Novel antigen-binding domain and synthetic antigen receptor incorporating the same

Abstract

The present disclosure relates to compositions and methods for producing synthetic antigen receptors or SARs (e.g., SIRs, zSIRs, cTCRs, ab-TCRs, AABD-TCRs, IFFs, TACs, etc.) and antibodies (e.g., bispecific antibodies, DARTs, etc.) comprising one or more novel antigen-binding domains. The SARs described include single-chain immunoreceptors (e.g., first-, second-, and third-generation chimeric antigen receptors, TFPs, Tri-TACs, etc.) and multi-chain immunoreceptors (e.g., SIRs, zSIRs, cTCRs, ab-TCRs, AABD-TCRs, αβTFPs, yclTFPs, recombinant TCRs, etc.). SARs can utilize antigen-binding domain characteristics to redirect immune cell specificity and reactivity to one or more selected targets. [Reference diagram] Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 35 of U.S. Provisional Application No. 62 / 990,396, filed March 16, 2020, the disclosure of which is incorporated herein by reference. [Technical Field]

[0002] The present disclosure relates to compositions and methods for producing synthetic antigen receptors or SARs (e.g., SIRs, zSIRs, cTCRs, ab-TCRs, AABD-TCRs, TFPs, TACs, etc.) and antibodies (e.g., bispecific antibodies, DARTs, etc.) comprising one or more novel antigen-binding domains. The SARs described include single-chain immunoreceptors (e.g., first-, second-, and third-generation chimeric antigen receptors, TFPs, Tri-TACs, etc.) and multi-chain immunoreceptors (e.g., SIRs, zSIRs, cTCRs, ab-TCRs, AABD-TCRs, αβTFPs, γdTFPs, recombinant TCRs, etc.). SARs can utilize antigen-binding domain characteristics to redirect immune cell specificity and reactivity to one or more selected targets. When expressed in immune cells, SARs as described herein confer the ability to recognize target antigens in an MHC-dependent or MHC-independent manner. The present disclosure describes useful configurations of SARs having two or more antigen-binding domains. This disclosure describes antigen-binding domains useful for constructing monospecific, bispecific, and / or multispecific SARs, useful configurations of such antigen-binding domains, and vector constructs. This disclosure also describes a novel method for selecting optimal antigen-binding domains for incorporation into SARs. This disclosure further relates to improving the quality of SAR-expressing T cells by expanding them in the presence of SMAC mimetics and / or NIK agonists. This disclosure also describes methods for producing SARs using devices or containers containing gas-permeable membranes under normoxic and hypoxic conditions. An exemplary device with a gas-permeable membrane is the GRex flask. This disclosure also describes novel tags that can be added to SAR constructs and used to identify, isolate, and eliminate SAR-expressing cells. This disclosure also provides the use of thermostable luciferases (e.g., firefly (Photinus pyralis) (lucPpy) and Photuris pennsylvanica (lucPpe) luciferases) for in vivo bioluminescence imaging applications. The present disclosure also provides novel antigen-binding domains and SARs incorporating same.

[0003] Incorporation by Reference of Sequence Listing This application is accompanied by a Sequence Listing entitled "Sequence-Listing_ST25.txt", formatted for IBM-PC, MS-Windows operating systems, created on March 16, 2021, and containing 101,792,917 bytes of data. [Background technology]

[0004] CARs are synthetic immune receptors that redirect T cells to selectively kill tumor cells. Unlike physiological T cell receptors (TCRs), which engage HLA-peptide complexes, CARs engage molecules that do not require peptide processing or HLA expression for recognition. Initial first-generation CARs were constructed by fusing an scFv (single-chain fragment variable)-based antigen-binding domain to the inactive CD8 transmembrane domain, linked to a cytoplasmic signaling domain derived from the CD3-ζ or Fc receptor γ chain. To overcome the lack of T cell costimulation, first-generation CARs were further modified by incorporating the cytoplasmic signaling domain of a T cell costimulatory receptor.

[0005] Despite the success of CAR-T cells, this approach has several limitations, including toxicity, such as cytokine release syndrome (CRS) and neurotoxicity. The inclusion of a costimulatory domain in a CAR construct can result in nonphysiological tonic signaling via the receptor, which may contribute to toxicity and lack of persistence. CAR-Ts with scFvs as antigen-binding domains can also form membrane clusters and generate tonic signaling by crosslinking the heavy and light chains of different scFvs. Furthermore, the scFvs used in CAR-Ts are usually derived from mouse mAbs, which raises potential immunogenicity concerns. Without wishing to be bound by theory, these issues are likely to be compounded when using scFvs to design CAR-Ts with bispecific, multispecific, bivalent, or biparatopic antigen-binding moieties.

[0006] To overcome some of the design limitations of conventional second-generation CARs, several alternative designs, collectively referred to as next-generation CARs, have been described, including Ab-TCRs (WO2017 / 070608A1, incorporated herein by reference), TCR receptor fusion proteins or TFPs (WO2016 / 187349A1, incorporated herein by reference), synthetic immune receptors (SIRs) (see WO2018 / 102795A1, incorporated herein by reference), trifunctional T cell antigen couplers (Tri-TACs) (WO2015 / 117229A1, incorporated herein by reference), and zSIRs (see WO2019232503, incorporated herein by reference). The AABD-TCR platform is described in the present disclosure. Generally, these alternative CAR designs lack a costimulatory domain. These alternative CAR designs can include a single chain (e.g., single-chain SIR, or εTFP, γTFP, and δTFP) or two or more chains (e.g., dual-chain SIR, cTCR, zSIR, αβTFP, and γδTFP). However, the problem of nonspecific aggregation of scFv chains and the resulting tonic signaling also impacts the design of next-generation CARs comprising scFv chains for antigen binding. This problem is further compounded when scFv fragments are used to design multi-chain synthetic antigen receptors, such as SIRs, cTCRs, and Ab-TCRs, with bispecific, bivalent, biparatopic, multispecific, multivalent, or multiparatopic antigen-binding moieties.

[0007] The present disclosure provides a solution to the above problem through the design of synthetic antigen receptors (SARs). The term synthetic antigen receptor (SAR) described herein includes receptors having the backbone of single-chain immunoreceptors (e.g., first-, second-, and third-generation chimeric antigen receptors, TFP, Tri-TAC, etc.) and multi-chain immunoreceptors (e.g., SIR, zSIR, cTCR, ab-TCR, AABD-TCR, αβTFP, γdTFP, recombinant TCR, and HLA-independent TCR, etc.). Summary of the Invention

[0008] The present disclosure describes compositions and configurations for producing synthetic antigen receptors (SARs). In one embodiment, the SAR comprises a polypeptide comprising two or more antigen-binding domains. In another embodiment, the SAR comprises a polypeptide that binds to two or more antigens. In yet another embodiment, the SAR comprises a polypeptide that binds to two or more epitopes of one or more antigens.

[0009] In some embodiments, the present disclosure provides a SAR polynucleotide encoding a polypeptide comprising an autonomous antigen-binding domain (AABD) or a fragment thereof connected in-frame to vL, vH, Va, Vb, Vg, Vd, Ig domain, Ig-like domain, or a combination thereof via one or more optional linkers. In some embodiments, the AABD is any domain that can bind to an antigen in an autonomous manner, i.e., in the absence of another domain. In some embodiments, the AABD is a non-scFv domain. In some embodiments, the AABD does not include the vL and vH fragments that form an Fv domain. In certain exemplary embodiments, the AABD is a vHH, a single variable domain antibody, FHVH (fully human vH domain), SVH (single vH domain), SVL (single vL domain), a non-immunoglobulin antigen binding scaffold (e.g., centilin, affibody, DARPIN, D domain, etc.), a ligand binding domain of a receptor, a receptor binding domain of a ligand, an autoantigen, an adaptor binding domain, an epitope, a mimotope, a single variable domain of a T cell receptor (svd-TCR) or a fragment thereof.

[0010] In one embodiment, the present disclosure provides a SAR polynucleotide encoding a polypeptide comprising at least one autonomous antigen-binding domain, or a fragment thereof, connected in-frame to a vL, vH, Va, Vb, Vg, Vd, Ig domain, Ig-like domain, or combinations thereof via one or more optional linkers. In one embodiment, the SAR polynucleotide encodes a polypeptide comprising two or more autonomous antigen-binding domains linked in-frame via optional linkers.

[0011] In some embodiments, the SAR polynucleotide encodes at least one polypeptide comprising one or more autonomous antigen-binding domains or fragments thereof connected in frame to a first module comprising a vL, vH, Va, Vb, Vg, Vd, Ig domain, or Ig-like domain, or a combination thereof, via one or more optional domains, wherein the first module is connected to a second module comprising a transmembrane domain via an optional linker. In some embodiments, the optional domain is a linker domain. In some embodiments, the second module comprises a binding peptide, a transmembrane domain, and an intracellular domain. In some embodiments, the second module comprises a TCR constant chain or a fragment thereof. In some embodiments, the second module comprises a TCR alpha, TCR beta 1, TCR beta 2, TCR gamma, TCR delta, or pre-TCR alpha constant chain or a fragment thereof.

[0012] In some embodiments, the SAR polynucleotide encodes a polypeptide comprising an autonomous antigen-binding domain connected in-frame to a polypeptide comprising a transmembrane domain via an optional domain. In some embodiments, the optional domain is a connecting peptide. In some embodiments, the transmembrane domain belongs to a T cell receptor constant chain (e.g., the constant chain of TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, pretc. R-α, etc.). In some embodiments, the connecting peptide belongs to a T cell receptor constant chain (e.g., the connecting peptide of TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, pretc. R-α, etc.).

[0013] In one embodiment, the SAR polynucleotide encodes a polypeptide comprising an autonomous antigen-binding domain connected in frame to a polypeptide comprising a connecting peptide and a transmembrane domain. In one embodiment, the transmembrane domain belongs to a T cell receptor constant chain (e.g., the constant chain of TCR alpha, TCR beta 1, TCR beta 2, TCR gamma, TCR delta, pretc. R-alpha, etc.). In one embodiment, the connecting peptide belongs to a T cell receptor constant chain (e.g., the connecting peptide of TCR alpha, TCR beta 1, TCR beta 2, TCR gamma, TCR delta, pretc. R-alpha, etc.).

[0014] In one embodiment, the SAR polynucleotide encodes a polypeptide comprising an autonomous antigen-binding domain or fragment thereof connected in-frame to or near the N-terminus of a vL, vH, Va, Vb, Vg, Vd, Ig domain, Ig-like domain, or combinations thereof via one or more optional domains. In one embodiment, the optional domain is a linker domain.

[0015] In some embodiments, the SAR polynucleotide encodes a polypeptide comprising one or more autonomous antigen-binding domains or fragments thereof connected in-frame via one or more optional domains to or near the N-terminus of a module comprising a vL, vH, Va, Vb, Vg, Vd, Ig domain, Ig-like domain, or a combination thereof. In some embodiments, the optional domain is a linker domain.

[0016] In some embodiments, the SAR polynucleotide encodes at least one polypeptide comprising one or more autonomous antigen-binding domains or fragments thereof connected in-frame via one or more optional domains to the N-terminus or near the N-terminus of a first module comprising a vL, vH, Va, Vb, Vg, Vd, Ig domain, or Ig-like domain, or a combination thereof, wherein the first module is connected to a second module comprising a transmembrane domain via an optional linker. In some embodiments, the optional domain is a linker domain. In some embodiments, the second module comprises a binding peptide, a transmembrane domain, and an intracellular domain. In some embodiments, the second module comprises a TCR constant chain or fragment thereof. In some embodiments, the second module comprises a TCR alpha, TCR beta 1, TCR beta 2, TCR gamma, TCR delta, or pre-TCR alpha constant chain or fragment thereof.

