), and each chain is linked to the two chains via an optional linker comprising the CD16 transmembrane domain. In embodiments, one or both chains lack a cytoplasmic domain. In embodiments, one or both chains comprise a partial cytoplasmic domain.

[0284] In embodiments, at least one chain of the dual-chain CD16 SAR comprises a CD16 extracellular domain comprising both immunoglobulin-like domains (i.e., D1 and D2) linked to a CD16 transmembrane domain via a CD16 hinge domain. The dual-chain CD16 SAR may further comprise an AABD attached, via an optional linker, to or near the N-terminus of a vL, vH, Vα, Vβ, Vγ, or Vδ chain comprising the antigen-binding domain of the SAR.

[0285] In embodiments, the present disclosure provides a dual-chain CD16 SAR comprising a mutant CD16 chain (e.g., S213Y mutation and / or a deleted cytoplasmic domain), wherein one or both chains comprise a partial or entire region of CD16. In embodiments, the present disclosure provides a dual-chain CD16-SAR comprising a mutant CD16 chain (e.g., S213Y mutation and / or lacking the cytoplasmic domain), wherein one or both chains comprise a partial or entire region of the CD16 extracellular domain. In embodiments, the present disclosure provides a dual-chain CD16 SAR comprising a mutant CD16 chain (e.g., S213Y mutation and / or a deleted cytoplasmic domain), wherein one or both chains comprise a partial or entire region of the CD16D1 domain. In embodiments, the present disclosure provides a dual-chain CD16 SAR comprising a mutant CD16 chain (e.g., having an S213Y mutation and / or lacking the cytoplasmic domain), wherein one or both chains comprise a partial or entire region of the CD16 D2 domain, or a functional variant or homolog thereof. In embodiments, the present disclosure provides a dual-chain CD16 SAR comprising a mutant CD16 chain (e.g., having an S213Y mutation and / or lacking the cytoplasmic domain), wherein one or both chains comprise a partial or entire region of the CD16 hinge domain, or a functional variant or homolog thereof. In embodiments, the present disclosure provides a dual-chain CD16 SAR comprising a mutant CD16 chain (e.g., having an S213Y mutation and / or lacking the cytoplasmic domain), wherein one or both chains comprise the CD16 transmembrane domain, or a functional variant or homolog thereof.

[0286] In embodiments, one or both chains of the dual-chain CD16 SAR comprise a CD16 extracellular domain comprising both immunoglobulin-like domains (i.e., D1 and D2) linked to a mutant CD16 transmembrane domain via a CD16 hinge domain. In embodiments, the CD16 transmembrane domain comprises a S213Y mutation. In embodiments, one or both chains of the dual-chain CD16 SAR comprise a CD16 extracellular domain comprising both immunoglobulin-like domains (i.e., D1 and D2) linked to a CD16 transmembrane domain via a CD16 hinge domain but lacking the CD16 cytoplasmic domain. In embodiments, one or both chains of the dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain also retain the ability to bind to the Fc region of an antibody, antibody fragment, or bispecific / trispecific inducer and mediate antibody-dependent cellular cytotoxicity. In embodiments, one or both chains of a double-chain CD16SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprise a partial CD16 extracellular domain containing a second immunoglobulin-like domain (i.e., D2) linked to the CD16 transmembrane domain via a CD16 hinge domain. In embodiments, one or both chains of such a double-chain CD16SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain contain only the D2 domain of CD16 and lack the D1 domain, thereby lacking the ability to bind to the Fc portion of an antibody or antibody fragment. In embodiments, one or both chains of a double-chain CD16SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprise a partial or entire CD16 hinge domain linked to the CD16 transmembrane domain. In embodiments, one or both chains of such a double-chain CD16SAR lack both the D1 and D2 domains and therefore lack the ability to bind to the Fc portion of an antibody or antibody fragment.

[0287] In embodiments, both chains of a dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprise an antigen-binding domain. In embodiments, only one of the chains of a dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises an antigen-binding domain. In embodiments, one chain of a dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises a non-native antigen-binding domain, and the second chain binds to the Fc portion of an antibody or antibody fragment or bispecific / trispecific engager via the CD16 extracellular domain.

[0288] In an embodiment, one chain of the dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises an antigen-binding domain consisting of a vL domain, and the second chain of the dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises an antigen-binding domain consisting of a vH domain. In an embodiment, both chains of the dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprise antigen-binding domains of the same class (i.e., scFv, vHH, FHVH, single-domain antibody, non-immunoglobulin antigen-binding scaffold, ligand, or receptor, etc.). In an embodiment, each chain of the dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises a vHH domain. In an embodiment, each chain of the dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises a FHVH domain. In an embodiment, both chains of a dual-chain CD16 SAR having a mutated CD16 transmembrane domain and / or lacking the cytoplasmic domain comprise antigen-binding domains of different classes (i.e., scFv, vHH, FHVH, single-domain antibody, non-immunoglobulin antigen-binding scaffold, ligand, or receptor, etc.). In an embodiment, one chain of a dual-chain CD16 SAR having a mutated CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises an antigen-binding domain derived from a vHH domain, while the second chain comprises an antigen-binding domain derived from a FHVH domain.

[0289] The two chains of a double-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain can target the same antigen (e.g., CD19) or different antigens (e.g., CD19 and CD20). The two chains of a double-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain can target two different epitopes of a single antigen (e.g., CD19) or two different antigens (e.g., CD19 and CD20). Each chain of the double-chain SAR may bind to one antigen or multiple antigens (e.g., two, three, four, etc.). Each chain of the double-chain CD16 SAR may further include an adaptor (e.g., RZIP, EZIP, NKG2D-YA, NKG2D-FA, etc.).

[0290] In another embodiment, one or both of the CD16 chains of the dual-chain CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain may further comprise a cytoplasmic costimulatory domain. In another embodiment, one or both of the CD16 chains of the dual-chain CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain may further comprise a cytoplasmic co-receptor domain. Examples of costimulatory domains include costimulatory domains of CD8a, CD8b, CD4, etc. In another embodiment, one or both of the CD16 chains of the dual-chain CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain may further comprise a cytoplasmic signaling molecule. Examples of signaling molecules include LAT-200-262del (SEQ ID NO: 50029), Lck, Lck-T316I (SEQ ID NO: 50031), SLP-76-224-244del (SEQ ID NO: 50030), ZAP-70 and variants thereof and variants thereof. In embodiments, the SAR is a z-CD16-SAR comprising one chain comprising a vL, Vα, or Vδ domain operably linked via an Ig-like linker (e.g., IgCL or TCR constant domain), a polypeptide encoding the CD3z hinge, transmembrane, and cytoplasmic domain, and a second polypeptide linker comprising a vH, Vβ, or Vγ domain operably linked via an Ig-like linker (e.g., IgG1-CH1 or TCRα constant domain), a polypeptide encoding the CD16 hinge and transmembrane domain, and a cytoplasmic domain comprising a costimulatory domain (e.g., 4-1BB, CD28, etc.) or a co-receptor domain (e.g., CD8a, CD8b, CD4, etc.) or a signaling molecule (e.g., Lck, Lck-T316I, LAT-200-262del, SLP-76-224-244del, or ZAP70, etc.). In some embodiments, the cytoplasmic rib, co-receptor, or signaling molecule is attached to the C-terminus of the CD16 transmembrane domain. In some embodiments, the cytoplasmic rib, co-receptor, or signaling molecule is attached to the C-terminus of the CD16 cytoplasmic domain. It should be understood that the antigen-binding domain can be switched.Thus, the vL, Vα, and Vδ domains can be linked to the second chain, and similarly, the vH, Vβ, and Vγ domains can be linked to the first chain of such a dual-chain SAR. In another aspect, the first chain may also include a cytoplasmic costimulatory domain (e.g., 41BB), a coreceptor (e.g., CD8), or a signaling molecule (e.g., Lck or LAT) C-terminal to the CD3z transmembrane domain. In another embodiment, the CD3z chain is replaced by FcRγ. Examples of zSARs and zCD16 SARs are shown in Tables 2A-2E. In embodiments, the Va and Vb in the constructs in Tables 2A-2E can be replaced with vL and vH fragments derived from an antibody. Similarly, the vL and vH fragments can be replaced with Va and Vb fragments. The order of Va, Vb, vL, and vH can be reversed. Similarly, one or more CD3z fragments can be replaced with the corresponding FcRy fragment. The Ig linker domain is derived from an immunoglobulin or TCR constant chain. One or both CD3z cytoplasmic domains may have the dQ101 mutation.

[0291] The two chains of CD16A-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain described herein may be encoded by a single polynucleotide chain, translated into a single polypeptide chain, and then cleaved into different proteins. The two chains of CD16A-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain described herein may be expressed using two different promoters and encoded by two separate polynucleotide chains. The two chains of CD16A-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain described herein may be encoded by a single vector. The two chains of CD16A-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain described herein may also be encoded by two different vectors. The nucleic acid molecule encoding CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain may include one or more leader sequences (also known as signal peptides). In one embodiment, each functional unit of CD16A-SAR (e.g., a CD16 chain and an antigen-binding domain linked to a Furine-SGSG cleavable linker) can be preceded by a leader sequence that directs CD16A-SAR to the cell surface as a type I transmembrane protein. In one embodiment, the antigen-binding domain of CD16-SAR faces the extracellular side. In some embodiments, the leader sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 301 to 303 and the amino acid sequence of SEQ ID NOs: 8681 to 8683. In some embodiments, a short nucleic acid sequence (3 to 9 nucleic acids) containing a restriction enzyme site is located between different subunits of CD16A-SAR having a mutant CD16 transmembrane domain and / or between different subunits of CD16A-SAR lacking a cytoplasmic domain, for example, between the signal sequence and the antigen-binding domain of CD16-SAR, or between the antigen binding domain and the CD16 chain.

[0292] The various SARSs disclosed herein are modular in design. Thus, the sequence encoding one of the signaling adaptors, including the CD16 variant chain or CD16SAR, can be replaced with a sequence encoding a different signaling module. Exemplary signaling modules are provided in Table 7 of the provisional patent application. Similarly, the antigen-binding domain can be replaced with another antigen-binding domain.

[0293] In one embodiment, the present disclosure provides a novel platform of synthetic antigen receptors comprising two chains, one of which incorporates a partial or entire region of CD16 with a mutated CD16 transmembrane domain and / or lacking the cytoplasmic domain.