[0017] In one embodiment, the SAR polynucleotide encodes two polypeptide chains, each chain comprising zero, one, two, or more autonomous antigen-binding domains or fragments thereof connected in-frame via one or more optional domains to at or near the N-terminus of a first module comprising a vL, vH, Va, Vb, Vg, Vd, Ig domain, or Ig-like domain, or a combination thereof, wherein the first module is connected via an optional linker to a second module comprising a binding peptide, a transmembrane domain, and an optional intracellular domain, and wherein the first and second polypeptide chains form a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling module when expressed in a T cell.

[0018] In certain embodiments, the SAR polynucleotide encodes a polypeptide comprising an autonomous antigen-binding domain joined in-frame to a polypeptide comprising a hinge domain and a transmembrane domain.

[0019] In some embodiments, the SAR polynucleotide encodes a polypeptide comprising an autonomous antigen-binding domain or a fragment thereof connected in frame to a polypeptide comprising a hinge domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain comprises a primary activation domain. In some embodiments, the intracellular signaling domain comprises a primary activation domain and one or more costimulatory domains. In some embodiments, the intracellular signaling domain comprises one or more costimulatory domains. In some embodiments, the intracellular signaling domain lacks an activation domain. In some embodiments, the intracellular signaling domain comprises an activation domain containing one or more ITAM motifs.

[0020] The present disclosure also provides polynucleotides encoding any of the SARs of the present disclosure together with accessory modules. Nucleic acid sequences of exemplary polynucleotides encoding SARs are provided in Tables 25-36 and 41-50. In some embodiments, the SAR components and any accessory modules (e.g., therapeutic controls) are encoded by a single polynucleotide molecule. In some embodiments, two or more SAR components (e.g., the two strands of a duplex SAR) and accessory modules are encoded by separate polynucleotides.

[0021] In various embodiments, the polynucleotide molecules encoding the SARs described herein comprise one or more antigen-specific coding domains that encode one or more antigen-specific domains. In some embodiments, the antigen-specific coding domains of the SARs encode one or more V L In some embodiments, the antigen-specific coding domain of the SAR comprises one or more V (or vL) fragments. HIn some embodiments, the antigen-specific coding domain encodes one or more scFVs (or multiple scFvs) specific for an antigen on a target cell, e.g., a cancer cell. In some embodiments, the antigen-specific coding domain encodes one or more Fv fragments. In some embodiments, the antigen-specific coding domain encodes one or more Fab fragments. In some embodiments, the antigen-specific coding domain of the SAR comprises one or more (Fab')2 fragments. In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes an autonomous antigen-binding domain (AABD). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more single domain antibodies (SDABs) or antibody fragments, such as, for example, a single vH domain (SVH) or a single vL domain (SVL). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes a single vH domain (i.e., FHVH) that is fully human in origin. In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more camelid VH domains. HHIn some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes a humanized vHH domain. In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes a variable domain from a T cell receptor (e.g., Va / Vα, Vb / Vβ, Vg / Vγ, and Vd / Vδ). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes a single-chain TCR (scTCR). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes a single variable domain TCR (svd-TCR). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more ligand-binding domains of a receptor. In some embodiments, the antigen-specific coding domain encodes one or more non-immunoglobulin scaffolds, such as, for example, DARPINs, affibodies, affilins, adnectins, affitins, obodies, repebodies, finomers, alphabodies, avimers, atrimers, sentinels, pronectins, anticalins, Kunitz domains, armadillo repeat proteins, or D domains (α3D domains). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more adaptor binding domains (e.g., RZIP, EZIP, E4, K4, NKG2D-AF, NKG2D-YA, etc.). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more Fc-binding domains (e.g., Fc-binding regions of CD16, CD32, or CD64, etc.). In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more autoantigens. In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more epitope tags or mimotopes. In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more ligand-binding domains of a receptor.In some embodiments, the antigen-specific coding domain of the SAR polynucleotide encodes one or more receptor-binding domains of a ligand. In some embodiments, the antigen-specific coding domain of the SAR comprises at least one Fv (e.g., vL and vH fragments) and one or more AABDs (e.g., SVH, SVL, vHH, svd-TCR, DARPIN, affibody, centilin, D domain, epitope, mimotope, EZIP, RZIP, E4, K4, etc.). In some embodiments, the antigen-specific coding domain of the SAR comprises at least one TCR variable domain (e.g., Va / Vb or Vg / Vd fragment of TCR) and one or more AABDs (e.g., SVH, SVL, FHVH, vHH, svd-TCR, DARPIN, affibody, centilin, D domain, epitope, mimotope, EZIP, RZIP, E4, K4, etc.). In some embodiments, the antigen-specific coding domain of a SAR comprises one or more (e.g., 2, 3, 4, 5, 6, or more) AABDs (e.g., SVH, SVL, vHH, FHVH, svd-TCR, DARPIN, affibody, centilin, D domain, epitope, mimotope, EZIP, RZIP, E4, K4, etc.). In some embodiments, a SAR comprises AABDs of the same type (e.g., two vHHs, three DARPINs, two centilins, etc.). In some embodiments, a SAR polynucleotide encodes AABDs of different types (e.g., one vHH and one DARPIN; one DARPIN, one vHH domain, and one centilin, etc.). In some embodiments, a SAR polynucleotide encodes an AABD of fully human, humanized, chimeric, or non-human origin. In some embodiments, a SAR polynucleotide encodes one or more peptide linkers. In some embodiments, the SAR polynucleotide encodes one or more flexible linkers (e.g., Gly-Ser linkers). In some embodiments, the SAR polynucleotide encodes one or more protease-cleavable linkers (e.g., a linker that is cleaved by a cellular protease, e.g., MMP14, e.g., a linker encoded by SEQ ID NO: 1218).In some embodiments, the SAR polynucleotide encodes one or more Ig linkers or Ig-like linkers. In some embodiments, the SAR polynucleotide encodes one or more SAR chains. In some embodiments, two or more SAR chains are encoded by one or more polynucleotides. In some embodiments, two or more SAR chains are encoded by polynucleotides separated by a cleavable linker (e.g., P2A, T2A, F2A, etc.), optionally preceded by a nucleic acid sequence encoding a furin cleavage site. In some embodiments, the SAR polynucleotide is partially or fully codon-optimized.

[0022] In certain embodiments, the present disclosure provides a SAR polypeptide encoded by any of the SAR polynucleotides described in the preceding section.

[0023] In some embodiments, provided herein are polypeptides comprising any of the SARs of the present disclosure, together with an optional accessory module. The amino acid sequences of exemplary SAR polypeptides are set forth in Tables 25-36 and 41-50. In some embodiments, two or more SAR components and an optional accessory module (e.g., a therapeutic control) comprise a single polypeptide molecule. In some embodiments, two or more SAR components (e.g., the two chains of a double-chain SAR) and an accessory module comprise separate polypeptides. In various embodiments, a SAR polypeptide comprises one or more antigen-specific domains. In some embodiments, the antigen-specific domain of a SAR comprises one or more V L In some embodiments, the antigen-specific domain of the SAR comprises one or more V (or vL) fragments. H(or vH) fragment. In some embodiments, the antigen-specific domain comprises one or more scFVs (or multiple scFvs) specific for an antigen on a target cell, e.g., a cancer cell. In some embodiments, the antigen-specific domain comprises one or more Fv fragments. In some embodiments, the antigen-specific domain comprises one or more Fab fragments. In some embodiments, the antigen-specific domain of the SAR comprises one or more (Fab')2 fragments. In some embodiments, the antigen-specific domain of the SAR polypeptide comprises an autonomous antigen-binding domain (AABD). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more single domain antibodies (SDABs) or antibody fragments, such as a single vH domain (SVH) or a single vL domain (SVL). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises a single vH domain of fully human origin (i.e., FHVH). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more camelid VH fragments. HHIn some embodiments, the antigen-specific domain of the SAR polypeptide comprises a humanized vHH domain. In some embodiments, the antigen-specific domain of the SAR polypeptide comprises a variable domain from a T cell receptor (e.g., Va / Vα, Vb / Vβ, Vg / Vγ, and Vd / Vδ). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises a single-chain TCR (scTCR). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises a single variable domain TCR (svd-TCR). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more ligand-binding domains of a receptor. In some embodiments, the antigen-specific domain comprises one or more non-immunoglobulin scaffolds, such as DARPIN, affibody, affilin, adnectin, affitin, obody, lipibody, finomer, alphabody, avimer, atrimer, centilin, pronectin, anticalin, Kunitz domain, armadillo repeat protein, or D domain (α3D domain). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more adaptor binding domains (e.g., RZIP, EZIP, E4, K4, NKG2D-AF, NKG2D-YA, etc.). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more Fc-binding domains (e.g., Fc-binding regions of CD16, CD32, or CD64, etc.). In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more autoantigens. In some embodiments, the antigen-specific domain of the SAR polypeptide comprises one or more epitope tags or mimotopes. In some embodiments, the antigen-specific domain of a SAR polypeptide comprises one or more ligand-binding domains of a receptor. In some embodiments, the antigen-specific domain of a SAR polypeptide comprises one or more receptor-binding domains of a ligand.In some embodiments, the antigen-specific domain of the SAR comprises at least one Fv (e.g., vL and vH fragments) and one or more AABDs (e.g., SVH, SVL, vHH, svd-TCR, DARPIN, affibody, centilin, D domain, epitope, mimotope, EZIP, RZIP, E4, K4, etc.). In some embodiments, the antigen-specific domain of the SAR comprises at least one TCR variable domain (e.g., Va / Vb or Vg / Vd fragment of TCR) and one or more AABDs (e.g., SVH, SVL, FHVH, vHH, svd-TCR, DARPIN, affibody, centilin, D domain, epitope, mimotope, EZIP, RZIP, E4, K4, etc.). In some embodiments, the antigen-specific domain of a SAR comprises one or more (e.g., 2, 3, 4, 5, 6, or more) AABDs (e.g., SVH, SVL, vHH, FHVH, svd-TCR, DARPIN, affibody, centilin, D domain, epitope, mimotope, EZIP, RZIP, E4, K4, etc.). In some embodiments, a SAR comprises AABDs of the same type (e.g., two vHH, three DARPIN, two centilin, etc.). In some embodiments, a SAR comprises AABDs of different types (e.g., one vHH and one DARPIN; one DARPIN, one vHH domain, and one centilin, etc.). In some embodiments, a SAR polypeptide comprises an AABD of fully human, humanized, chimeric, or non-human origin. In some embodiments, a SAR polypeptide comprises one or more linkers. In some embodiments, the SAR polypeptide comprises one or more flexible linkers (e.g., a Gly-Ser linker). In some embodiments, the SAR polypeptide comprises one or more protease-cleavable linkers (e.g., a linker that is cleaved by a cellular protease, e.g., MMP14, e.g., a linker encoded by SEQ ID NO: 1218). In some embodiments, the SAR polypeptide comprises one or more Ig linkers or Ig-like linkers. In some embodiments, the SAR comprises one or more chains.In some embodiments, the two or more chains of the SAR comprise one or more polypeptides, hi some embodiments, the two or more chains of the SAR comprise polypeptides separated by a cleavable linker (e.g., P2A, T2A, F2A, etc.), optionally preceded by a nucleic acid sequence encoding a furin cleavage site.

[0024] The present disclosure provides SAR polynucleotides and polypeptides having exemplary SAR compositions shown in Tables 25-36 and 41-50. Because SARs are modular in design, one of skill in the art can generate additional SARs with novel compositions by substituting different modules and testing them using the assays provided in the present disclosure. Exemplary component modules of SARs are shown in Tables 2-24.