[0294] In another embodiment, the disclosure provides a dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or one or both chains lacking a cytoplasmic domain that includes a partial or entire region of the CD16 extracellular domain. In an embodiment, the disclosure provides a dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or lacking a cytoplasmic domain, where one or both chains includes a partial or entire region of the CD16 hinge domain. In an embodiment, the disclosure provides a dual-chain CD16 SAR having a mutant CD16 transmembrane domain and / or one or both chains lacking a cytoplasmic domain that includes the CD16 transmembrane domain. In an embodiment, the disclosure provides a dual-chain CD16 SAR where one or both chains include a partial or entire deletion of the CD16 cytoplasmic domain.

[0295] The present disclosure provides that the vL fragment of an antibody can bind to a CD16 chain having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain, and the vH fragment can bind to another signaling chain, such as CD3z, FcRγ, NKp30, NKp44, NKp46, TCR alpha constant chain, TCR beta constant chain, TCR gamma constant chain, or TCR delta constant chain. The present disclosure provides that the vH fragment of an antibody can bind to a CD16 chain having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain, and the vL fragment can bind to another signaling chain, such as CD3z, FcRγ, NKp30, NKp44, NKp46, TCR alpha constant chain, TCR beta constant chain, TCR gamma constant chain, or TCR delta constant chain. When two such chains (e.g., vL-mutant CD16 and vH-CD3z) are coexpressed in the same cell, the vL and vH fragments can bind to their cognate antigen and transduce a T cell signal. In particular, T cells expressing CD16-heterodimer SARs with mutant CD16 transmembrane domains and / or lacking the cytoplasmic domain can activate NFAT signaling, induce IL2 production, promote T cell proliferation, promote T cell activation, and exert cytotoxicity when exposed to cell lines expressing the cognate target antigen. In another embodiment, NK cells expressing CD16-SARs with mutant CD16 transmembrane domains and / or lacking the cytoplasmic domain can induce IL2 production, promote NK cell proliferation, promote NK cell activation, or exert cytotoxicity when exposed to cell lines expressing the cognate target antigen. The expression and activity of CD16-heterodimer SARs can be further increased by incorporating linkers between vL / vH and CD16 and other signaling chains (e.g., CD3z, FcRγ, NKp30, NKp44, NKp46, etc.). In particular, the IgCL and Ig-CH1 domains derived from antibodies serve as useful linkers between vL / vH fragments lacking mutant CD16 transmembrane and / or cytoplasmic domains and CD16 fragments.Additional Ig-like domains are known in the art and can serve as useful linkers in alternative embodiments of the disclosure. The present disclosure also provides that the vL / vH fragments in the above SARs can be substituted with Vα and Vβ domains from a TCR to generate uTCR-SARs.

[0296] Also provided herein are clonal iPS cells genetically engineered to contain a CD16 SAR with a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain, among other edits as contemplated and described herein. In embodiments, the CD16 SAR with a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain is a high-affinity CD16 SAR or a high-affinity non-cleavable CD16 SAR (hnCD16-SAR). The genetically engineered iPS cells can be differentiated into effector cells (such as high-affinity CD16 SAR or hnCD16-SAR) that contain a CD16 SAR with a mutant CD16 transmembrane domain and / or a CD16 SAR lacking the cytoplasmic domain, which are then introduced into the iPS cells. In some embodiments, the induced effector cells containing a CD16 SAR with a mutant CD16 transmembrane domain and / or a CD16 SAR lacking the cytoplasmic domain are NK cells. In some embodiments, the induced effector cells comprising a CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain are T cells. In embodiments, a CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain (e.g., a high-affinity CD16SAR or hnCD16-SAR) binds not only to an ADCC antibody or fragment thereof expressed in iPS cells or their derivative cells, but also to bi-, tri-, or multispecific engagers or binders that recognize the CD16 or CD64 extracellular binding domain of the CD16SAR. Thus, the present application includes providing induced effector cells or cell populations thereof with an extracellular binding domain of CD64, or CD16 having FI76V and S197P, having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain, preloaded with one or more ADCC antibodies preselected through binding to the extracellular domain of CD16-SAR expressed on the induced effector cells, in an amount sufficient for therapeutic use in treating diseases of said CD16-SAR.In embodiments, the antigen-binding domain of a CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises an AABD, scFv, Fv, the extracellular domain of a receptor, a ligand, or another non-immunoglobulin antigen-binding module. In embodiments, a CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises an antigen-binding domain attached to or near the N-terminus of the Fc-binding domain of CD16 or CD64. In embodiments, a CD16-SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain further comprises an antigen-binding domain (e.g., an AABD, e.g., an FHVH, chVH, aVH, vHH, darpin, centyrin, affibody, etc.) attached to or near the N-terminus of the Fc-binding domain of CD16 or CD64.

[0297] In embodiments, the disclosed CD16-SAR comprises the Fc binding region of CD32 or CD64 fused in frame to CD16 having a mutant transmembrane and / or cytoplasmic domain or a mutant thereof. In emapy embodiments, the order of the different modules in a CD16SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain may include, from NH2 to C-terminus: antigen-binding domain(n)-CD32-Fc-binding domain-CD16 transmembrane domain; where n = 1, 2, 3, or more. antigen binding domain (n)-CD32-Fc binding domain-CD16 mutant transmembrane domain-CD16 cytoplasmic domain; where n = 1, 2, 3, or more. antigen binding domain(n)-CD32-Fc binding domain-CD16 mutant transmembrane domain; where n = 1, 2, 3, or more.

[0298] In an EMAP embodiment, the order of the different modules in a CD16SAR having a mutated CD16 transmembrane domain and / or lacking the cytoplasmic domain may include, from NH2 to C-terminus:

[0299] antigen-binding domain(n)-CD64-Fc-binding domain-CD16 transmembrane domain; where n = 1, 2, 3, or more.

[0300] antigen binding domain (n)-CD64-Fc binding domain-CD16 mutant transmembrane domain-CD16 cytoplasmic domain; where n = 1, 2, 3, or more.

[0301] antigen binding domain(n)-CD64-Fc binding domain-CD16 mutant transmembrane domain; where n = 1, 2, 3, or more.

[0302] Unlike primary NK cells, mature T cells from primary sources (i.e., native / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that mature T cells expressing exogenous CD16-SAR constructs with mutant CD16 transmembrane domains and / or lacking the cytoplasmic domain exhibited cell surface expression of CD16 SAR and were able to transduce cellular signals (e.g., NFAT signaling) upon exposure to target antigen-expressing cells.

[0303] The present disclosure provides derivative T cells comprising an exogenous CD16-based SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain. In some embodiments, the CD16-based SAR having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain comprises a wild-type sequence of CD16. In some embodiments, the hnCD16 having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain contained in the derivative T cells comprises F176V (158V) and S197R (or S197P). In some other embodiments, the hnCD16 having a mutant CD16 transmembrane domain and / or lacking the cytoplasmic domain contained in the derivative T cells may comprise a complete or partial ectodomain derived from CD64, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain.

[0304] In addition to primary NK cells and T cells, CD16 SARs having a mutant CD16 transmembrane domain and / or lacking the disclosed cytoplasmic domain can be expressed in immortalized cell lines. For example, immortalized cell lines suitable for expressing CD16 SARs having a mutant CD16 transmembrane domain and / or lacking the disclosed cytoplasmic domain include NK92 and NK92MI cell lines. Furthermore, CD16 SARs having a mutant CD16 transmembrane domain and / or lacking the disclosed cytoplasmic domain can be expressed in pluripotent hematopoietic stem cells (e.g., CD34+ stem cells), which can be differentiated to generate CD16 SARs expressing blood cells belonging to different lineages.

[0305] Cells with mutant CD16 transmembrane domains and / or cells lacking the cytoplasmic domain can also express accessory modules encoding cytokines (e.g., membrane-anchored IL2, membrane-anchored IL15, etc.), suicide switches, and survival switches. Examples of accessory modules are provided in SEQ ID NOs: (DNA): 656-667, 904-928, 968-969 and SEQ ID NOs: (PRT): 9036-9047, 9284-9308, 9348-9349.

[0306] The synthetic immunoreceptor (SIR) architecture consists of an antibody vL fragment bound to one TCR constant chain and a vH fragment bound to a second, complementary TCR constant chain that can form a heterodimer with the first. A major issue with SIR design is the steric hindrance between the vL / vH domain and specific residues present in the TCR constant chain, since the vL / vH domains are artificially grafted onto the TCR constant chain. This steric hindrance between the TCR constant chain and the vL / vH domains can interfere with the interaction between the vL and vH domains of the SIR, potentially reducing binding affinity and signaling through the receptor. Because the steric hindrance is due to interactions between specific residues in the vL and vH fragments and those in the TCR constant chain, it varies depending on the sequences of the vL and vH fragments used to generate the SIR. This issue is also seen in other non-TCR antigen-binding domains that can be used to construct SIRs.

[0307] On-target and off-tumor toxicity, cytokine release syndrome, and neurotoxicity are major issues in cell therapy and can be fatal. A potential solution is the generation of affinity-tuned SIRs with varying levels of affinity for target antigens. The standard approach to creating affinity-tuned CAR constructs is to use CDR mutagenesis to select low-affinity antigen-binding domains (e.g., scFvs). However, this approach is costly and time-consuming. CDR mutagenesis of antigen-binding domains can also lead to the acquisition of new binding affinities, potentially resulting in on-target and off-tumor toxicity.

[0308] The present invention provides a solution to the above problem by providing a panel of hybrid chains that can be used to construct SARs (e.g., SIRs). One or two of these hybrid chains can replace one or both wild-type TCR chains when there is steric hindrance between the antigen-binding domain (e.g., vL, vH, scFv, vHH, etc.) used in constructing the SAR and one or both wild-type TCR chains. Hybrid TCR chains can also be used to rapidly generate a large panel of SAR (e.g., SIR) constructs with various affinity levels, saving cost, time, and effort. Furthermore, the HC-SAR approach does not require the generation of a large panel of new antigen-binding domains, thereby avoiding the risk of acquiring new antigen-binding specificities and extratumor toxicity.