[0025] The present disclosure also provides constructs useful for making the SARs of the present disclosure. The present disclosure provides SAR polynucleotides and polypeptides having the modular domain structures and constructs of exemplary SARs shown in Tables 25-36 and 41-50. In certain embodiments, the present disclosure provides at least one recombinant polynucleotide encoding at least one synthetic antigen receptor (SAR), wherein the at least one SAR comprises: a) a first polypeptide chain comprising a vH, Va, Vg, or Ig linker domain fragment operably linked, via an optional linker, to a first T cell receptor constant chain fragment comprising a first transmembrane domain of a first TCR subunit; and b) a second polypeptide chain comprising a vL, Vb, Vd, or Ig linker domain operably linked, via an optional linker, to a second T cell receptor constant chain fragment comprising a second transmembrane domain of a second TCR subunit; wherein the one or more polypeptide chains comprise a single vH domain operably linked, via an optional linker, to or near the N-terminus of the vH, vL, Va, Vb, Vg, Vd, or Ig linker domain. a single domain antibody; a svd-TCR, a non-immunoglobulin antigen-binding scaffold such as a DARPIN, an affibody, an affilis, an adnectin, an afftin, an obody, a lipibody, a finomer, an alphabody, an avimer, an atrimer, a centrinin, a pronectin, an anticalin, a Kunitz domain, an armadillo repeat protein, and a D domain; a ligand-binding domain of a receptor; a receptor-binding domain of a ligand; an autoantigen; one or more autonomous antigen-binding domains (AABDs) selected from the group consisting of an adaptor-binding domain, an Fc-binding domain, an epitope tag, a mimotope, or an equivalent; and the first TCR constant chain fragment and the second TCR constant chain fragment form a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling module when expressed in a T cell.

[0026] In certain embodiments, the present disclosure demonstrates that a configuration useful for generating bispecific or multispecific CARs comprises the attachment of one or more AABDs (e.g., non-scFV-based antigen binding domains) to or near the N-terminus of the vL and / or vH domains comprising the scFvs of such CARs via optional linkers.

[0027] In certain embodiments, the present disclosure provides constructs useful for SARs (e.g., bispecific or multispecific SARs) comprising one or more AABDs operably linked, via an optional linker, to the N-terminus or near the N-terminus of a single-chain CAR (e.g., second-generation CAR, third-generation CAR), single-chain TFP (e.g., TFPε, TFPγ, TFPδ), SIR, cTCR, Ab-TCR, αβTFP, γδTFP, or vL, vH, scFv, vHH, FHVH, Va, Vb, Vd, Vg fragment comprising a TCR. In another embodiment, the present disclosure provides constructs useful for making SARs (e.g., bispecific or multispecific SARs), wherein the AABD-TCR scaffold comprises one or more AABDs operably linked to a TCR signaling module via an intervening Ig linker domain.

[0028] The present disclosure describes constructs useful for generating bispecific or multispecific SIRs, Ab-TCRs, TFPαβ, TFPγδ or TCRs that comprise one or more AABDs (e.g., non-scFV-based antigen-binding domains) at or near the N-terminus of the vL and / or vH domains comprising the Fv of such SIRs, optionally via a linker.

[0029] The present disclosure further provides one or more vectors comprising a nucleic acid encoding any of the SAR polypeptides and accessory modules described in the preceding section. The SARs of the present disclosure can be encoded by a single vector or by two or more vectors.

[0030] The present disclosure further provides genetically modified cells comprising vectors comprising polynucleotides encoding the SARs and accessory modules of the present disclosure.

[0031] The present disclosure further provides therapeutic methods using genetically modified cells comprising vectors comprising polynucleotides encoding the SARs and accessory modules of the present disclosure.

[0032] The present disclosure also provides novel antibodies, antibody fragments, vHHs, and single human vH domains (e.g., FHVHs and chVHs) capable of binding to different antigens. The target antigens, names, and SEQ ID NOs of these novel antigen-binding domains, along with the SEQ ID NOs of their CDR1-CDR3, are listed in Table 39. These novel antigen-binding domains can be used to construct SARs and / or SAR adaptors of the present disclosure. Furthermore, these novel antigen-binding domains can be used to generate other therapeutic and diagnostic biologics and cell-based therapies, including antibodies (e.g., bispecific and trispecific antibodies, antibody-drug conjugates, radiolabeled antibodies, fluorochrome-labeled antibodies, scFvs, bispecific and trispecific T / NK cell engagers, etc.), using methods described herein and known in the art. The present disclosure also provides polynucleotides, polypeptides, vectors, pharmaceutical compositions, cells, and kits (ktis) comprising and / or expressing the novel antigen-binding domains of the present disclosure.

[0033] The present disclosure further relates to SARs, polynucleotides encoding SAR polypeptides, vectors comprising polynucleotides encoding SAR polypeptides, and isolated cells expressing the SARs of the present disclosure. The SARs of the present disclosure can be expressed in immune cells (e.g., T cells, NK cells, etc.) or stem cells capable of giving rise to immune cells (e.g., hematopoietic stem cells or induced pluripotent stem cells (iPSCs)). The SARs can be expressed in autologous or allogeneic stem cells. Cells expressing the SARs may have reduced or eliminated expression of one or more components of the TCR / CD3 signaling complex or their downstream signaling mediators. Cells expressing the SARs may have reduced or eliminated expression of HLA molecules, such as through downregulation or knockout of β2-macroglobulin. Cells for expressing the SARs of the present disclosure can be obtained from autologous or allogeneic donors. Cells for expressing the SARs of the present disclosure can be obtained from autologous or allogeneic donors administered with a mobilizing agent (e.g., a CXCR4 antagonist, G-CSF, GM-CSF, etc.). The cells of the present disclosure may express one SAR or two or more SARs. In an exemplary embodiment, one of these SARs may produce an immune cell effector function (e.g., cytotoxicity), while the other SAR may provide costimulation. SAR-expressing cells may target a single antigen or two or more antigens. SAR-expressing cells may target the same epitope or different epitopes of a single antigen. In one aspect, SAR-expressing cells recognize a single antigen and may be preferentially or exclusively expressed in hematopoietic cells. Exemplary antigens preferentially or exclusively expressed in hematopoietic cells include CD19, CD20, CD22, BCMA, CS1, CD30, CD33, MPL, CD138, CD38, CD79b, BAFF-R, CD123, etc. In one aspect, SAR-expressing cells recognize a single antigen and may be preferentially or exclusively expressed in non-hematopoietic cells. Exemplary antigens that are preferentially or exclusively expressed on non-hematopoietic cells include mesothelin (MSLN), Her2, EGFR, EGFRviii, Muc16, PSMA, IL13Ra2, and the like.In one aspect, SAR-expressing cells recognize two or more novel antigens, at least one of which is preferentially or exclusively expressed in hematopoietic cells, and at least one of which is expressed in non-hematopoietic cells.The SAR-expressing cells of the present disclosure can be used for therapy, particularly for the treatment of cancer.Methods for treating diseases, such as cancer, are also within the scope of the present invention.

[0034] In one embodiment, the present invention provides isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets BCMA. In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting BCMA are set forth in SEQ ID NOs: 7409-8374. In an exemplary embodiment, the sequences of the isolated polypeptides targeting BCMA are set forth in SEQ ID NOs: 18099-19064 (Table 36). In some embodiments, the vL, vH, and scFv fragments targeting BCMA are set forth in Table 3 and set forth in SEQ ID NOs: 235-248, 477-490, and 719-732, respectively. The amino acid sequence numbers of the vL, vH, and scFv fragments targeting BCMA are also set forth in Table 3 and set forth in SEQ ID NOs: 10925-10938, 10978-11180, and 11220-11422, respectively. In some embodiments, SVH and vHH fragments that target BCMA are listed in Table 4 and set forth in SEQ ID NOs: 852-858, 888-891, 893, 895, and 901-902. In some embodiments, centilin that targets BCMA is listed in Table 7 and set forth in SEQ ID NO: 983. Exemplary monospecific, bispecific, and multispecific SARs incorporating an AABD comprising an SVH, vHH, and centilin are listed in Tables 25-36. Further provided herein are vectors encoding nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets BCMA. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets BCMA.

[0035] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets PSMA (prostate-specific membrane antigen). In an exemplary embodiment, the sequence of the isolated nucleic acid fragment targeting PSMA is set forth in SEQ ID NOs: 9686-10030. In an exemplary embodiment, the sequence of the isolated polypeptide targeting PSMA is set forth in SEQ ID NOs: 20376-20720 (Table 36). In some embodiments, vL, vH, and scFv fragments targeting PSMA are set forth in Table 3 and set forth in SEQ ID NOs: 268-272, 510-514, and 752-756, respectively. The amino acid sequence numbers of vL, vH, and scFv fragments targeting PSMA are also set forth in Table 3 and set forth in SEQ ID NOs: 10958-10962, 11200-11204, and 11442-11446, respectively. In some embodiments, SVH fragments that target PSMA are listed in Table 4 and set forth in SEQ ID NOS: 830-833. In some embodiments, centilin that targets PSMA is listed in Table 7 and set forth in SEQ ID NOS: 977-979. Exemplary monospecific, bispecific, and multispecific SARs incorporating an AABD comprising an SVH, a vHH, and centilin are listed in Tables 25-36. Further provided herein are vectors comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets PSMA. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets PSMA. Also provided are PSMA-targeting SARs whose expression and affinity are optimized to target cancer cells that express high levels of PSMA (e.g., more than 1.5-fold higher than normal cells) and spare normal healthy cells that express low to moderate levels of PSMA. In an exemplary embodiment, a SAR is provided that responds to cancer cells that express PSMA at levels at least 1.5 times the levels found in normal healthy prostate epithelium or immortalized cell lines derived from normal prostate epithelial cells.The expression level of PSMA can be measured by techniques known in the art, including, but not limited to, immunohistochemistry, Western blotting, Northern blotting, and quantitative PCR. In one embodiment, the present disclosure provides a novel SAR that targets cells expressing high levels of PSMA but not cells expressing low levels of PSMA. The reference cells expressing high levels of PSMA are the LNCaP cell line, and the cells expressing low levels of PSMA are the PC3 and / or Huh-7 cell lines. In one embodiment, the present disclosure provides a SAR expressed in JNG cells that, when co-cultured with LNCaP cells, exhibits GFP induction that is two-fold or more (e.g., three-fold, four-fold, five-fold, ten-fold, etc.) greater than the GFP induction observed when co-cultured with PC3 or Huh-7 cell lines in a Jurkat NFAT-GFP assay. In certain embodiments, the present disclosure provides a SAR expressed in JNG cells that, when co-cultured with LNCaP cells, exhibits GFP induction in a Jurkat NFAT-GFP assay that is greater than 10% (e.g., 20%, 30%, 40%, or 50%) of the GFP induction seen when co-cultured with PC3 or Huh-7 cell lines.