[0309] Finally, the HC-SAR (e.g., HC-SIR) approach also offers a strategy for preventing TCR mispairing that leverages our understanding of TCR biogenesis. Without being limited by any theory, functional surface expression of a TCR complex typically involves the recruitment of an accessory CD3 protein by the invariant domains of the two polypeptide chains of the TCR. Disruption of the TCR-CD3 interaction can inhibit both surface expression and signaling of the TCR complex. Therefore, it may be useful to completely prevent mispairing between the introduced and endogenous chains, thereby eliminating the risk of autoimmunity and maximizing surface expression of therapeutic TCR heterodimers. Furthermore, modifications made to the introduced TCR chains may be useful to minimize or avoid foreign sequences; at the very least, the absence of foreign sequences may minimize immunogenicity and prevent the development of antibodies against the introduced TCR. Note that hybrid chain SIRs according to some embodiments herein can minimize or prevent mispairing while simultaneously including host organism sequences (e.g., a fully human TCR for a human host), minimizing immunogenicity to the HC-SAR.

[0310] A number of suitable HC-SAR configurations are provided in accordance with several embodiments herein.

[0311] HC-SAR (e.g., HC-SIR) chains can include various domains, such as a constant domain (C), a connecting peptide (ConnP), a transmembrane domain (TM), and a cytoplasmic tail (CP or IC). The constant domain of the SIR includes an immunoglobulin-like (Ig-like) linker domain. Examples of Ig-like linker domains derived from TCR and immunoglobulin constant domains are provided in SEQ ID NOS: (DNA): 581-614, 19751-19756) and (PRT): 8960-8994, 20371-20376. In embodiments, the TCR constant domain and antibody constant domain (e.g., HC-SIR) used in the construction of the HC-SAR include the aforementioned N- and C-terminal deletion mutants, in which between 1 and 50 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19) are deleted. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50). In some embodiments, the constant domains of the TCR chains contain mutations that result in the formation of disulfide bonds between the two TCR chains. Exemplary mutations include T48C in the TCRa constant domain (Ig-like linker) and S57C in the TCRb constant domain (Ig-like linker domain).

[0312] Some configurations for HC-SAR (e.g., HC-SIR) chains according to some embodiments of the present specification are set forth in Tables 2F-2J herein. It is contemplated that synthetic versions and variants of any or all of the indicated domains may be useful in HC-SIRs according to some embodiments of the present specification. Thus, unless otherwise specified, it is contemplated that the C, ConnP, TM, and CP (IC) domains encompass naturally occurring and synthetic versions of the indicated domains. In some embodiments, the HC-SIRs comprise synthetic variants of naturally occurring human domains. In some embodiments, the HC-SIRs are chimeras of naturally occurring human regions and synthetic variants of non-human species. In some embodiments, the HC-SAR (e.g., HC-SIR) comprises synthetic variants of naturally occurring mouse domains. In some embodiments, the HC-SAR (e.g., HC-SIR) is a chimera of naturally occurring human domains and synthetic variants of mouse domains. In some embodiments, the HC-SIRs are chimeras of naturally occurring human regions and synthetic variants of non-human species.

[0313] According to some embodiments herein, the HC-SAR (e.g., HC-SIR) may be entirely from one organism. Optionally, the HC-SAR (e.g., HC-SIR) may be a chimera comprising a combination of domains from two different organisms (e.g., human and mouse). For example, a chimeric SIR comprising a murine human TCR constant chain is described in WO 2018 / 102795 A1, which is hereby incorporated by reference in its entirety. Without being limited by any theory, it is believed that a fully human and / or chimeric HC-SAR (e.g., HC-SIR) is less likely to be immunogenic in a human host than a mouse HC-SIR. In some embodiments, a chimeric HC-SAR (e.g., HC-SIR) is provided. In some embodiments, the domains of the HC-SAR (e.g., HC-SIR) are entirely human. In some embodiments, the domains of the HC-SAR (e.g., HC-SIR) are entirely derived from a non-human species. In some embodiments, the domains of the HC-SAR are entirely mouse. In some embodiments, the HC-SAR is a chimera of human and non-human domains. In some embodiments, the H--SAR is a chimera of human and mouse domains.

[0314] According to alternative 1, the HC-SAR comprises two polypeptide chains: in the first chain, the antibody vL domain and the constant domain (or Ig-like linker domain) of the α chain (TCR chain), the connecting peptide of the α and β chains, the transmembrane domain and the cytoplasmic tail (TCR β chain), and, reciprocally, in the second chain, the antibody vH domain and the constant domain (or Ig-like linker domain) of the β chain, the connecting peptide, the transmembrane domain and the cytoplasmic tail of the alpha chain. Schematically, this can be diagrammed as follows: first chain: vL-Calpha-ConnPbeta-TMbeta-CPbeta; second chain: vH-Cbeta-ConnPalpha-TMalpha-CPalpha.

[0315] According to alternative 2, the HC-SAR comprises two polypeptide chains: in the first chain, the vL domain of the antibody, the constant domain (or Ig-like linker domain) and connecting peptide of the α chain, the transmembrane domain and cytoplasmic tail of the β chain, and, reciprocally, in the second chain, the vH domain of the antibody, the constant domain (or Ig-like linker domain) and connecting peptide of the β chain, combined with the transmembrane domain and cytoplasmic tail of the α chain. Schematically, this can be diagrammed as follows: first chain: vL-Calpha-ConnPalpha-TMbeta-CPbeta; second chain: vH-Cbeta-ConnPbeta-TMalpha-CPalpha.

[0316] According to alternative 3, the HC-SAR comprises two polypeptide chains: in the first chain, the vL domain, constant domain (or Ig-like linker domain) of the gamma chain (TCR gamma chain), and the connecting peptide, transmembrane domain, and cytoplasmic tail of the delta chain (TCR delta chain), and in the second chain, the vH domain, constant domain (or Ig-like linker domain) of the delta chain and connecting peptide, transmembrane domain, and cytoplasmic tail of the gamma chain. Schematically, this can be diagrammed as follows: first chain: vL-Cgamma-ConnPdelta-TMdelta-CPdelta; second chain: vH-Cdelta-ConnPgamma-TMgamma-CPgamma.

[0317] According to option 4, the HC-SAR comprises two polypeptide chains: in the first chain, the vL, constant domain (or Ig-like linker domain) and connecting peptide of the gamma chain, the transmembrane domain and cytoplasmic tail of the delta chain, and in the second chain, the vH, constant domain (or Ig-like linker domain) and connecting peptide of the delta chain, the transmembrane domain and cytoplasmic tail of the gamma chain. Schematically, this can be diagrammed as follows: first chain: vL-Cgamma-ConnPgamma-TMdelta-CPdelta; second chain: vH-Cdelta-ConnPdelta-TMgamma-CPgamma.

[0318] According to alternative 5, the HC-SAR comprises two polypeptide chains: in the first chain, the antibody vL domain and the constant domain (or Ig-like linker domain) of the alpha chain, the connecting peptide of the delta chain, the transmembrane domain and the cytoplasmic tail, and, mutually, in the second chain, the antibody vH domain and the constant domain (or Ig-like linker domain) of the beta chain, the connecting peptide, the transmembrane domain and the cytoplasmic tail of the gamma chain. Schematically, this can be diagrammed as follows: first chain: vL-Calpha-ConnPdelta-TMdelta-CPdelta; second chain: vH-Cbeta-ConnPgamma-TMgamma-CPgamma.

[0319] According to alternative 6, the HC-SAR comprises two polypeptide chains: in the first chain, the antibody vL domain and the constant domain (or Ig-like linker domain) of the α chain, the connecting peptide of the delta chain, the transmembrane domain, and the cytoplasmic tail, and, respectively, in the second chain, the antibody vH domain and the constant domain (or Ig-like linker domain), the connecting peptide of the γ or β chain, the transmembrane domain, and the cytoplasmic tail. Schematically, this can be diagrammed as follows: first chain: vL-Calpha-ConnPdelta-TMdelta-CPdelta; second chain: vH-Cgamma-ConnPgamma-TMgamma-CPgamma or vH-Cbeta-ConnPbeta-TMbeta-CPbeta.

[0320] According to alternative 7, the HC-SAR comprises two polypeptide chains: in the first chain, the antibody vL domain and constant domain (or Ig-like linker domain) of the delta chain, the connecting peptide of the alpha chain, the transmembrane domain, and the cytoplasmic tail, and, respectively, in the second chain, the antibody vH domain and constant domain (or Ig-like linker domain), the connecting peptide of the gamma or beta chain, the transmembrane domain, and the cytoplasmic tail. Schematically, this can be diagrammed as follows: first chain: vL-Cdelta-ConnPalpha-TMalpha-CPalpha; second chain: vH-Cgamma-ConnPgamma-TMgamma-CPgamma or vH-Cbeta-ConnPbeta-TMbeta-CPbeta.

[0321] According to alternative 8, the HC-SAR comprises two polypeptide chains: in the first chain, the antibody vL domain and the constant domain (or Ig-like linker domain) of the β chain, the connecting peptide of the γ chain, the transmembrane domain and the cytoplasmic tail, and, reciprocally, in the second chain, the antibody vH domain and the constant domain (or Ig-like linker domain), the connecting peptide, the transmembrane domain and the cytoplasmic tail of the alpha or delta chain. Schematically, this can be diagrammed as follows: first chain: vL-Cbeta-ConnPgamma-TMgamma-CPgamma; second chain: vH-Calpha-ConnPalpha-TMalpha-CPalpha or vH-Cdelta-ConnPdelta-TMdelta-CPdelta

[0322] According to alternative 9, the HC-SAR comprises two polypeptide chains: in the first chain, the antibody vL domain and the constant domain (or Ig-like linker domain) of the gamma chain, the connecting peptide of the beta chain, the transmembrane domain, and the cytoplasmic tail, and, reciprocally, in the second chain, the antibody vH domain and the constant domain (or Ig-like linker domain), the connecting peptide, the transmembrane domain, and the cytoplasmic tail of the alpha or delta chain. Schematically, this can be diagrammed as follows: first chain: vL-Cgamma-ConnPbeta-TMbeta-CPbeta; second chain: vH-Calpha-ConnPalpha-TMalpha-CPalpha or vH-Cdelta-ConnPdelta-TMdelta-CPdelta

[0323] According to alternative 10, the HC-SAR is composed of two polypeptide chains: in the first chain, the vL domain of the antibody and the constant domain (or Ig domain) of the antibody chain (e.g., IgCL), the connecting peptide of the gamma chain, the transmembrane domain and cytoplasmic tail of the beta chain, and in the second chain, the vH domain of the antibody, the constant domain (or Ig domain) of the antibody (e.g., IgG1-CH1 or IgG1-CH4), the connecting peptide of the delta chain, the transmembrane domain of the alpha chain, and the cytoplasmic tail. Schematically, this can be diagrammed as follows: first chain: vL-IgCL-ConnPgamma-TMbeta-CPbeta; second chain: vH-IgG1-CH1-ConnPdelta-TMalpha-CPalpha.

[0324] According to alternative 11, the HC-SAR comprises two polypeptide chains: in the first chain, the vL domain of an antibody, the constant domain (or Ig domain) of an antibody chain (e.g., IgCL), the connecting peptide of the gamma chain, the transmembrane domain and cytoplasmic tail of the beta chain, and, reciprocally, in the second chain, the vH domain of an antibody, the constant domain (or Ig domain) of an antibody (e.g., IgG1-CH1 or IgG1-CH4), the connecting peptide, the transmembrane domain of the alpha chain, and the cytoplasmic tail. Schematically, this can be diagrammed as follows: first chain: vL-IgCL-ConnPgamma-TMbeta-CPbeta; second chain: vH-IgG1-CH1-ConnPalpha-TMalpha-CPalpha.

[0325] According to alternative 12, the HC-SAR is composed of two polypeptide chains: in the first chain, the vL domain of the antibody and the constant domain (or Ig domain) of the antibody chain (e.g., IgCL), the gamma chain of the beta chain, the transmembrane domain, and the connecting peptide of the cytoplasmic tail; and in the second chain, the vH domain of the antibody, the constant domain (or Ig domain) of the antibody (e.g., IgG1-CH1 or IgG1-CH4), the connecting peptide of the delta chain, the transmembrane domain, and the cytoplasmic tail. Schematically, this can be diagrammed as follows: first chain: vL-IgCL-ConnPgamma-TMbeta-CPbeta; second chain: vH-IgG1-CH1-ConnPdelta-TMdelta-CPdelta.

[0326] According to alternative 13, the HC-SAR is composed of two polypeptide chains: in the first chain, the connecting peptide of the antibody vL domain and the constant domain (or Ig domain) of the antibody chain (e.g., IgCL), the beta chain of the gamma chain, the transmembrane domain, and the cytoplasmic tail, and in the second chain, the connecting peptide of the antibody vH domain, the constant domain (or Ig domain) of the antibody (e.g., IgG1-CH1 or IgG1-CH4), the connecting peptide, the transmembrane domain of the alpha or delta chain, and the cytoplasmic tail. Schematically, this can be diagrammed as follows: first chain: vL-IgCL-ConnPbeta-TMgamma-CPgamma; second chain: vH-IgG1-CH1-ConnPalpha-TMalpha-CPalpha or vH-IgG1-CH1-ConnPdelta-TMdelta-CPdelta.

[0327] It is understood that the vL and vH domains in the above designs can be switched, such that the first chain contains a vH domain and the second chain contains a vL domain. The vL and vH domains together create an Fv domain that binds to a target antigen with high specificity. It is understood that the IgCL and IgG1-CH1 domains in the above designs can be switched. Numerous IgG-CH domains are known (e.g., SEQ ID NOs: 8963-8976) and can replace the IgG1-CH1 domain (SEQ ID NO: (PRT): 8962). It is also understood that the TCR constant domain can switch one or both Ig linker domains. Thus, the first chain may contain a TCR constant domain (e.g., SEQ ID NOs: 8977-8994) and the second chain may contain an Ig linker domain (e.g., IgG1-CH1).

[0328] It is also understood that in any of the above designs, the CP domain can be switched. Thus, CPdelta can be switched to CPbeta.

[0329] In embodiments, the vL and vH antibody domains of the HC-SAR described above are replaced by one or more non-TCR antigen-binding domains, such as scFv, vHH, single variable domain antibodies, FHVH (fully human vH domain), SVH (single vH domain), SVL (single vL domain), non-immunoglobulin antigen-binding scaffolds (e.g., centrin, affibody, DARPIN, D domain, etc.), ligand-binding domains of receptors, autoantigens, adaptor-binding domains, epitopes, mimotopes, etc.

[0330] In embodiments, the present disclosure provides at least one recombinant polynucleotide encoding at least one hybrid chain synthetic antigen receptor (HC-SIR), or a functional variant thereof, and at least one SAR or functional variant thereof comprising a heterodimer of a T cell receptor (TCR) constant chain, the heterodimer having one or more non-TCR antigen-binding domains. In embodiments, at least one TCR constant chain of the HC-SAR is a hybrid TCR chain. In embodiments, the hybrid TCR chain comprises at least one domain selected from the group consisting of a TCR chain constant domain (C), a TCR chain connecting peptide (ConnP), a TCR chain transmembrane (TM) domain, and a heterologous TCR chain cytoplasmic domain (CP). In embodiments, the hybrid TCR chain comprises at least one domain selected from the group consisting of a TCR chain constant domain (C), a TCR chain connecting peptide (ConnP), and a TCR chain cytoplasmic domain (CP) heterologous to the TCR chain transmembrane (TM) domain. In embodiments, the hybrid TCR chain comprises at least one domain selected from the group consisting of a TCR chain constant domain (C), a TCR chain transmembrane (TM) domain, and a TCR chain cytoplasmic domain (CP) heterologous to a TCR chain connecting peptide (ConnP). In embodiments, the HC-SAR lacks a TCR chain variable domain.

[0331] In one embodiment, at least one domain of the HC-SAR that is heterologous to the TD of the hybrid TCR chain is derived from any of the following: (a) a TCR alpha, TCR beta, TCR gamma, TCR delta, or pre-TCR alpha chain lacking the TCR chain transmembrane domain present in the hybrid TCR chain; b) a TCR chain having a transmembrane domain with less than 50% sequence identity to the TM of the hybrid TCR chain; c) a TCR constant chain or functional variant from a different species; d) any combination of (a), (b), and (c).

[0332] In embodiments, a plurality of domains selected from the group consisting of a constant domain (C), a connecting peptide (ConnP), and a cytoplasmic domain (CP) are heterologous to the transmembrane (TM) domain. In embodiments, ConnP is a) heterologous to the TM domain, b) the constant region (C); c) both a) and b).

[0333] In embodiments, the TM domain of the HC-SAR encodes a peptide having a sequence selected from the group of a) a peptide having a sequence selected from SEQ ID NOs: 31985-88 and 31992, and / or a) a peptide having a sequence selected from the group of transmembrane domains having SEQ ID NOs: 40606-40670 and 40737-40758, or a functional variant or homolog having 1, 2, 3, 4, 5, or 6 amino acid substitutions excluding residues 7, 12, 17, and 21; and / or b) a transmembrane domain of SEQ ID NOs: 40671-40708, or a functional variant or homolog having 1, 2, 3, 4, 5, or 6 amino acid substitutions excluding residues 6, 12, and 16.

[0334] In embodiments, the hybrid chain constant domain (C) is derived from an immunoglobulin. In embodiments, the constant domain of at least one hybrid chain is derived from an immunoglobulin. In embodiments, the constant domains of both hybrid chains are derived from an immunoglobulin.

[0335] In an embodiment, both TCR constant chains of the HC-SAR are hybrid chains.

[0336] In an embodiment, the first hybrid chain of the HC-SAR comprises a first TCR chain transmembrane domain, a non-TCR antigen-binding domain, and a second TCR chain constant domain or a functional variant or fragment thereof, but does not comprise the first TCR chain constant domain. In an embodiment, the second hybrid TCR chain comprises a second TCR chain transmembrane domain, any non-TCR antigen-binding domain, and a first TCR chain constant domain or a functional variant or fragment thereof, but does not comprise the second TCR chain constant domain. In an embodiment, the first TCR chain constant domain comprises a TCR α chain or γ chain constant domain, or a functional variant or fragment thereof. In an embodiment, the first TCR chain transmembrane domain comprises a TCR α chain or γ chain transmembrane domain, or a functional variant or fragment thereof. In an embodiment, the second TCR chain constant domain comprises a TCR beta chain or delta chain constant domain, or a functional variant or fragment thereof. In an embodiment, the second TCR chain transmembrane domain comprises a TCR beta chain or delta chain transmembrane domain, or a variant thereof. In embodiments, the first TCR chain constant domain comprises a TCR alpha or delta chain constant domain, or a functional variant or fragment thereof. In embodiments, the first TCR chain transmembrane domain comprises a TCR alpha or delta chain transmembrane domain, or a functional variant thereof. In embodiments, the second TCR chain constant domain comprises a TCR beta or gamma chain constant domain, or a functional variant or variant thereof. In embodiments, the second TCR chain transmembrane domain comprises a TCR beta or gamma chain transmembrane domain, or a variant thereof.

[0337] In embodiments, the first hybrid TCR chain further comprises a second TCR chain-linking peptide or a functional variant or fragment thereof, but does not comprise the first TCR chain-linking peptide. In embodiments, the second TCR hybrid chain further comprises a first TCR chain-linking peptide or a functional variant or fragment thereof, but does not comprise the second TCR chain-linking peptide. In embodiments, the first TCR chain-linking peptide comprises a TCR alpha or gamma chain-linking peptide or a functional variant or fragment thereof, and the second TCR chain-linking peptide comprises a TCR beta or delta chain-linking peptide or a functional variant or fragment thereof. In embodiments, the first TCR chain-linking peptide comprises a TCR alpha or delta chain-linking peptide or a functional variant or fragment thereof, and the second TCR chain-linking peptide comprises a TCR beta or gamma chain-linking peptide or a functional variant or fragment thereof.

[0338] In an embodiment, the HC-SAR comprises: a) a first polypeptide chain comprising a first antigen-binding domain comprising a vH antibody domain, a TCR constant domain or a functional variant or fragment thereof, and a first T cell receptor domain (TCRD) comprising a first transmembrane domain of a first TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a vL antibody domain, a TCR constant domain or a functional variant or fragment thereof, and a second TCRD comprising a second transmembrane domain of a second TCR subunit, wherein the vH antibody domain of the first antigen-binding domain and the vL antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to a target antigen. In another embodiment, the HC-SAR comprises: a) a first polypeptide chain comprising a first antigen-binding domain comprising a vL antibody domain, a functional variant of a first TCR subunit or a fragment thereof, and a T cell receptor domain (TCRD) comprising a first transmembrane domain of a second TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a vH antibody domain, a TCR constant domain or a functional variant thereof or a functional variant or fragment of a second TCR subunit, and a second TCRD comprising the second transmembrane domain of the first TCR subunit; in embodiments, the vH antibody domain of the first antigen-binding domain and the vL antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to a target antigen. In embodiments, (i) the first TCR subunit is a TCRγ chain and the second TCR subunit is a TCRδ or TCRα chain, or (ii) the first TCR subunit is a TCRδ chain and the second TCR subunit is a TCRγ or TCRβ chain, or (iii) the first TCR subunit is a TCRα chain and the second TCR subunit is a TCRβ or TCRγ chain, or (iv) the first TCR subunit is a TCRβ chain that is a TCR or TCR chain and the second TCR subunit is a TCRα or TCRβ chain.In embodiments, the first polypeptide chain further comprises a TCR-linked peptide or a functional variant or fragment thereof, and the second polypeptide chain further comprises a TCR-linked peptide or a functional variant or fragment thereof. In embodiments, one or both of the linking peptides are heterologous to a) the transmembrane domain and b) the constant region; c) in both a) and b) embodiments, the first TCRD and the second TCRD form a T cell receptor module (TCRM) that can recruit at least one TCR-associated signaling module. In embodiments, the TCRM is a non-canonical TCRM. In embodiments, the TCRM is an interspecies non-canonical TCRM.