[0036] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets mesothelin (MSLN). In an exemplary embodiment, the sequence of the isolated nucleic acid fragment targeting MSLN is set forth in SEQ ID NOs: 10307-10720. In an exemplary embodiment, the sequence of the isolated polypeptide targeting MSLN is set forth in SEQ ID NOs: 20997-21410 (Table 36). In some embodiments, the vL, vH, and scFv fragments targeting MSLN are set forth in Table 3 and set forth in SEQ ID NOs: 277-282, 519-524, and 761-766, respectively. The amino acid sequence numbers of the vL, vH, and scFv fragments targeting MSLN are also set forth in Table 3 and set forth in SEQ ID NOs: 10967-10972, 11209-11214, and 11451-11456, respectively. In some embodiments, vHH fragments targeting MSLN are listed in Table 4 and set forth in SEQ ID NOs: 876-877. Exemplary monospecific, bispecific, and multispecific SARs incorporating vLs, vHs, scFvs, and vHHs targeting MSLN are listed in Tables 25-36. Further provided herein are vectors comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets MSLN. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets MSLN. Also provided are SARs targeting MSLN, whose expression and affinity are optimized to target cancer cells expressing high levels of MSLN (e.g., more than 1.5-fold higher than normal cells) and spare normal healthy cells expressing low to moderate levels of MSLN. In one exemplary embodiment, the present invention provides a SAR that responds to cancer cells that express MSLN at a level at least 1.5 times the level of MSLN found in normal healthy peritoneal epithelial cells or immortalized cell lines derived from normal healthy peritoneal epithelial cells. The expression level of MSLN can be measured by techniques known in the art, including but not limited to immunohistochemistry, Western blotting, Northern blotting, and quantitative PCR.In one embodiment, the present disclosure provides a novel SAR that targets cells expressing high levels of MSLN but not cells expressing low levels of MSLN. The reference cells expressing high levels of MSLN are SKOV-3 cell lines, and the cells expressing low levels of MSLN are MCF-7 or LNCaP cell lines. In one embodiment, the present disclosure provides a SAR expressed in JNG cells, which, when co-cultured with SKOV-3 cells, shows GFP induction that is two-fold or more (e.g., three-fold, four-fold, five-fold, ten-fold, etc.) greater than the GFP induction observed when co-cultured with MCF-7 or LNCaP cell lines in a Jurkat NFAT-GFP assay. In certain embodiments, the present disclosure provides a SAR expressed in JNG cells that, when co-cultured with SKOV-3 cells, exhibits GFP induction in a Jurkat NFAT-GFP assay that is greater than 10% (e.g., 20%, 30%, 40%, or 50%) of the GFP induction when co-cultured with MCF-7 or LNCaP cell lines.

[0037] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets Her2. In an exemplary embodiment, the sequence of the isolated nucleic acid fragment targeting Her2 is set forth in SEQ ID NOs: 8858-8995. In an exemplary embodiment, the sequence of the isolated polypeptide targeting HER2 is set forth in SEQ ID NOs: 19548-19616 (Table 36). In some embodiments, the vL, vH, and scFv fragments targeting HER2 are set forth in Table 3 and set forth in SEQ ID NOs: 256-257, 498-499, and 740-741, respectively. The amino acid sequence numbers of the vL, vH, and scFv fragments targeting HER2 are also set forth in Table 3 and set forth in SEQ ID NOs: 10946-10947, 11188-11189, and 11430-11431, respectively. In some embodiments, vHH fragments targeting HER2 are listed in Table 4 and set forth in SEQ ID NOS: 864-866. In some embodiments, DARPINS fragments targeting HER2 are listed in Table 4 and set forth in SEQ ID NOS: 972-973. Exemplary monospecific, bispecific, and multispecific SARs incorporating vL, vH, scFv, vHH, and DARPINS targeting HER2 are listed in Tables 25-36. Further provided herein are vectors comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets HER2. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets HER2. Also provided are SARs targeting Her2, whose expression and affinity are optimized to target cancer cells expressing high levels of Her2 (e.g., more than 1.5-fold higher than normal cells) and spare normal healthy cells expressing low to moderate levels of Her2. In an exemplary embodiment, a SAR is provided that responds to cancer cells that express Her2 at levels at least 1.5 times the Her2 levels found in normal healthy breast epithelial cells or immortalized cell lines derived from normal healthy breast epithelium.The expression level of Her2 can be measured by techniques known in the art, including, but not limited to, immunohistochemistry, Western blotting, Northern blotting, and quantitative PCR. In one embodiment, the present disclosure provides a novel SAR that targets cells expressing high levels of Her2 but not cells expressing low levels of Her2. The reference cells expressing high levels of Her2 are the SKOV-3 cell line, and the cells expressing low levels of Her2 are the MBA-MD-231 cell line. In one embodiment, the present disclosure provides a SAR expressed in JNG cells that, when co-cultured with SKOV-3 cells, exhibits GFP induction that is two-fold or more (e.g., three-fold, four-fold, five-fold, ten-fold, etc.) greater than the GFP induction observed when co-cultured with the MBA-MD-231 cell line in a Jurkat NFAT-GFP assay. In certain embodiments, the present disclosure provides a SAR expressed in JNG cells that, when co-cultured with SKOV-3 cells, exhibits GFP induction in a Jurkat NFAT-GFP assay that is greater than 10% (e.g., 20%, 30%, 40%, or 50%) of the GFP induction seen when co-cultured with the MBA-MD-231 cell line.

[0038] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets CD229. In an exemplary embodiment, the sequence of the isolated nucleic acid fragment targeting CD229 is set forth in SEQ ID NOs: 9134-9409. In an exemplary embodiment, the sequence of the isolated polypeptide targeting CD229 is set forth in SEQ ID NOs: 19824-20099 (Table 36). In some embodiments, vL, vH, and scFv fragments targeting CD229 are set forth in Table 3 and set forth in SEQ ID NOs: 260-263, 502-505, and 744-747, respectively. The amino acid sequence numbers of vL, vH, and scFv fragments targeting CD229 are also set forth in Table 3 and set forth in SEQ ID NOs: 10950-10953, 11192-11195, and 11434-11437, respectively. Exemplary monospecific, bispecific, and multispecific SARs incorporating vL, vH, and scFv targeting CD229 are shown in Tables 25-36. Further provided herein are vectors comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets CD229. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets CD229.

[0039] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets ROR1. In an exemplary embodiment, the sequences of isolated nucleic acid fragments targeting ROR1 are set forth in SEQ ID NOs: 8375-8581. In an exemplary embodiment, the sequences of isolated polypeptides targeting ROR1 are set forth in SEQ ID NOs: 19065-19133 (Table 36). The amino acid sequences of vL, vH, and scFv fragments targeting CD229 are also set forth in Table 3. Further provided herein are vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets ROR1. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets ROR1.

[0040] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets CEA. In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting CEA are set forth in SEQ ID NOs: 8720-8857. In an exemplary embodiment, the sequences of the isolated polypeptides targeting CEA are set forth in SEQ ID NOs: 19410-19547 (Table 36). In some embodiments, the vL, vH, and scFv fragments targeting CEA are listed in Table 3. The amino acid sequences of the vL, vH, and scFv fragments targeting CEA are also listed in Table 3. Further provided herein are vectors comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets CEA. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets CEA.

[0041] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets Toso. In an exemplary embodiment, the sequences of isolated nucleic acid fragments targeting Toso are set forth in SEQ ID NOs: 8996-9064. In an exemplary embodiment, the sequences of isolated polypeptides targeting Toso are set forth in SEQ ID NOs: 19686-19747 (Table 36). In some embodiments, vL, vH, and scFv fragments targeting Toso are listed in Table 3. The amino acid sequences of vL, vH, and scFv fragments targeting Toso are also listed in Table 3. Further provided herein are vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets Toso. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets Toso.

[0042] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets the gp350 protein encoded by EBV. In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting EBV gp350 are set forth in SEQ ID NOs: 9410-9547. In an exemplary embodiment, the sequences of the isolated polypeptides targeting EBV gp350 are set forth in SEQ ID NOs: 20169-20237 (Table 36). In some embodiments, vL, vH, and scFv fragments targeting EBV gp350 are listed in Table 3. The amino acid SEQ ID NOs of vL, vH, and scFv fragments targeting EBV gp350 are also listed in Table 3. Further provided herein are vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets EBV gp350. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets EBV gp350. These SARs are useful for treating disorders associated with EBV infection.

[0043] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets the LMP1 protein encoded by EBV. In an exemplary embodiment, the sequences of the isolated nucleic acid fragments targeting EBV LMP1 are set forth in SEQ ID NOs: 9617-9685. In an exemplary embodiment, the sequences of the isolated polypeptides targeting EBV LMP1 are set forth in SEQ ID NOs: 20307-20375 (Table 36). In some embodiments, vL, vH, and scFv fragments targeting EBV LMP1 are listed in Table 3. The amino acid SEQ ID NOs of the vL, vH, and scFv fragments targeting EBV LMP1 are also listed in Table 3. Further provided herein are vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domain of the SAR targets EBV LMP1. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets EBV LMP1. These SARs are useful for treating disorders associated with EBV infection.

[0044] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domain of the SAR targets influenza A neuraminidase (NA) protein. In an exemplary embodiment, the sequence of the isolated nucleic acid fragment targeting influenza A NA is set forth in Table 36. In an exemplary embodiment, the sequence of the isolated polypeptide targeting influenza A NA is set forth in (Table 36). In some embodiments, vL, vH, and scFv fragments targeting influenza A NA are listed in Table 3. The amino acid sequence numbers of vL, vH, and scFv fragments targeting influenza A NA are also listed in Table 3. Further provided herein are vectors comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets influenza A NA. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets influenza A NA. These SARs are useful in the treatment of disorders associated with influenza A infection.

[0045] In one embodiment, provided herein is an isolated nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets the receptor-binding domain (S-RBD) of the SARS-CoV2 spike glycoprotein. In an exemplary embodiment, the sequence of the isolated nucleic acid fragment targeting the S-RBD is set forth in SEQ ID NOs: 25-26. In an exemplary embodiment, the sequence of the isolated polypeptide targeting the S-RBD is set forth in SEQ ID NOs: 23318-19. Further provided herein is a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets the S-RBD. Also provided herein is a genetically engineered cell (e.g., T cell, NK cell) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domain of the SAR targets the S-RBD. These SARs are useful for treating disorders associated with S-RBD infection.

[0046] In one embodiment, provided herein are isolated nucleic acids encoding monospecific, bispecific, and multispecific SARs, wherein the antigen-specific domains of the SARs target the antigens listed in Tables 25-36. In exemplary embodiments, the sequences of isolated nucleic acid fragments targeting these antigens are set forth in SEQ ID NOs: 1330-1332, 1848-10720. In exemplary embodiments, the sequences of isolated polypeptides targeting these different antigens are set forth in SEQ ID NOs: 12020-12022, 12539-21410 (Table 36). In some embodiments, vL, vH, and scFv fragments targeting these different antigens are set forth in Table 3 and set forth in SEQ ID NOs: 46-282, 288-524, and 530-766, respectively. The amino acid sequence numbers of vL, vH, and scFv fragments targeting various antigens are also listed in Table 3 and are set forth in SEQ ID NOS: 10736-10972, 10978-11214, and 11220-11456, respectively. In some embodiments, exemplary AABDs (e.g., vHHs, SVHs, DARPINs, centilins, affibodies, ligands, receptors, Zip domains, protein tags, etc.) targeting various antigens are listed in Tables 5, 7-9 and are set forth in SEQ ID NOS: 830-902; 972-1023. Exemplary monospecific, bispecific, and multispecific SARs incorporating vLs, vHs, scFvs, and AABDs are listed in Tables 25-36 and 41-50. Further provided herein are vectors comprising nucleic acids encoding SARs, wherein the antigen-specific domains of the SARs target different antigens. Also provided herein are genetically engineered cells (e.g., T cells, NK cells) comprising a vector comprising a nucleic acid encoding a SAR, wherein the antigen-specific domains of the SAR target different antigens.

[0047] The present disclosure further relates to improving the quality of SAR-expressing T cells by expanding them in the presence of a SMAC mimetic.

[0048] The present disclosure further relates to improving the quality of SAR-expressing T cells by expanding them in the presence of a NIK (NF-κB-inducing kinase) agonist.

[0049] The SARs of the present disclosure may be manufactured using open, semi-closed, or closed system processes.

[0050] The SAR of the present disclosure can be manufactured using a device or container that includes a gas-permeable membrane. An exemplary device that includes a gas-permeable membrane is a GRex flask.

[0051] The present disclosure further teaches methods for producing SAR under hypoxic conditions.

[0052] The present disclosure further teaches methods for producing SAR under hypoxic conditions using a device or container comprising a gas-permeable membrane. An exemplary device comprising a gas-permeable membrane is a GRex flask.

[0053] The present disclosure also teaches a method for controlling the activity of SAR by incorporating at least one module that binds to albumin via a protease-cleavable linker. An exemplary albumin-binding module is an albumin-targeting vH or vH domain (e.g., SEQ ID NO: (DNA): 878 and SEQ ID NO: (PRT): 11568), which can be linked to SAR via an MMP9 target sequence (e.g., SEQ ID NOs: 11911-11912 and 11921).

[0054] The present disclosure further relates to polynucleotides encoding the SVHs of the present disclosure, vectors encoding such polypeptides, isolated cells expressing the SVHs, and isolated polypeptides encoding the SVHs of the present disclosure. Such cells and polypeptides can be used in therapy, particularly for the treatment of cancer, autoimmune, and infectious diseases. Methods for treating diseases, such as cancer, are also within the scope of the present disclosure.

[0055] In another aspect, the present disclosure relates to an isolated SAR comprising two or more antigen-binding domains and two transmembrane domains, wherein the antigen-binding domains are linked to one or more, e.g., at least two, human heavy chain variable chains (V H ) domain and no light chains are present.

[0056] In some embodiments, a SAR comprises two or more sets of two or more polypeptides, where the polypeptides of each SAR set are contiguous with each other (functional polypeptide unit 1) but not with the polypeptides of the other set (functional polypeptide unit 2).

[0057] In another aspect, the present disclosure relates to methods, e.g., ex vivo methods, for producing cells or cell populations for use in adaptive immunotherapy comprising transforming a cell or cell population with a SAR of the present disclosure.

[0058] In another aspect, the disclosure relates to methods for generating SAR-expressing T cells (e.g., CAR-T or SIR-T or TCR-T or ab-TCR-T, AABD-TCR-T, or TFP-T, TCR-T cells, etc.) by expansion in the presence of a SMAC mimetic. In some embodiments, the SAR-T cells are expanded ex vivo in the presence of a SMAC mimetic for 2 to 50 days.

[0059] In another aspect, the disclosure relates to methods for improving the efficacy of SAR-expressing T cells (e.g., CAR-T or SIR-T or TCR-T or ab-TCR-T, AABD-TCR-T, or TFP-T, TCR-T cells, etc.) by expanding them in the presence of a SMAC mimetic. In some embodiments, the SAR-T cells are expanded ex vivo in the presence of a SMAC mimetic for 2 to 50 days.

[0060] In another aspect, the present disclosure relates to methods for enhancing the cytotoxicity of SAR-expressing T cells (e.g., CAR-T or SIR-T or TCR-T or ab-TCR-T, AABD-TCR-T, or TFP-T, TCR-T cells, etc.) by expanding them in the presence of a SMAC mimetic. In some embodiments, the SAR-T cells are expanded ex vivo in the presence of a SMAC mimetic for 2 to 50 days.

[0061] In another aspect, the present disclosure relates to a method for generating SAR-expressing T cells (e.g., CAR-T, SIR-T, TCR-T, ab-TCR-T, or TFP-T cells, etc.) by expansion in the presence of a NIK agonist. In some embodiments, the SAR-T cells are expanded ex vivo in the presence of a NIK agonist for 2 to 50 days.

[0062] In another aspect, the present disclosure relates to methods for improving the efficacy of SAR-expressing T cells (e.g., CAR-T or SIR-T or TCR-T or ab-TCR-T, AABD-TCR-T, or TFP-T, TCR-T cells, etc.) by expanding them in the presence of a NIK agonist. In some embodiments, the SAR-T cells are expanded ex vivo in the presence of a NIK agonist for 2 to 50 days.

[0063] In another aspect, the present disclosure relates to methods for improving the cytotoxicity of SAR-expressing T cells (e.g., CAR-T or SIR-T or TCR-T or ab-TCR-T, AABD-TCR-T, or TFP-T, TCR-T cells, etc.) by expanding them in the presence of a NIK agonist. In some embodiments, the SAR-T cells are expanded ex vivo in the presence of a NIK agonist for 2 to 50 days.

[0064] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a cell or cell population of the present disclosure.

[0065] Bioluminescence imaging is often used to monitor the in vivo activity of cell therapy products. The present disclosure describes the use of thermostable luciferases (e.g., LucPPE and its engineered variants, such as LucPPe-146-1H2 (SEQ ID NO: (DNA) 17 and SEQ ID NO: (PRT) 11997), LucPPe-133-1B2 (SEQ ID NO: (DNA) 18), LucPPe-78-0B10 (SEQ ID NO: 19), LucPPe49-7C6A (SEQ ID NO: 20), and LucPpL-81-6G1 (SEQ ID NO: 21)) for in vivo bioluminescence imaging.

[0066] The present disclosure also provides novel vL, vH, and scFv fragments that can be used to construct single- and dual-chain SARs, including second-generation CARs, single- and dual-chain SIRs, single- and dual-chain cTCRs, Ab-TCRs, AABD-TCRs, TFPs, TACs, and the like. Exemplary vL, vH, and scFv fragments and their target antigens are listed in Table 3. Exemplary monospecific, bispecific, and multispecific SARs based on the vL, vH, and scFv fragments of hu-mROO5-1 are shown in Table 35. Because SARs are modular, the vL, vH, and scFv fragments of hu-mROO5-1 can be replaced with vL, vH, and scFv fragments targeting other antigens to develop SARs targeting those antigens. Table 36 lists the names of the initial constructs and the sequence numbers of other constructs in the series in which the vL, vH, and scFv fragments of hu-mROO5-1 are replaced with other vL, vH, and scFv fragments. The order of the constructs in each lineage is the same as the order of the constructs in the hu-mROO5-1 lineage listed in Table 35, and therefore the name and SEQ ID NO of a particular lineage construct can be determined by reference to Tables 35 and 36. For example, Table 35 shows that the last construct in the hu-mROO5-1 lineage (Lineage 1) is CD8SP-CD38-USC1-FHVH-32184-G4Sx3-R1-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-USC1-vHH-2HCD26-G4Sx3v2-Bst-hu-mROO5-1-vH-[hTCRa-T48C], having nucleic acid and amino acid SEQ ID NOs: 7408 and 18098. Table 36 shows that in lineage 5, hu-mROO5-1-vL is replaced with BCMA-huUSC76-vL and hu-mROO5-1-vH is replaced with BCMA-huUSC76-I58S-vH. The nucleic acid SEQ ID NOs of the various constructs in this lineage range from 7616 to 7684, and the amino acid SEQ ID NOs of the constructs in this lineage range from 18306 to 18374.Because the order of the various constructs in Line 5 is the same as the order of the constructs in Line 1 shown in Table 35, the nucleic acid and amino acid SEQ ID NOs for the construct CD8SP-CD38-USC1-FHVH-32184-G4Sx3-R1-BCMA-huUSC76-vL-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-USC1-vHH-2HCD26-G4Sx3v2-Bst-BCMA-huUSC76-I58S-vH-[hTCRa-T48C] can be determined to be 7684 and 18374, respectively. The same method can be used to determine the SEQ ID NOs for any of the SARs of the lineages listed in Table 36.

[0067] In certain embodiments, the present disclosure provides at least one recombinant polynucleotide encoding at least one synthetic antigen receptor (SAR) polypeptide, the at least one SAR polypeptide comprising: (A) a first module comprising one or more autonomous antigen-binding domains (AABDs) or fragments thereof selected from the group consisting of a single vH domain (SVH) or fragment thereof; a single vL domain (SVL) or fragment thereof; a vHH domain or fragment thereof; a single-domain antibody or fragment thereof; a single variable domain of a TCR (svd-TCR) or fragment thereof; a non-immunoglobulin antigen-binding scaffold selected from a DARPIN, an affibody, an affilis, an adnectin, an affitin, an obody, a lipibody, a finomer, an alphabody, an avimer, an atrimer, a centilin, a pronectin, an anticalin, a Kunitz domain, an armadillo repeat protein, and a D domain, or a fragment of any of the above; a ligand-binding domain of a receptor or fragment thereof; a receptor-binding domain of a ligand; an autoantigen or fragment thereof; an adaptor-binding domain or fragment thereof; and an Fc-binding domain or fragment thereof; (B) a vL(V L ), vH(V H(C) an optional third module comprising an extracellular domain, a binding peptide, or a hinge domain; (D) a fourth module comprising a transmembrane domain; and (E) an optional fifth module comprising one or more intracellular signaling domains; wherein the first, second, optional third, fourth, and optional fifth modules are operably linked via one or more optional linkers.

[0068] The present disclosure also provides a recombinant polynucleotide encoding a SAR polypeptide, wherein the one or more AABDs are attached via an optional linker to at or near the N-terminus of: (A) a first polypeptide chain comprising a vH, Va, Vg, or Ig linker domain fragment operably linked via an optional linker to a first T cell receptor constant chain fragment comprising a first binding peptide operably linked to a first transmembrane domain of a first TCR subunit; and (B) a second polypeptide chain comprising a vL, Vb, Vd, or Ig linker domain operably linked via an optional linker to a second T cell receptor constant chain fragment comprising a second binding peptide operably linked to a second transmembrane domain of a second TCR subunit; wherein the first TCR constant chain fragment and the second TCR constant chain fragment form a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling module when expressed in a T cell.

[0069] In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the encoded first antigen-binding domain vH, Va, or Vg and the second antigen-binding domain vL, Vb, or Vd form an antigen-binding module that specifically binds to a target antigen.

[0070] In one embodiment, the disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the AABD, vL, vH, Va, Vb, Vg, Vd and / or Ig linker domains are fully human, humanized, chimeric or non-human domains.

[0071] In one embodiment, the disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the encoded Ig linker domain comprises a polypeptide having SEQ ID NO: 11832-11865, or a fragment or variant thereof having at least 70% sequence identity to a polypeptide having a sequence of SEQ ID NO: 11832-11865. In one embodiment, the disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the encoded optional linker domain comprises a polypeptide having SEQ ID NO: 11832-11865, 11714-11730, or a fragment or variant thereof having at least 70% sequence identity to a polypeptide having a sequence of SEQ ID NO: 11832-11865, 11714-11730, or a domain of 25-500 amino acids in length. In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide comprising part or all of the extracellular, transmembrane, and intracellular domains of a polypeptide that can be recruited to a T cell receptor module (TCRM). In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide comprising the extracellular, transmembrane, and intracellular domains of a polypeptide selected from the group consisting of CD3δ, CD3ε, CD3γ, and CD3ζ, or a fragment thereof having at least 70% sequence identity to a polypeptide having the sequence of any one of SEQ ID NOs: 11903-11906. In certain embodiments, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the encoded T cell receptor constant chain fragment comprises a polypeptide having the sequence of any one of SEQ ID NOs: 11732-11830, or a fragment thereof having at least 70% sequence identity to the polypeptide.In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR, wherein the encoded T cell receptor constant chain comprises a binding peptide having the sequence of any one of SEQ ID NOs: 11867-11875 or a fragment thereof having at least 70% sequence identity to the binding peptide. In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide comprising a transmembrane domain having the sequence of any one of SEQ ID NOs: 11877-11881 or 23332-23334 or a fragment thereof having at least 70% sequence identity to the transmembrane domain. In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the intracellular domain comprises a cytosolic domain or a fragment thereof having at least 70% sequence identity to a polypeptide having the sequence of any one of SEQ ID NOs: 11883-11886, 11785, or 23335-23337.

[0072] In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide having a backbone of a first generation CAR, a second generation CAR, a third generation CAR, a single-chain SIR, a 1.5-chain SIR, a dual-chain SIR, a zSIR, a single-chain cTCR, a 1.5-chain cTCR, a dual-chain cTCR, an Ab-TCR, an AABD-TCR, TFPε, TFPγ, TFPδ, TFPαβ, TFPγδ, Tri-TAC, a single-chain TCR, a dual-chain TCR, or an HLA-independent TCR.