[0339] In an embodiment, the present disclosure provides an HC-SAR that specifically binds to a target antigen, comprising: a) a first polypeptide chain comprising a first antigen-binding domain comprising a vH antibody domain, a constant antibody domain, or a functional variant or fragment thereof, and a first T cell receptor domain (TCRD) comprising a first transmembrane domain of a first TCR subunit; and b) a second antigen-binding domain comprising a vL antibody domain, a TCR constant domain, or a functional variant or fragment thereof, and a second TCRD comprising a second transmembrane domain of a second TCR subunit, wherein the VH antibody domain of the first antigen-binding domain and the VL antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to the target antigen; or c) a first polypeptide chain comprising a first antigen-binding domain comprising a vL antibody domain, a constant antibody domain or a functional variant or fragment thereof, and a T cell receptor domain (TCRD) comprising a first transmembrane domain of a first TCR subunit, and d) a second polypeptide chain comprising a second antigen-binding domain comprising a vH antibody domain, a TCR constant domain or a functional variant or fragment thereof, and a second TCRD comprising a second transmembrane domain of a second TCR subunit. In embodiments, the vH antibody domain of the first antigen-binding domain and the vL antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to a target antigen. In embodiments, (i) the first TCR subunit is a TCR gamma chain and the second TCR subunit is a TCR delta or TCR alpha chain, or (ii) the first TCR subunit is a TCR delta chain and the second TCR subunit is a TCR gamma or TCR beta chain, or (iii) the first TCR subunit is a TCR alpha chain and the second TCR subunit is a TCR beta or TCR gamma chain, or (iv) the first TCR subunit is a TCR beta chain and the second TCR subunit is a TCR alpha or TCR delta chain. In embodiments, the first polypeptide chain further comprises a TCR-linked peptide or a functional variant or fragment thereof, and the second polypeptide chain further comprises a TCR-linked peptide or a functional variant or fragment thereof. In embodiments, one or both of the connecting peptides are heterologous to a) the transmembrane domain and b) the constant region; c) in both a) and b) embodiments, the first TCRD and the second TCRD form a T cell receptor module (TCRM) that can recruit at least one TCR-associated signaling module. In embodiments, the TCRM is a non-canonical TCRM. In embodiments, the TCRM is an interspecies non-canonical TCRM.

[0340] In embodiments, the present disclosure provides an HC-SAR in which both TCR constant chains comprise an antibody constant domain linked in-frame to the connecting peptide (ConnP), TM (transmembrane), and CP (cytoplasmic, also called intracellular, or IC) domains of the TCR chains. In embodiments, the ConnP, TM, and IC domains of such an HC-SAR are heterologous to each other, i.e., they are not derived from the same TCR constant chain.

[0341] In an embodiment, the present disclosure provides an HC-SAR that specifically binds to a target antigen, comprising: a) a first polypeptide chain comprising a first antigen-binding domain comprising a vH antibody domain, a first constant antibody domain or a functional variant or fragment thereof, and a first T cell receptor domain (TCRD) comprising a first ConnP, TM, and CP domain of a first TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a vL antibody domain, a second constant antibody domain or a functional variant or fragment thereof, and a second TCRD comprising a second ConnP, TM, and CP domain of a second TCR subunit, wherein the vH antibody domain of the first antigen-binding domain and the vL antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to the target antigen; or c) a first polypeptide chain comprising a first antigen-binding domain comprising a vL antibody domain, a first constant antibody domain or a functional variant or fragment thereof, and a first T cell receptor domain (TCRD) comprising a first ConnP, TM, and CP domain of a first TCR subunit; and d) a second polypeptide chain comprising a second antigen-binding domain comprising a vH antibody domain, a second constant antibody domain or a functional variant or fragment thereof, and a second TCRD comprising a second ConnP, TM, and CP domain of a second TCR subunit, the vH antibody domain of the first antigen-binding domain and the vL antibody domain of the second antigen-binding domain forming an antigen-binding module that specifically binds to a target antigen. In embodiments, the first TCRD and the second TCRD form a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling module. In embodiments, the TCRM is a non-canonical TCRM. In embodiments, the TCRM is an interspecies non-canonical TCRM.

[0342] In embodiments, the present disclosure provides HC-TCRs in which one or both TCR constant chains comprise an antibody constant domain joined in-frame to the connecting peptide (ConnP), TM (transmembrane), and CP (cytoplasmic, also called intracellular, or IC) domains of the TCR chain. In embodiments, the ConnP, TM, and IC domains of such HC-TCRs are heterologous to each other, i.e., they are not derived from the same TCR constant chain.

[0343] In one embodiment, the present disclosure provides an HC-TCR that specifically binds to a target peptide antigen, comprising: a) a first polypeptide chain comprising a first TCR antigen-binding domain comprising a Vα or Vγ domain; a first T cell receptor domain (TCRD) comprising a first ConnP; and the TM and CP domains of a first TCR subunit. b) a second polypeptide chain comprising a second TCR antigen-binding domain comprising a Vβ or Vδ domain; a second constant antibody domain or a functional variant or fragment thereof; and a second TCRD comprising a second ConnP, TM, and CP domains of a second TCR subunit. In one embodiment, the Vα or Vγ domain of the first antigen-binding domain and the Vβ or Vδ antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to the target peptide antigen in an MHC-dependent or MHC-independent manner. In another embodiment, the first TCRD and the second TCRD form a T cell receptor module (TCRM) that can recruit at least one TCR-associated signaling module. In some embodiments, the TCR is a non-standard TCR. In some embodiments, the TCR is an interspecies non-standard TCR. Examples of TCR antigen-binding domains are variable domains (e.g., V and V) of TCRs that can bind to peptides derived from NY-ESO-1, MAGE-A4, WT-1, and PRAME in the presence of αβ HLA-A2 molecules (Table 4). Such HC-TCRs are exemplified by SEQ ID NOs: (DNA): 40512-40513.

[0344] In certain embodiments, Ig-like linker domains derived from TCR and antibody (immunoglobulin) constant domains are selected from the group consisting of SEQ ID NOS: (DNA): 581-614; 19751-19756) and SEQ ID NOS: (PRT): 8960-8994, 20371-20376. In certain embodiments, the antibody constant domains include functional variants and fragments of the foregoing having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, etc.) sequence identity, including variants derived from non-human species. In certain embodiments, the first or second constant antibody domain is an Ig-liker domain (e.g., IgG1-CH1; SEQ ID NOS: 582-596) and the second or first constant antibody domain is an Ig linker domain (e.g., IgG-CL; SEQ ID NOS: 581). In embodiments, the TCR constant domain is an Ig-like linker domain represented by SEQ ID NOs: (DNA): 597-614, 19751-19756 and SEQ ID NOs: (PRT): 8977-8994, 20371-20376. In embodiments, the antibody constant domain includes functional variants and fragments of the foregoing having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, etc.) sequence identity, including variants from non-human species. In embodiments, antibody and TCR constant domains contain 1 to 50 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50).

[0345] In embodiments, the linking peptide is derived from TCR alpha, TCR beta, TCR gamma, TCR delta, or pre-TCR alpha. Examples of linking peptides are represented by SEQ ID NOs: (PRT): 8995-9002.

[0346] In an embodiment, the connecting peptide of the HC-SAR is derived from TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. Examples of connecting peptides are represented by SEQ ID NOs: (PRT):8995-9002. In an embodiment, the connecting peptide is derived from the same TCR chain as the transmembrane domain. For example, both the connecting peptide and the transmembrane domain are derived from the human TCRα chain. In an embodiment, the connecting peptide is derived from the same TCR chain as the TCR constant domain. For example, both the connecting peptide and the TCR constant domain are derived from the human TCR chain α. In an embodiment, the connecting peptide is derived from a different TCR chain as the transmembrane domain. For example, the connecting peptide is derived from the human TCRβ chain, and the transmembrane domain is derived from the human TCR chain α. In an embodiment, the connecting peptide is derived from a different TCR chain as the TCR constant domain. For example, the connecting peptide is derived from the human TCRβ chain, and the TCR constant domain is derived from the human TCRα chain.

[0347] In embodiments, the ConnP, TM and CP domains of at least one TCR subunit are heterologous. In embodiments, the ConnP and TM domains of at least one TCR subunit are heterologous. In embodiments, the ConnP, TM and CP domains of both TCR subunits are heterologous. In embodiments, the ConnP and TM domains of both TCR subunits are heterologous.

[0348] In one embodiment, the first TCR subunit comprises a TCRβ ConnP and a TCRγ TM, and the second TCR subunit is a TCRα or TCRδ. In one embodiment, the first TCR subunit comprises a TCRγ ConnP and a TCRβ TM, and the second TCR subunit is a TCRα or TCRδ. In one embodiment, the first TCR subunit comprises a TCRα ConnP and a TCRδ TM, and the second TCR subunit is a TCRβ or TCRγ. In one embodiment, the first TCR subunit comprises a TCRδ ConnP and a TCRα TM, and the second TCR subunit is a TCRβ or TCRγ.

[0349] In one embodiment, the first TCR subunit comprises a TCRβ ConnP and a TCRγ TM, and the second TCR subunit comprises a TCRα ConnP and a TCRδ TM or a TCRδ ConnP and a TCRα TM. In one embodiment, the first TCR subunit comprises a TCRγ ConnP and a TCRβ TM, and the second TCR subunit comprises a TCRα ConnP and a TCRδ TM or a TCRδ ConnP and a TCRα TM. In one embodiment, the first TCR subunit comprises a TCRα ConnP and a TCRδ TM, and the second TCR subunit comprises a TCRβ ConnP and a TCRγ TM or a TCRγ ConnP and a TCRβ TM. In an embodiment, the first TCR subunit comprises a TCRδ ConnP and a TCRα TM and the second TCR subunit comprises a TCRβ ConnP and a TCRγ TM or a TCRγ ConnP and a TCRβ TM.

[0350] In one embodiment, the TM (transmembrane) domains of TCRα, TCRβ, TCRγ, TCRδ, and preTCRα are represented by SEQ ID NOs: (PRT) 31985-31988 and 31992, respectively. In an embodiment, the TM domains of TCRα, TCRδ, and preTCRα are peptides of SEQ ID NOs: (PRT): 40606-40638, 40639-40670, 40737-40758, respectively, or functional variants or homologs having one to six conservative amino acid substitutions excluding residues 7, 12, 17, and 21. In an embodiment, the TM domains of TCRβ and TCRγ are peptides having a sequence selected from SEQ ID NOs: 40671-40708 and 40709-40736, respectively, or functional variants or homologs having one to six amino acid substitutions excluding residues 6, 12, and 16.

[0351] Examples of HC-SARs are set forth in SEQ ID NOs (DNA): 40162-40171 and 55078-55335.

[0352] In embodiments, the vL and vH antibody domains of the above-mentioned HC-SAR are replaced by one or more non-TCR antigen-binding domains, such as scFv, vHH, single variable domain antibodies, FHVH (fully human vH domain), SVH (single vH domain), SVL (single vL domain), non-immunoglobulin antigen-binding scaffolds (e.g., centrin, affibody, DARPIN, D domain, etc.), ligand-binding domain of a receptor, receptor-binding domain of a ligand, autoantigen, adaptor-binding domain, epitope, mimotope, etc.

[0353] In embodiments, the present disclosure provides a SAR comprising a heterodimer of two chains, each chain comprising a C, a TM, a ConnP, and a CP, and at least one domain of the SAR being replaced by a corresponding domain from another species (e.g., human, mouse, monkey, etc.). In embodiments, the SAR comprises at least one non-TCR antigen-binding domain bound to at least one of the two chains. In embodiments, at least one chain of the SAR comprises at least one transmembrane domain selected from SEQ ID NOs: 31985-88 and 31992. In embodiments, both chains of the SAR comprise transmembrane domains selected from SEQ ID NOs: 31985-88 and 31992. In embodiments, at least one domain of the HC-SAR is of human origin. In embodiments, at least one domain of the HC-SAR is of non-human origin.

[0354] In some embodiments, any domain of the HC-SAR architecture described herein is fully human. In some embodiments, the domain of the HC-SAR is derived entirely from a non-human species. In some embodiments, the domain of the HC-SAR is fully mouse. In some embodiments, the HC-SAR is a chimera of a human domain and a non-human domain. In some embodiments, the HC-SAR is a chimera of a human domain and a mouse domain.

[0355] In embodiments, the HC-SAR forms a T cell receptor module (TCRM) that, when expressed in a T cell, can recruit at least one TCR-associated signaling module.

[0356] In one embodiment, the HC-SAR forms a **non-canonical T-cell receptor module (TCRM)**. Examples of non-canonical T-cell receptor modules include modules comprising signaling chains including: a) the TCRα and TCRγ transmembrane domains and / or connecting peptides; b) the TCRβ and TCRδ transmembrane domains and / or connecting peptides. Examples of HC-SARs that form non-canonical T-cell receptor modules are set forth in SEQ ID NOs: 32254-32265, 32268-32269, 40142, 40145, and 40146-40161. In one embodiment, the HC-SAR forms an interspecies non-canonical T-cell receptor module. Examples of HC-SARs that form such non-canonical T-cell receptor modules are set forth in SEQ ID NOs: 32266-32267.

[0357] In embodiments, the TCR constant chain comprising any of the disclosed SARs (e.g., HC-SAR, SIR, Ab-TCR, zSIR, zCD16-SIR, etc.) comprises one or more of the following features: (i) mutations that enhance constant chain dimerization and reduce their pairing with endogenous T cell receptor chains; (ii) one or both TCR constant chains are human codon-optimized; and / or (iii) the TCR constant chains are derived from a human, mouse, or dog.

[0358] In one embodiment, the non-TCR antigen-binding domain comprising any of the SARs of the present disclosure (e.g., HC-SAR, SIR, Ab-TCR, zSIR, zCD16-SIR, etc.) is selected from the following group: an antibody; an antibody fragment selected from Fv, Fab, and (Fab')2; an antibody heavy chain variable region (vH domain); an antibody light chain variable region (vL domain); a single chain variable fragment (scFv); a single domain antibody (SDAB); a VHH domain from Camelidae; a monomeric variable region of an antibody; and may further be selected from the following non-immunoglobulin antigen-binding scaffolds (and binding fragments thereof): DARPIN, D domain (DD), affibody, affilin, adnectin, affitin, obody, lipibody, finomer, alphabody, avimer, atrimer, centilin, pronectin, anticalin, Kunitz domain, armadillo repeat protein; also, any of the following, or binding fragments thereof: extracellular domain of a receptor; ligand; bispecific antibody; autoantigen; an HLA molecule or a fragment thereof; a β2M molecule or a fragment thereof; an HLA / peptide complex.

[0359] In embodiments, the disclosed SARs, including HC-SARs and SIRs, comprise the variable regions of the heavy and light chains of an antibody or fragment thereof, such that, upon expression, one of the heavy and light chains of the antibody or fragment thereof is...

Claims

1. A recombinant polynucleotide encoding at least one hybrid chain synthetic antigen receptor (HC-SAR) or a functional variant thereof, wherein the at least one SAR or functional variant thereof comprises a heterodimer of two T cell receptor (TCR) constant chains, the heterodimer comprising one or more non-TCR antigen-binding domains, the non-TCR antigen-binding domains being operably linked to the TCR constant chain via an optional linker, and at least one TCR constant chain of the HC-SAR is a hybrid TCR chain, the hybrid TCR chain comprising at least one domain selected from the group consisting of a TCR chain constant domain (C), a TCR chain connecting peptide (ConnP), a TCR chain transmembrane (TM) domain, and a TCR chain cytoplasmic (CP) domain, wherein the at least one domain is of heterologous origin.

2. 2. The HC-SAR of claim 1, wherein at least one domain heterologous to the TD of the hybrid TCR chain is derived from a group of domains heterologous to the TD of the hybrid TCR chain: a. a TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα chain lacking the TCR chain transmembrane domain present in the hybrid TCR chain; b. A TCR chain having a TM domain with less than 50% sequence identity to the TM of the aforementioned hybrid TCR chain; c. TCR constant chain or functional variant thereof of 757 species origin; d. Any combination of a-c.

3. An HC-SAR as described in claim 1, wherein multiple domains selected from the group of domains consisting of a TCR chain constant domain (C), a TCR chain connecting peptide (ConnP), a transmembrane (TM) domain, and a connecting domain (CP) are heterologous.

4. The HC-SAR of claim 1, wherein ConnP is heterologous to any of the following: a. TM domain; b. constant domain (C); c. a and b.

5. The HC-SAR of claim 1, wherein the TM domain encodes: a. a sequence selected from SEQ ID NOs: 31985-88 and 31992; and / or b. A sequence selected from the following group of sequences: i) the TM domain of SEQ ID NOs: 40606-40670 and 40737-40758, or functional variants or homologs containing 1-6 amino acid substitutions excluding the remaining 7, 12, 17, and 21 amino acids; ii) The TM domain of SEQ ID NOs: 40671 to 40708, or functional variants or homologs containing 1 to 6 amino acid substitutions excluding the remaining 6, 12, and 16.

6. HC-SAR according to claim 5, the first hybrid TCR chain further comprises a second TCR chain-linking peptide or a functional variant or fragment thereof, but does not comprise the first TCR chain-linking peptide; the second hybrid TCR chain further comprises a first TCR chain-linking peptide or a functional variant or fragment thereof, but does not comprise a second TCR chain-linking peptide; the first TCR chain-linking peptide consists of a TCR alpha chain or TCR gamma chain-linking peptide or a functional variant or fragment thereof; the second TCR chain-linking peptide consists of a TCR beta chain or TCR delta chain-linking peptide or a functional variant or fragment thereof; or the first TCR chain-linking peptide consists of a TCR alpha chain or TCR delta chain-linking peptide or a functional variant or fragment thereof; The second TCR chain-linking peptide is characterized in that it consists of a TCR β chain or TCR γ chain-linking peptide or a functional variant or fragment thereof.

7. HC-SAR according to claim 6, the first hybrid chain comprises a first TCR chain transmembrane domain, a non-TCR antigen-binding domain, and a second TCR chain constant domain or a functional variant or fragment thereof, but does not comprise the first TCR chain constant domain; the second hybrid TCR chain comprises a second TCR chain transmembrane domain, an optional non-TCR antigen-binding domain, and a first TCR chain constant domain or a functional variant or fragment thereof, but does not comprise a second TCR chain constant domain; wherein the first TCR chain constant domain consists of a constant domain of a TCR alpha chain or a TCR gamma chain or a functional variant or fragment thereof; the second TCR chain constant domain consists of a TCR beta chain or a TCR delta chain constant domain or a functional variant or fragment thereof; the second TCR chain transmembrane domain consists of a TCR beta or TCR delta chain transmembrane domain or a variant thereof; Alternatively, the first TCR chain constant domain consists of a TCR alpha chain or a TCR delta chain constant domain or a functional variant or fragment thereof; the first TCR chain transmembrane domain consists of a TCR alpha chain or a TCR delta chain transmembrane domain or a functional variant thereof; the second TCR chain constant domain consists of a TCR β chain or a TCR γ chain constant domain or a functional variant thereof; The second TCR chain transmembrane domain is characterized by consisting of a TCR β chain or TCR γ chain transmembrane domain or a mutant thereof.

8. HC-SAR according to claim 1, characterized in that both TCR constant chains are hybrid chains.

9. HC-SAR according to claim 1, characterized in that the constant domain of at least one hybrid chain is derived from an immunoglobulin or both hybrid chains are derived from an immunoglobulin.