[0073] In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the SAR can bind to at least one antigen and / or at least one epitope of one or more antigens. In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide of the disclosure, wherein two or more encoded AABDs bind to at least one antigen and / or at least one epitope of one or more antigens.

[0074] In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the encoded one or more antigen-binding domains bind to at least one antigen selected from the antigens listed in Table B.

[0075] In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the encoded SAR polypeptide is selected from the group consisting of: (i) a sequence set forth in any one of SEQ ID NOs: 10978-11214, or a sequence having at least 70% identity thereto, or a sequence having at least 70% identity in three complementarity-determining regions (CDRs) to any one or more of SEQ ID NOs: 10978-11214 and 23148-23161. or a sequence having less than three substitutions in three CDRs of the sequence shown in any one or more of SEQ ID NOs: 10978 to 11214 and 23148 ​​to 23161, or a sequence that binds to the same target antigen or the same epitope of the target antigen as the sequence shown in any one or more of SEQ ID NOs: 10978 to 11214 and 23148 ​​to 23161 and encodes a polypeptide that binds to the antigen; (ii) a sequence shown in any one of SEQ ID NOs: 10736 to 10972, or or a sequence having at least 70% identity with any one or more of SEQ ID NOs: 10736 to 10972 and 23136 to 23147, or a sequence having at least 70% identity in three complementarity determining regions (CDRs) with any one or more of SEQ ID NOs: 10736 to 10972 and 23136 to 23147, or a sequence having less than three substitutions in three CDRs of any one or more of SEQ ID NOs: 10736 to 10972 and 23136 to 23147, or (iii) a light chain variable region (vL) comprising a sequence that binds to the same target antigen or the same epitope of the target antigen as the sequence set forth in any one or more of SEQ ID NOs: 10736 to 10972 and 23136 to 23147 and encodes a polypeptide that binds to the antigen; (iv) a single-chain variable fragment (scFv) comprising a sequence set forth in any one of SEQ ID NOs: 11220 to 11456, or a sequence having at least 70% identity thereto, and encoding a polypeptide that binds to the antigen;(iv) SEQ ID NOs: A sequence having at least 70% identity to any one of SEQ ID NOs: 11524 to 11525, 11530 to 11531, 11549 to 11575, 11576 to 11592, and 23163 to 23173, or any one or more of SEQ ID NOs: 11520 to 11592 and 23163 to 23173, and / or any one or more of SEQ ID NOs: 11524 to 11525, 11530 to 11531, 11549 to 11575, 11576 to 11592, and 23163 to 23173. a sequence having at least 70% identity in three complementarity determining regions (CDRs) with a sequence set forth in any one or more of SEQ ID NOs: 11524 to 11525, 11530 to 11531, 11549 to 11575, 11576 to 11592, and 23163 to 23173, or a sequence having less than three substitutions in three CDRs of a sequence set forth in any one or more of SEQ ID NOs: 11524 to 11525, 11530 to 11531, 11549 to 11575, 11576 to 11592, and 23163 to 23173, or a sequence having the same target as a sequence set forth in any one or more of SEQ ID NOs: 11524 to 11525, 11530 to 11531, 11549 to 11575, 11576 to 11592, and 23163 to 23173, (v) a camelid VHH domain comprising a sequence that binds to an antigen or the same epitope of a target antigen and encodes a polypeptide that binds to the antigen; (v) a non-immunoglobulin encoded by a polynucleotide of any one of SEQ ID NOs: 11662 to 11673, or a sequence having at least 70% identity to any one or more of SEQ ID NOs: 11662 to 11673, or a sequence that binds to the same target antigen or the same epitope of the target antigen as any one or more of SEQ ID NOs: 11662 to 11673. a scaffold; (vi) a ligand-binding domain of a receptor comprising a sequence set forth in any one of SEQ ID NOs: 11674-11691, or a sequence having at least 70% identity thereto and encoding a polypeptide that binds to a homolog thereof; (vii) a receptor-binding domain of a ligand comprising a sequence set forth in any one of SEQ ID NOs: 11692-11702, 22391-22392, 22402-22404, or a sequence having at least 70% identity thereto and encoding a polypeptide that binds to a homolog thereof;(viii) a sequence having at least 70% identity to any one of SEQ ID NOs: 11519 to 11523, 11526 to 11529, 11532 to 11548, 11644 to 11645, and 23174, or any one or more of SEQ ID NOs: 11519 to 11523, 11526 to 11529, 11532 to 11548, 11644 to 11645, and 23174, and / or SEQ ID NOs: 11519 to 11523, 11526 to 11529, 11532 to 11548, 11644 to 11645, and 23174 A sequence having 70% identity in three complementarity determining regions (CDRs) with any one or more of the sequences shown in SEQ ID NOs: 11519 to 11523, 11526 to 11529, 11532 to 11548, 11644 to 11645, and 23174, or a sequence having less than three substitutions in three CDRs of any one or more of the sequences shown in SEQ ID NOs: 11519 to 11523, 11526 to 11529, 11532 to 11548, 11644 to 11645, and 23174; or a single vH domain comprising a sequence that binds to the same target antigen or the same epitope of a target antigen as a sequence set forth in any one or more of SEQ ID NOs: 11519-11523, 11526-11529, 11532-11548, 11644-11645, and 23174, and encodes a polypeptide that binds to the antigen; (ix) an adaptor binding domain comprising a sequence set forth in any one of SEQ ID NOs: 11704-11712, or 22383, or a sequence having at least 70% identity thereto, and encoding a polypeptide that binds to the adaptor; (x) an autoantigen comprising a sequence set forth in any one of SEQ ID NOs: 11687, 22406-22407 to 11712, or 22383, or a sequence having at least 70% identity thereto, and encoding a polypeptide that binds to the autoantibody or autoantibody-producing cells;(xi) a TCR variable region (Va, Vb) comprising a sequence set forth in any one of SEQ ID NOs: 22396 to 2239 and 11653 to 11660, or a sequence having at least 70% identity thereto, or a sequence having at least 70% identity in three complementarity determining regions (CDRs) with any one or more of SEQ ID NOs: 22396 to 22397 and 11653 to 11660, or a sequence having less than three substitutions in three CDRs of any one or more of SEQ ID NOs: 22396 to 22397 and 11653 to 11660, or a sequence encoding a polypeptide that binds to the same target antigen or the same epitope of a target antigen as a sequence set forth in any one or more of SEQ ID NOs: 22396 to 22397 and 11653 to 11660, and that binds to the antigen; Vb, Vg, or Vd); and (xii) a single variable TCR domain (svd-TCR) comprising a sequence set forth in any one of SEQ ID NOs: 22399 to 22400, or a sequence having at least 70% identity thereto, or a sequence having 70 to 99% identity in three complementarity determining regions (CDRs) to a sequence set forth in any one or more of SEQ ID NOs: 22399 to 22400, or a sequence having less than three substitutions in the three CDRs of a sequence set forth in any one or more of SEQ ID NOs: 22399 to 22400, or a sequence that binds to the same target antigen or the same epitope of a target antigen as a sequence set forth in any one or more of SEQ ID NOs: 22399 to 22400 and encodes a polypeptide that binds to the antigen;

[0076] In one embodiment, the present disclosure provides at least one recombinant polynucleotide encoding at least one SAR polypeptide, At least one of the target antigens is expressed on blood lineage cells and at least one of the target antigens is expressed on solid tumor cells.

[0077] In one embodiment, the present disclosure is co-expressed with a therapeutic control, wherein the therapeutic control is selected from the group consisting of truncated epidermal growth factor receptor (tEGFR), truncated epidermal growth factor receptor viii (tEGFRviii), truncated CD30 (tCD30), truncated BCMA (tBCMA), truncated CD19 (tCD19), CD34, thymidine kinase, cytosine deaminase, nitroreductase, xanthine guanine phosphoribosyltransferase, human caspase 8, human caspase 9, inducible caspase 9 (iCaspase 9), purine nucleoside phosphatase, phospholipase A (PGA), phospholipase B (PGB), phospholipase C (PGC), phospholipase D (PGD), phospholipase E (PGD), phospholipase B (PGD), phospholipase C (PGD ... Sulfonylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), gamma-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, doxorubicin-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), 41BBL, CD40L, K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV-1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4

[0013] Provided is a recombinant expression system comprising a recombinant polynucleotide of the present disclosure selected from the group consisting of: CTLA4-ipilimumab-scFv, CTLA4-ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, CD3z, CD3z-GGGS-41BB, CD3-BBz, CD3-CD28z, CD3-CD28-Lck fusion protein, shRNA targeting Brd4, chimeric antigen receptor (CAR), hTERT, heparinase, CAR, inhibitory CAR, and combinations thereof.

[0078] In one embodiment, the present disclosure provides at least one vector comprising a recombinant polynucleotide comprising a SAR, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a baculoviral vector, a sleeping beauty transposon vector, and a piggybac transposon vector.

[0079] In one embodiment, the present disclosure provides at least one SAR polypeptide encoded by at least one recombinant polynucleotide comprising a SAR. In one embodiment, the present disclosure provides a recombinant cell or cell population expressing at least one recombinant polynucleotide encoding a SAR of the present disclosure. In another embodiment, the present disclosure provides a cell or cell population expressing a SAR, wherein the cell is an immune effector cell, a stem cell capable of giving rise to an immune effector cell, or an induced pluripotent stem cell (iPSC) capable of giving rise to an immune effector cell. The cell or cell population can be autologous or allogeneic.

[0080] In one embodiment, the present disclosure provides a cell or cell population in which the functional expression of an endogenous TCR is impaired or eliminated and which exhibits functional expression of a SAR polypeptide. The present disclosure also provides a cell or cell population in which the SAR mRNA and translated polypeptide are expressed from an expression cassette located at the locus of an endogenous T cell gene. The present disclosure also provides a cell population in which at least one polynucleotide is under the control of a promoter and / or regulatory element of an endogenous T cell gene. The present disclosure provides a cell or cell population in which the endogenous T cell gene locus is the TRAC locus, the TRBC locus, the TRGC locus, and / or the TRDC locus. In one embodiment, the present disclosure provides a cell or cell population in which the location of the SAR polynucleotide disrupts or eliminates the endogenous expression of a TCR comprising an endogenous TCR α chain and / or an endogenous TCR β chain, or an endogenous TCR γ chain and / or an endogenous TCR δ chain in T cells. In one embodiment, the present disclosure provides a cell or cell population wherein disruption or impairment of expression of an endogenous TCR results in enhanced expression and / or activity of a non-naturally occurring immune receptor compared to its expression and / or activity in a T cell with a wild-type endogenous TCR, and wherein the SAR has a framework of one or more of an SIR, a cTCR, and / or an Ab-TCR.

[0081] In one embodiment, the disclosure provides a cell or cell population in which the SAR polypeptide has a TFP backbone and disruption or loss of expression of an endogenous TCR results in impaired expression and / or activity of the SAR compared to its expression and / or activity in a T cell with a wild-type endogenous TCR.

[0082] In one embodiment, the present disclosure provides a cell or cell population, wherein the SAR polypeptide has a TFP backbone, and the cell or cell population further expresses a sequence encoding a TCR constant chain, wherein the TCR constant chain is i) a TCR alpha constant chain or a fragment thereof, or ii) a TCR beta constant chain or a fragment thereof, or iii) a TCR gamma constant chain or a fragment thereof, or iv) a TCR delta constant chain or a fragment thereof, or a combination of i) and ii) or iii) and iv), and wherein expression of the sequence encoding the TCR constant chain restores the expression and / or activity of the SAR polypeptide.