10. HC-SAR according to claim 1, the first polypeptide chain comprises a first antigen-binding domain having a vH antibody domain, a first T cell receptor domain (TCRD) having a TCR constant domain or a functional variant or fragment thereof, and a first transmembrane domain of a first TCR subunit; the second polypeptide chain comprises a second antigen-binding domain having a vL antibody domain, a TCR constant domain or a functional variant or fragment thereof, and a second TCRD having a second transmembrane domain of a second TCR subunit; the vH antibody domain of the first antigen-binding domain and the vL antibody domain of the second antigen-binding domain form an antigen-binding module that specifically binds to a target antigen; or the first polypeptide chain comprises a first antigen-binding domain having a vL antibody domain, a TCR constant domain of a first TCR subunit or a functional variant or fragment thereof, and a TCRD comprising a first transmembrane domain of a second TCR subunit; the second polypeptide chain comprises a second antigen-binding domain having a vH antibody domain, a TCR constant domain of a second TCR subunit or a functional variant or fragment thereof, and a TCRD comprising the second transmembrane domain of the first TCR subunit; The vH and vL domains form an antigen-binding module that specifically binds to a target antigen; (i) the first TCR subunit is a TCR gamma chain and the second TCR subunit is a TCR delta chain or a TCR alpha chain; (ii) the first TCR subunit is a TCR δ chain and the second TCR subunit is a TCR γ chain or a TCR β chain; (iii) the first TCR subunit is a TCR α chain and the second TCR subunit is a TCR β chain or a TCR γ chain; or (iv) the first TCR subunit is a TCR β chain and the second TCR subunit is a TCR α chain or a TCR δ chain; and optionally, each of the first and second polypeptide chains further comprises a TCR-linked peptide or a functional variant or fragment thereof, the linking peptide or functional variant or fragment being located N-terminal to the transmembrane domain; and optionally, one or both connecting peptides are heterologous to any of the following: a) transmembrane domain; b) constant domain; c) Both a) and b).

11. HC-SAR according to claim 1, the first polypeptide chain comprises a first antigen-binding domain having a vH antibody domain, a constant antibody domain or a functional variant or fragment thereof, and a TCR domain (TCRD) having a transmembrane domain of a first TCR subunit; the second polypeptide chain comprises a second antigen-binding domain having a vL antibody domain, a TCR constant domain or a functional variant or fragment thereof, and a TCRD having a transmembrane domain of a second TCR subunit; the vH and vL antibody domains form an antigen-binding module that specifically binds to a target antigen; or the first polypeptide chain comprises a first antigen-binding domain having a vL antibody domain, a constant antibody domain or a functional variant or fragment thereof, and a TCRD having a transmembrane domain of a first TCR subunit; the second polypeptide chain comprises a second antigen-binding domain having a vH antibody domain, a TCR constant domain or a functional variant or fragment thereof, and a TCRD having a transmembrane domain of a second TCR subunit; (i) the first TCR subunit is a TCR gamma chain and the second TCR subunit is a TCR delta chain or a TCR alpha chain; (ii) the first TCR subunit is a TCR δ chain and the second TCR subunit is a TCR γ chain or a TCR β chain; (iii) the first TCR subunit is a TCR α chain and the second TCR subunit is a TCR β chain or a TCR γ chain; (iv) the first TCR subunit is a TCR β chain and the second TCR subunit is a TCR α chain or a TCR δ chain; and optionally, each of the first and second polypeptide chains further comprises a TCR-linked peptide or a functional variant or fragment thereof, the linking peptide or functional variant or fragment being located N-terminal to the transmembrane domain; and optionally, one or both connecting peptides are heterologous to any of the following: a) transmembrane domain; b) constant domain; c) Both a) and b).

12. HC-SAR according to claim 1, the first polypeptide chain comprises a first antigen-binding domain having a vH antibody domain, a first constant antibody domain or a functional variant or fragment thereof, a TCR domain (TCRD) comprising the ConnP, TM, and CP domains of a first TCR subunit; the second polypeptide chain comprises a second antigen-binding domain having a vL antibody domain, a second constant antibody domain or a functional variant or fragment thereof, a TCR domain (TCRD) comprising the ConnP, TM, and CP domains of a second TCR subunit; the vH and vL antibody domains form an antigen-binding module that specifically binds to a target antigen; or the first polypeptide chain comprises a first antigen-binding domain having a vL antibody domain, a first constant antibody domain or a functional variant or fragment thereof, a TCRD comprising the ConnP, TM and CP domains of a first TCR subunit; the second polypeptide chain comprises a second antigen-binding domain having a vH antibody domain, a second constant antibody domain or a functional variant or fragment thereof, a TCRD comprising the ConnP, TM and CP domains of a second TCR subunit; And optionally, a) the ConnP, TM and CP domains of at least one TCR subunit are heterologous; b) the ConnP and TM domains of at least one TCR subunit are heterologous; c) ConnP, TM and CP of both TCR subunits are heterologous; d) The ConnP and TM domains of both TCR subunits are heterologous.

13. HC-SAR according to claim 1, 10, 11 or 12, the first TCR subunit contains ConnP of TCRβ and TM of TCRγ, and the second TCR subunit is TCRα or TCRδ; or the first TCR subunit comprises a ConnP of TCRγ and a TM of TCRβ, and the second TCR subunit is TCRα or TCRδ; or the first TCR subunit comprises a ConnP of TCRα and a TM of TCRδ, and the second TCR subunit is TCRβ or TCRγ; or the first TCR subunit comprises a ConnP of TCRδ and a TM of TCRα, and the second TCR subunit is TCRβ or TCRγ; Alternatively, the first TCR subunit comprises a TCRβ ConnP and a TCRγ TM, and the second TCR subunit comprises a TCRα ConnP and a TCRδ TM or a TCRδ ConnP and a TCRα TM; Alternatively, the first TCR subunit comprises a TCRγ ConnP and a TCRβ TM, and the second TCR subunit comprises a TCRα ConnP and a TCRδ TM or a TCRδ ConnP and a TCRα TM; or the first TCR subunit comprises a TCRα ConnP and a TCRδ TM, and the second TCR subunit comprises a TCRβ ConnP and a TCRγ TM or a TCRγ ConnP and a TCRβ TM; Alternatively, the first TCR subunit comprises a TCRδ ConnP and a TCRα TM, and the second TCR subunit comprises a TCRβ ConnP and a TCRγ TM or a TCRγ ConnP and a TCRβ TM.

14. HC-SAR according to claim 1, characterized in that the TCR constant chain has one or more of the following characteristics: Contains mutations that enhance constant chain dimerization and prevent pairing with endogenous T cell receptor chains; One or both TCR constant chains are human codon-optimized; and / or of human, murine, or canine origin.

15. 2. The HC-SAR of claim 1, wherein the non-TCR antigen-binding domain is selected from the following group of binding domains: (a) Antibody; (b) an antibody fragment selected from Fv, Fab, and (Fab')2; (c) the heavy chain variable region (vH domain) of the antibody; (d) the light chain variable region (vL domain) of an antibody; (e) single-chain variable fragment (scFv); (f) Single chain antibody (SDAB); (g) Camelidae VHH domain; (h) a monomeric variable region of an antibody; (i) a non-immunoglobulin antigen-binding scaffold or fragment thereof selected from a DARPIN, a D domain, an affibody, an affilin, an adnectin, an affitin, an obody, a lipibody, a phinomer, an alphabody, an avimer, an atrimer, a centailin, a pronectin, an anticalin, a Kunitz domain, or an armadillo repeat protein; (j) the extracellular domain of a receptor or a fragment thereof; (k) a ligand or a fragment thereof; (l) Bispecific antibodies; (m) self-antigen; (n) an HLA molecule or a fragment thereof; (o) a β2M molecule or a fragment thereof; (p) HLA / peptide complex.

16. In the HC-SAR of claim 1, one or more autonomous antigen-binding domains (AABDs) or fragments thereof are operatively linked to the N-terminus or N-terminally adjacent to one or more non-TCR antigen-binding domains via any linker.

17. 17. The HC-SAR of claim 16, wherein the one or more autonomous antigen-binding domains (AABDs) or fragments thereof are selected from the group of antigen-binding domains consisting of: (a) a single vH domain (SVH) or a fragment thereof; (b) a single vL domain (SVL) or a fragment thereof; (c) a vHH domain or a fragment thereof; (d) a single domain antibody or fragment thereof; (e) a single variable domain of a TCR (svd-TCR) or a fragment thereof; (f) a non-immunoglobulin antigen-binding scaffold or fragment thereof; (g) the ligand-binding domain of a receptor or a fragment thereof; (h) the receptor-binding domain of a ligand or a fragment thereof; (i) an autoantigen or a fragment thereof; (j) an adaptor binding domain or a fragment thereof; (k) adapters or fragments thereof; (l) an epitope or a fragment thereof, and (m) an Fc-binding domain or a fragment thereof;

18. 18. The HC-SAR of claim 17, wherein the non-immunoglobulin antigen-binding scaffold is selected from the group of binding scaffolds consisting of DARPIN, D domain (DD), affibody, affilin, adnectin, affitin, obodies, repebodies, finomers, alphabodies, avimers, atrimers, centilins, pronectins, anticalins, Kunitz domains, and armadillo repeat proteins.

19. 19. The HC-SAR of claim 18, wherein the one or more non-TCR antigen-binding domains and / or AABDs bind to one or more disease-associated antigens selected from the group of disease-associated antigens consisting of CD19, CD5, CD123, CD22, CD30, CD171, CS-1 (CRACC, SLAMF7, CD319, and 19A24), CD45, C-type lectin-like molecule-1 (CLL-1 or CLECL1), 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) or (GalNAcα-Ser / Thr)), 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 on acute leukemia or lymphoma but not on hematopoietic progenitor cells, Non-hematopoietic cancer, 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-llRa), prostate stem cell antigen (PSCA), 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), C D20, folate receptor α, receptor tyrosine protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), carbonic anhydrase IX (CAlX), tyrosinase, fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97;CD179a, anaplastic lymphoma kinase (ALK), mammary differentiation antigen (NY-BR-1), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), melanoma-associated antigen 1 (MAGE-A1), T cell 1 (MelanA or MARTI), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIRl), C-type lectin domain family 12 member A (CLEC12A), EGF-like module-containing mucin-like hormone-like receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), immunoglobulin lambda-like polypeptide 1 (IGLLl), biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, CDH19, CD200R, Slea (CA19.9, Sialyl Lewis Antigen) fucosyl-GM1, PTK7, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1, ALK, TCR-γ-δ, NKG2D, CD32(FCGR2A), CSPG4-HMW-MAA, Tim1- / HVCR1, CSF2RA(GM-CSFR-α), TGFbetaR2, VEGFR2 / KDR, Lewis Ag, TCR-α chain, TCR-β1 chain, TCR-β2 chain, TCR-γ chain, TCR-delta chain, FITC, leukemia 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, guanylate cyclase C (GCC), KSHV-K8.1 protein, KSHV-gH protein, autoantibodies against moglein 3 (Dsg3), autoantibodies against moglein 1 (Dsg1), HLA-A2, HLA-A2:01, HLA-B; HLA-C;HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR; HLA-G, IGE, CD99, Lym1, Lym2, RAS G12V, tissue factor 1 (TF1), AFP, GPRC5D, claudin18.2 (CLD18A2 or CLDN18A.2), STEAP1, STEAP2, LIV1, NECTIN-4, CRIPTO, GPA33, BST1 / CD157, small conductance chloride channel, TAJ / TNFRSF19, MPL (TPO-R), KIR3DL2, CD32b, CD229, Toso, BAFF-R, OR2H1, p95-Her2, huTAG2, immunoglobulin kappa light chain, immunoglobulin gamma light chain, SARS-CoV2 spike glycoprotein, SARS-CoV2 receptor binding domain, CSF1R, mutant p53, p53-R175H mutant, p53-R248Q mutant, NPM1c, PRAME1, melanoma-associated antigen 4 (MAGE-A4), gp100, IL23R, MYCN, and myelin oligodendrocyte glycoprotein (MOG).

20. 18. The HC-SAR according to claim 17, characterized in that the one or more non-TCR antigen-binding domains and / or AABDs are selected from the following group of antigen-binding domains: Light chain variable region (vL): SEQ ID NO: a polypeptide having at least 75% identity in the framework regions of any of the sequences of 339-354, 19766-19776, 32912-33120, and 32006-32068, and encoding a polypeptide that contains the complementarity-determining regions (CDRs) of said polypeptide and binds to an antigen; and the complementary heavy chain variable region (vH): SEQ ID NO: a CDR encoded by a polynucleotide encoding a polypeptide that binds to an antigen and has at least 75% identity in the framework regions of any of 363-378, 19785-19795, 33121-33329, or 32069-32131; Single chain variable fragment (scFv): SEQ ID NO: a CDR encoded by a polynucleotide encoding a polypeptide that binds to an antigen and has at least 75% identity in the framework regions of any of sequences 387-402, 19804-19814, 33330-33538, or 32132-32194; Camelidae VHH domain: SEQ ID NO: a sequence encoded by a polynucleotide having at least 75% identity in the framework regions of any of sequences 412-426, 32195-32213, and encoding a polypeptide that contains the CDR and binds to an antigen; Non-immunoglobulin scaffold: SEQ ID NO: 435-450, or a polynucleotide having at least 75% identity thereto, which encodes a polypeptide that binds to an antigen; Receptor: SEQ ID NO: 437, 438, 445-448, or a polynucleotide having at least 75% identity thereto, which encodes a gametophyte-binding polypeptide; Ligand: SEQ ID NO: 439, or encoded by a polynucleotide having at least 75% identity thereto and encoding a gametophyte-binding polypeptide; Polynucleotides encoding a light chain variable region (vL) comprising one or more light chain complementarity determining regions 1-3 (LC-CDR1-3) for a selected target antigen: SEQ ID NOs: 20989～21015、41591～41861；21024～21050、41862～42132；21059～21085、42133～42403； and a polynucleotide encoding a heavy chain variable region (vH) comprising one or more heavy chain complementarity determining regions 1-3 (HC-CDR1-3) for a selected target antigen: SEQ ID NOs: 21094～21120、42404～42674；21129～21155、42675～42945；21164～21190、42946～43216。

21. 18. The HC-SAR of claim 17, wherein the one or more non-TCR antigen-binding domains and / or AABDs are selected from the group consisting of: a variable light (vL) domain comprising a complementary heavy variable (vH) domain comprising any one of SEQ ID NOs: 8719-8734, 20386-20396, 40759-41029, or a sequence with at least 85% identity in the framework regions, or a sequence with up to 10 conservative amino acid substitutions in the framework regions, and the CDRs of any of the foregoing polypeptides, wherein the non-native TCR antigen binding domain binds to the antigen; and A single domain antibody comprising a sequence set forth in any of SEQ ID NOs: 8792-8806, 41572-41590, 43217-43318, a vHH domain, a SVH, and / or a FHVH domain, a sequence having at least 85% identity in the framework regions, or a sequence having up to 10 conservative amino acid substitutions in the framework regions, comprising the CDRs of any of the aforementioned polypeptides and binding to the antigen; A non-immunoglobulin antigen-binding domain having a sequence set forth in any of SEQ ID NOs: 43364-43375, 43406-43410, a sequence having at least 85% identity thereto, or a sequence having up to 10 conservative amino acid substitutions thereto, which binds to the antigen; an scFv domain comprising light chain complementary determining regions 1-3 (LC-CDR1-3) of a selected target antigen set forth in any of SEQ ID NOs: 20989-21015, 41591-41861; a complementary heavy chain variable region (vH) comprising one or more heavy chain complementary determining regions 1-3 (HC-CDR1-3) of a selected target antigen set forth in any of SEQ ID NOs: 21024-21050, 41862-42132, 21059-21085, 42133-42403, and 21094-21120 and 42404-42674; 21129-21155, 42675-42945, and 21164-21190, 42946-43216; an scFv fragment having a sequence selected from the group consisting of SEQ ID NOS: 8767-8782, 20424-20434, 41301-41571, a sequence having at least 85% identity in the framework regions, or a sequence having up to 10 conservative amino acid substitutions in the framework regions, comprising the CDRs of any of the foregoing polypeptides and binding to its antigen; one or more receptors comprising the amino acid sequence of any of SEQ ID NOs: 43377-43392, a sequence having at least 85% identity, or a sequence having up to 10 conservative amino acid substitutions; One or more ligands comprising any of SEQ ID NOs: 43394-43404, a sequence having at least 85% identity, or a sequence having up to 10 conservative amino acid substitutions; the extracellular domain of CD16A, NKG2D, CD4, PD1, or PD1-like protein 3 (Dsg3); one or more extracellular domains of hTPO, mTPO, CGH α chain, CGH β chain, FH β chain, LH β chain, TSH β chain, APRIL, or any combination thereof; a light chain variable region (vH) comprising one or more light chain complementarity determining regions (LC-CDR1-3) comprising one or more of the light chain complementarity determining regions 1-3 (HC-CDR1-3) of a selected target antigen set forth in any of SEQ ID NOs: 20989-21015, 41591-41861, 21024-21050, 41862-42132, 21059-21085, 42133-42403, and one or more light chain complementarity determining regions (vH) for a selected target antigen set forth in any of SEQ ID NOs: 21094-21120, 42404-42674, 21129-21155, 42675-42945, 21164-21190, 42946-43216; and Any combination of them.

22. HC-SAR according to claim 1, characterized in that both of the TCR constant chains are hybrid chains.

23. HC-SAR according to claim 1, wherein the constant domain of at least one hybrid chain is derived from an immunoglobulin, or both hybrid chains are derived from an immunoglobulin.

24. 2. The HC-SAR of claim 1, wherein the one or more non-TCR antigen-binding domains are selected from the group of antigen-binding domains consisting of: The heavy and light chains of an antibody or fragment thereof are variable regions specific for a given target antigen, such that, upon expression, one of the heavy and light chains of the antibody or fragment thereof is attached to one of the two chains of a T-cell constant chain, and the heavy and light chains of the other antibody or fragment thereof are attached to the other of the two chains of the T-cell constant chain. Upon expression of two single-chain variable fragments (scFvs) specific for one or more predefined target antigens, one of the scFvs is attached to one of the two chains of the T-cell constant chain and the other of the scFvs is attached to the other of the two chains of the T-cell constant chain. two antibody fragments specific for one or more predefined target antigens, wherein, upon expression, one of the antibody fragments is attached to one of the two chains of the T-cell constant chain and the other of the antibody fragments is attached to the other of the two chains of the T-cell constant chain; two single domain antibody (SDAB) fragments specific for one or more predefined target antigens, i.e., two Camelidae vHH domains specific for one or more predefined target antigens, such that, upon expression, one of the SDAB fragments is attached to one of the two chains of the T-cell constant chain and the other SDAB fragment is attached to the other of the two chains of the T-cell constant chain; and, upon expression, one of the vHH domains is attached to one of the two chains of the T-cell constant chain and the other vHH domain binds to the other of the two chains of the T-cell constant chain. two non-immunoglobulin antigen-binding scaffolds specific for one or more predefined target antigens, i.e., when expressed, one of the non-immunoglobulin antigen-binding scaffolds is attached to one of the two chains of the T-cell constant chain and the other of the non-immunoglobulin antigen-binding scaffold domains is attached to the other of the two chains of the T-cell constant chain; two receptors or fragments thereof specific for one or more predefined target antigens, such that, upon expression, one of said receptors or fragments thereof is attached to one of said two chains of said T-cell constant chain and the other of said receptors or fragments thereof is attached to the other of said two chains of said T-cell constant chain; two ligands or fragments thereof specific for one or more predetermined target antigens, such that, upon expression, one of the ligands or fragments thereof is attached to one of the two chains of the T-cell constant chain and the other of the ligands or fragments thereof is attached to the other of the two chains of the T-cell constant chain; two structurally distinct antigen-binding fragments specific for one or more predefined target antigens, whereby upon expression one of the antigen-binding fragments is attached to one of the two chains of the T-cell constant chain and the other of the antigen-binding fragments is attached to the other of the two chains of the T-cell constant chain; two antigen-binding fragments, one or both of which are bispecific or multispecific, and upon expression, one of the antigen-binding fragments is attached to one of the two chains of the T-cell constant chain and the other of the antigen-binding fragments is attached to the other of the two chains of the T-cell constant chain; two autoantigens or fragments thereof, such that, upon expression, one of said autoantigens or fragments thereof is attached to one of said two chains of said T-cell constant chain and the other of said autoantigens or fragments thereof is attached to the other of said two chains of said T-cell constant chain; two vLs or fragments thereof, such that, upon expression, one of the vLs or fragments thereof is attached to one of the two chains of the T-cell constant chain and the other of the vLs or fragments thereof is attached to the other of the two chains of the T-cell constant chain; and Two vHs or fragments thereof, such that, upon expression, one of the vHs or fragments thereof is attached to one of the two chains of the T-cell constant chain and the other of the vHs or fragments thereof is attached to the other of the two chains of the T-cell constant chain.

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