[0083] In one embodiment, the present disclosure provides a method for producing a SAR-expressing immune effector cell, comprising introducing at least one vector comprising a SAR or at least one recombinant polynucleotide comprising a SAR into an immune effector cell or a hematopoietic stem or progenitor cell capable of giving rise to an immune effector cell under conditions such that the SAR polypeptide is expressed.

[0084] In one embodiment, the present disclosure provides a method for expanding SAR-expressing immune effector cells in gas-permeable flasks under normoxic or hypoxic conditions.

[0085] In one embodiment, the present disclosure provides a method, wherein said SAR-expressing immune effector cells are expanded in the presence of a SMAC mimetic or a NIK agonist compound.

[0086] In one embodiment, the present disclosure provides a method of providing anti-disease immunity in a subject, comprising administering to the subject an effective amount of immune effector cells or stem cells capable of giving rise to immune effector cells, wherein the cells are autologous or allogeneic T cells, or autologous or allogeneic NKT cells, or autologous or allogeneic hematopoietic stem cells or autologous or allogeneic iPSCs capable of giving rise to immune effector cells.

[0087] In one embodiment, the present disclosure provides a method of treating or preventing a disease associated with expression of a disease-associated antigen in a subject, the method comprising administering to the subject an effective amount of immune effector cells comprising a synthetic antigen receptor (SAR) polypeptide, wherein the SAR polypeptide binds to one or more disease-associated antigens selected from the antigens listed in Table B, and the disease associated with expression of the disease-associated antigen is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer-related indication associated with expression of the disease-associated antigen.

[0088] In one embodiment, the present disclosure provides SEQ ID NOs: 1849-10720, 21609-21625, 21626-21659, 21662-21792, 21808, 21813-21840, 21843-21879, 21882-21892, 21894-21899, 21901-21947, 21949-21963, 21967-21972, 21985-22085, 22087-22088 99, 22104-22207, 22209-22250, 22252-22254, 22256-22262, 22264-22273, 22275-22284, 22286-22296, and 22307-22312, or a sequence having at least 75% identity to the above nucleotide sequence. In one embodiment, the present disclosure provides a method for the preparation of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12539-21410, 22445-22460, 22462-22495, 22498-22614, 22624, 22626-22655, 22658-22685, 22687-22694, 22697-22707, 22709-22714, 22716-22762, 22764-22778, 22782-22787, 22800-22809, 22810-22811, 22812-22813, 22814-22815, 22816-22817, 22818-22819, 22820-22821, 22822-22824, 22826-22827, 22828-22829, 22830-22831, 22832-22833, 22834-22835, 22836-22837, 22838-22839, 22840-22841, 22842-22843, 22844-22845, 22846-22847, 22848-22849, 22850-22851, 22852-22853, 22854-22855, 22856-22857, 22858 and 23122-23127, or a sequence having at least 75% identity to the above amino acid sequences.

[0089] In one embodiment, the present disclosure provides a composition comprising at least one SAR polynucleotide, SAR polypeptide molecule, SAR vector, or SAR-expressing cell and a pharmaceutically acceptable excipient. In one embodiment, the present disclosure provides a kit comprising at least one SAR polynucleotide, SAR polypeptide molecule, SAR vector, or SAR-expressing cell.

[0090] The present disclosure is further illustrated in the following non-limiting drawings. [Brief explanation of the drawings]

[0091] [Figure 1] FIG. 1 shows a schematic representation of the monospecific, bispecific and multispecific SAR of different duplexes. [Figure 2] FIG. 2 shows a schematic representation of monospecific, bispecific and multispecific SARs of different duplexes comprising different forms of AABDs (eg, vHH, SVH, aVH, affibody, centirin, etc.). DETAILED DESCRIPTION OF THE INVENTION

[0092] Detailed Description The present invention will be further described below. In the following sections, various aspects of the present invention are defined in more detail. Each aspect thus defined can be combined with any other one or more aspects, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other one or more features indicated as being preferred or advantageous.

[0093] Unless otherwise stated or implied from context, the following terms and phrases have the meanings provided below. Unless otherwise stated or apparent from context, the following terms or phrases do not exclude the meaning that those terms or phrases have acquired in the relevant art. Definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims.

[0094] 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.

[0095] As used herein, the term "comprising" is used in reference to compositions, methods, and their respective components that are useful in embodiments but may include unspecified elements, whether or not useful. In general, those of skill in the art will understand that the terms used herein are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including, but not limited to," etc.).

[0096] Generally, the nomenclatures used in connection with and techniques of cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and, unless otherwise indicated, as described in the various general and more specific references cited and discussed herein. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2013)). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and experimental procedures and techniques of, immunology, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0097] The term "about," when referring to a measurable value, such as an amount, temporal duration, or the like, is meant to encompass a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the particular value, as such variations are appropriate for performing the disclosed methods or describing the compositions herein. Furthermore, any value or range (e.g., less than 20 or similar terminology) explicitly includes any integer between or up to such value. Thus, for example, "1 to 5 variations" explicitly includes 1, 2, 3, 4, and / or 5 variations. The term "at least" refers to the minimum value within a range. When a percentage is preceded by "at least," increments contemplated within the range are from 0.1% to, for example, 100% (in the case of identity / homology).

[0098] 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. Antigen-binding domains can be derived from different sources, such as antibodies (full-length heavy chains, Fab fragments, single-chain Fv (scFv) fragments, bivalent single-chain antibodies, or diabodies), non-immunoglobulin-binding proteins, ligands, or receptors. However, numerous alternatives exist, such as linked cytokines (leading to recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains from naturally occurring receptors, soluble protein / peptide ligands of receptors (e.g., on tumor cells), peptides, and vaccines to promote immune responses, each of which can be used in various embodiments of the present disclosure. In some embodiments, as will be appreciated by those skilled in the art, virtually any molecule that binds to a given cognate or antigen with high affinity can be used as an ASD. In some embodiments, the antigen-binding domain comprises a T-cell receptor (TCR) or a portion thereof. In exemplary embodiments, the target antigens and SEQ ID NOs for various antigen-binding domains are set forth in Tables 3-7 herein. In exemplary embodiments, the target antigens and SEQ ID NOs for vL, vH, scFV, and their CDR regions are set forth in Tables 6A-C of patent application PCT / US18 / 53247 and Tables 3-4 of patent application PCT / US19 / 035096, the entire contents of which are incorporated herein by reference. The vL, vH, and scFv that can be used to construct the SARs of the present disclosure can be of "fully human," "humanized," "chimeric," or non-human origin. Exemplary fully human vL, vH, and scFv are represented by SEQ ID NOs: 10854, 11096, and 11338, respectively. Exemplary humanized vL, vH, and scFv are represented by SEQ ID NOs: 10839, 11082, and 11323, respectively.

[0099] The term "autonomous antigen-binding domain" or "AABD," as used herein, refers to an antigen-binding domain that can bind to an antigen autonomously, i.e., in the absence of another antigen-binding domain. An exemplary AABD is a single vH domain or an autonomous vH domain (aVH), generally a single human vH domain (SVH), that can bind to an antigen in the absence of a vL domain. An exemplary AABD is a single vL domain or an autonomous vL domain, generally a single human vL domain (SVL), that can bind to an antigen in the absence of a vH domain. Another exemplary AABD is a fully human vH domain (FHVH). AABD also refers to other antigen-binding domains that can bind to an antigen autonomously. In certain embodiments, the AABD is a non-scFv antigen-binding domain. Exemplary non-scFV-based autonomous antigen-binding domains include, but are not limited to, vHH domains, humanized vHH domains, svd-TCRs, and non-immunoglobulin antigen-binding scaffolds, such as DARPINs, affibodies, ZIP domains (e.g., RZIP, EZIP, E4, R4, etc.), affilins, adnectins, affitins, obodies, lipibodies, finomers, alphabodies, avimers, atrimers, centrins, pronectins, anticalins, Kunitz domains, armadillo repeat proteins or fragments thereof; receptors (e.g., Examples of such an antigen-binding domain include a ligand-binding domain of CD16-V158A, NKG2D, or a fragment thereof; a receptor-binding domain of 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., Strep tag, FLAG tag, etc.); and an autoantigen or a fragment thereof.

[0100] The present disclosure describes the use of AABDs, such as human VH domains, typically multiple human VH domains, as building blocks for generating monospecific, bispecific, and multispecific SARs. In certain embodiments, the present disclosure describes the use of AABDs, such as human VH domains, typically multiple human VH domains, as building blocks for generating monospecific, bispecific, and multispecific CARs, SIRs, cTCRs, Ab-TCRs, AABD-TCRs, TFPs, and recombinant TCRs.

[0101] The term "ABR" or "antigen-binding receptor" as used herein refers to any receptor having an antigen-binding domain. The antigen-binding domain of an ABR may comprise an scFv, vL, vH, VHH, antibody, antibody fragment (e.g., Fab), antibody-like portion, Vα, Vβ, cytokine, receptor, etc. In one embodiment, the ABR has a transmembrane or membrane-tethering domain that allows expression on the cell surface. Exemplary ABRs include first-generation CARs, second-generation CARs, TFP, TRI-TAC, or TAC, etc. The antigen-masking receptors described herein are also examples of ABRs.

[0102] The term "Ab-TCR" or "AbTCR" refers to a next-generation CAR platform such as that described in WO2017 / 070608A1, which is incorporated herein by reference. In one embodiment, the Ab-TCR comprises an antibody moiety that specifically binds to a target antigen fused to a TCR module capable of recruiting at least one TCR signaling module. Exemplary TCR modules that can be used to construct Ab-TCRs are set forth in SEQ ID NOS: 6009-6014 (Table 6) and WO2017 / 070608A1, which are incorporated herein by reference. The present disclosure provides bispecific, biparatopic, and multispecific SARs having an Ab-TCR scaffold comprising one or more AABDs. The AABD domains of the SARs of the present disclosure having an Ab-TCR scaffold can be fully human, humanized, or non-human. In one embodiment, the present disclosure provides an Ab-TCR comprising one or more fully human vH domains. In one embodiment, the present disclosure provides an Ab-TCR comprising one or more fully human vL domains.

[0103] The term "AABD-TCR" refers to the novel duplex SAR platform described in the present disclosure in which one or more AABDs are operably linked in-frame to a possible TCR module without the presence of an intervening vL, vH, Va, Vb, Vg, and / or Vd chain. In a preferred embodiment of the present disclosure, the AABD-TCR comprises an Ig-like linker domain present between the AABD and the TCR module. The TCR module of the AABD-TCR is capable of recruiting at least one TCR signaling module. Exemplary AABD-TCRs are represented by SEQ ID NOs: (DNA) 6521 and 6530. Additional AABD-TCRs are shown in Tables 33 and 34. The AABD domain of the AABD-TCR can be fully human, humanized, or non-human. In certain embodiments, the present disclosure provides an AABD-TCR comprising one or more fully human vH domains. In certain embodiments, the present disclosure provides an AABD-TCR comprising one or more fully human vL domains.

[0104] The term "accessory module" refers to PDL1, PDL2, CD80, CD86, crmA, p35, hNEMO-K277A (or NEMO-K277A), hNEMO-K277A-delta-V249-K555, mNEMO-K270A, K13-opt, IKK2-S177E-S181E (or IKK2-SS / EE), IKK1-S176E-S180E (or IKK1-SS / EE), MyD88-L265P, TCL-1a, MTCP-1, CMV-141, 41BBL, CD40L, vFLIP-K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV-1 Tax, HTLV-2 Tax, and HTLV-2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4

[0023] The accessory module refers to any one or more of: scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-ipilimumab-scFv, CTLA4-ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, shRNA targeting Brd4, IgSP-[hTRAC-opt2], IgSP-[hTRBC-opt2], and combinations thereof, expressed in immune cells (e.g., T cells, e.g., CAR-T cells or TCR-T cells) to enhance, regulate, or modify the activity of the immune cells. In some embodiments, the accessory module is co-expressed with an immune receptor such as a CAR or TCR to enhance, reduce, regulate, or modify the expression or activity of the CAR or TCR or a CAR- or TCR-expressing cell. The accessory module can be co-expressed with the CAR or TCR using a single vector or two or more different vectors. In a further embodiment, the accessory module comprises an FKBP (FK506 binding protein) fusion protein, such as FKBPx2-NEMO, the activity of which can be controlled by administration of a dimerizing molecule.In some embodiments, the accessory module is expressed on an antigen-presenting cell, such as a dendritic cell.

[0105] The term "affibody," as used herein, refers to an antibody mimetic molecule composed of alpha helices and lacking disulfide bonds. An exemplary affibody that targets Her3 is represented by SEQ ID NO: 11664. Other exemplary affibodies are known in the art.

[0106] As used herein, "affinity" refers to a measure of binding strength. Affinity may vary depending on the closeness of the steric and chemical fit between a binder and its target (e.g., between an antibody and an antigen containing an epitope specific to the binding domain), the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity generally refers to the "ability" of a binder to bind to its target. There are many methods in the art for measuring "affinity." For example, methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual-polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry. An exemplary method for determining binding affinity uses surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows the analysis of real-time biospecific interactions by detection of changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). As used herein, the term "specific binding" refers to binding of at least 10 -6 M. In a particular aspect, the antibody has a binding affinity of at least about 10 -7 M, generally 10 -8 M, 10 -9 M, 10 -10 M, 10 -11M, or 10 -12 It binds with an affinity of M.

[0107] 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 tetramers of immunoglobulin molecules. Antibodies can be "fully human," "humanized," "chimeric," or non-human.

[0108] As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.

[0109] As used herein, "human antibody" or "fully human antibody" means an antibody or antibody fragment having an amino acid sequence that corresponds to that of an antibody produced by a human and / or that is made using any of the techniques for making human antibodies known to those of skill in the art or disclosed herein. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide.

[0110] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences derive from one species and the constant region sequences derive from another species.

[0111] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, antibody fragments such as Fab, Fab', Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of the VH and CH1 domains, linear antibodies, single domain antibodies (sdAbs) such as either vL or vH, camelid vHH domains, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and multispecific antibodies formed from isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0112] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and usually determines the class to which the antibody belongs.

[0113] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0114] "Anticancer agent" refers to an agent that inhibits abnormal cell division and proliferation, inhibits the migration of neoplastic cells, inhibits invasiveness, or prevents the growth and metastasis of cancer. This term includes chemotherapeutic agents, biological agents (e.g., siRNA, engineered MLV that delivers cytotoxic genes, adenovirus, herpes virus, and other viral vectors), antibodies, and the like.

[0115] The term "anti-cancer 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 the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with cancer pathology. An "anti-cancer effect" can also be manifested by the ability of SAR to prevent the development of cancer in the first place.

[0116] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. The present disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, these nucleotide sequences arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be synthetically produced, derived from biological samples, or macromolecules other than polypeptides. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0117] The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using T cell receptors (TCRs). APCs process and present antigens to T cells.

[0118] The term "anti-infective effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in the titer of an infectious agent, a reduction in the colony count of an infectious agent, and an amelioration of various physiological symptoms associated with an infection. An "anti-infective effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent an infection from occurring in the first place.

[0119] The term "anti-tumor effect" or "anti-cancer 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 tumor cell number, a reduction in tumor cell proliferation, an inhibition of metastasis, or a reduction in tumor cell survival.

[0120] The term "association constant (Ka)" is defined as the equilibrium constant for the association of receptor and ligand.

[0121] "Autoantibody" refers to an antibody produced by B cells specific for a self-antigen.

[0122] The term "autoantigen" refers to an endogenous antigen that stimulates the production of autoimmune responses, such as the production of autoantibodies.Autoantigens also include normal tissue-derived autoantigens or antigens that are the target of cell-mediated or antibody-mediated immune responses, which may lead to the development of autoimmune diseases.Examples of autoantigens include, but are not limited to, desmoglein 1, desmoglein 3, and their fragments.

[0123] "Avidity" refers to the strength of the interaction between a binder and its target (e.g., the strength of the interaction between an antibody and its antigen target, a receptor and its cognate, etc.). Avidity can be weak or strong. Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays or Malibu-Glo assays) also reflects antibody affinity.

[0124] As used herein, the term "scaffold" or "configuration" refers to the configuration of different SARs (e.g., CARs, SIRs, cTCRs, Ab-TCRs, TFPs, etc.) and / or different components (e.g., antigen-binding domains, hinge domains, transmembrane domains, signaling domains) generally comprising any optional accessory modules. In exemplary embodiments, SARs may have a scaffold such as a first-generation CAR, a second-generation CAR, a dual-chain SIR, a 1.5-chain SIR, a dual-chain cTCR, a 1.5-chain cTCR, a zSIR, an Ab-TCR, an AABD-TCR, an εTFP, a γTFP, a δTFP, an αβTFP, a γδTFP, or a TCR. SARs with a specific scaffold may have further subtypes, each exhibiting a different scaffold. Thus, SARs having a double-chain SIR backbone may have further subtypes based on the number (monospecific, bispecific, multispecific, etc.) and nature (e.g., vL, vH, scFv, vHH, FHVH, DARPIN, etc.) of their antigen-binding domains. Each of the above subtypes can be considered a separate backbone. In an exemplary embodiment, a SAR in any of the backbones may further co-express an accessory module (e.g., PAC, K13-opt, MC159, iCaspase 9, etc.). Thus, a SAR having a double-chain SIR backbone may be co-expressed with K13-opt. In one embodiment, the SAR and the accessory module are encoded by a single nucleic acid molecule. In another embodiment, the SAR is encoded by a first nucleic acid molecule, and the accessory module is encoded by a second nucleic acid molecule. In some embodiments, the accessory module is encoded by two or more nucleic acid molecules depending on the number of components of the accessory module. The two or more components of the SAR may be separated by a cleavable linker, such as a 2A ribosomal skip sequence (eg, P2A, T2A, F2A, etc.).

[0125] Table A1-1: SAR structure / backbone. First-generation conventional CARs (conventional CAR I) have an activation domain (AD) domain (e.g., CD3z) and no costimulatory domain (CD). TCR fusion protein (TFP) is another example of conventional CAR I. Second-generation conventional CARs (conventional CAR 2 or CAR II) have one costimulatory domain (e.g., 4-1BB or CD28) and an intracellular activation domain (AD) domain (e.g., CD3z). Third-generation conventional CARs (conventional CAR 3 or CAR III) have two costimulatory domains (e.g., 4-1BB and CD28) and an intracellular activation domain (e.g., CD3z). Ab-TCR is a dual-chain receptor incorporating a vL-IgCL-linker-TCR domain (TCRD) and a vH-Ig-CH1-linker-TCR domain (TCRD), and is described in PCT / US2016 / 058305. AABD-TCRs are dual-chain receptors described herein and generally incorporate an AABD-IgCL-TCRD module and an IgCH1-TCRD module, or an IgCL-TCRD module and an AABD-IgCH1-TCRD module, with the TCRDs being effectively complementary (i.e., TCRα and TCRβ, or TCRγ and TCRδ). Bispecific and multispecific AABD-TCRs are also described herein and generally incorporate an (AABD)n-IgCL-TCRD module and an (AABD)n-IgCH1-TCRD module, with the TCRDs being effectively complementary (i.e., TCRα and TCRβ, or TCRγ and TCRδ). cTCRs are single-, 1.5-chain, or dual-chain receptors consisting of antigen-binding domains derived from vL and vH fragments fused to one or more TCR constant chains (TCR-C) comprising wild-type nucleic acid and amino acid sequences, resulting in activation of T cell signaling. Various configurations of cTCRs are described in PCT / US2017 / 064379, WO2018 / 102795A1 and Gross et al, Proc. Natl. Acad. Sci USA (1989) 86:10024-26. Synthetic immune receptors are next-generation CARs and are described in PCT / US2017 / 064379 or WO2018 / 102795A1.zSIR is a dual-chain receptor comprising two CD3z chains or fragments thereof with an optional linker, and is described in PCT / US2019 / 035096.

[0126] [Table 1]

[0127] Tables A1-1 to A1-9 show exemplary configurations of monospecific, bispecific, and multispecific SARs of the present disclosure. Abbreviations used: SP (signal peptide); AADB (autonomous antigen-binding domain); L (optional linker); LL (long linker), (AABD-L)n (n copies of AABD with optional linkers, n=0, 1, 2, 3, 4 or more), AABD1-4 (different AABDs targeting one or more antigens), V1 (vL, vH, Va, Vb, Vg, or Vd chain), Ig (Ig linker), ConP (connecting peptide), TM (transmembrane domain), IC (intracellular domain), Ca (constant chain of TCR alpha), Cb (constant chain of TCR beta), Cg (constant chain of TCR gamma), Cd (constant chain of TCR delta), scFv (single-chain variable region fragment), scTFv (constant chain of TCR alpha), two variable fragments, e.g., single-chain fragments comprising Va and Vb), dCa / dCb / dCg / dCd (N-terminally deleted constant chains of TCR alpha, beta, gamma, or delta lacking the Ig linker domain), TCR-ConP (binding peptide of the TCR alpha, beta, gamma, or delta constant chain), Ca-ConP (binding peptide of the TCR alpha constant chain), IgCL (Ig linker derived from an immunoglobulin light chain), IgCH1 (Ig linker derived from an immunoglobulin heavy chain), CD3εγδ ECD (extracellular domain of the CD3ε, gamma, or delta chain), CD (costimulatory domain), 4-1BB (costimulatory domain of 4-1BB), CD28 (costimulatory domain of CD28), CD3z (activation domain of CD3z).

[0128] [Table 2]

[0129] [Table 3]

[0130] [Table 4]

[0131] [Table 5]

[0132] [Table 6]

[0133] [Table 7]

[0134] [Table 8]

[0135] [Table 9]

[0136] As used herein, a "beneficial result" can include, but is not limited to, reducing or alleviating the severity of the condition, preventing the condition from worsening, curing the condition, preventing the condition from developing, reducing the patient's chance of developing the condition, and extending the patient's lifespan or life expectancy.

[0137] In another embodiment, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In another embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. A bispecific molecule may be a bispecific T cell-engaging antibody, wherein a first antigen-binding domain binds to an antigen expressed on T cells (e.g., CD3ε) and a second antigen-binding domain binds to an antigen expressed on disease-causing or disease-associated cells (e.g., cancer cells). Bispecific antibodies can be used to induce T cell-mediated cytotoxicity against cells expressing the target antigen recognized by their second antigen-binding domain. The novel antigen-binding domains described in this disclosure can be used to construct bispecific T cell engagers.

[0138] "Binds to the same epitope" refers to the ability of an antibody, scFv, or other antigen-binding domain to bind to a target antigen that has the same epitope as the exemplified antibody, scFv, or other antigen-binding domain. By way of example, the epitopes of the exemplified antibodies, scFv, or other binding agents and other antibodies can be determined using standard epitope mapping techniques. Epitope mapping techniques well known in the art include the epitope mapping protocol in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, NJ. For example, linear epitopes can be determined by simultaneously synthesizing multiple peptides on a solid support, where the peptides represent portions of protein molecules, and reacting the peptides with an antibody while still operably linked to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al. (1986) Mol. Immunol. 23: 709-715. The epitope bound by the antigen-binding domain of CAR can also be determined by epitope binning assay. Epitope binning is a competitive immunoassay used to characterize and then select a library of monoclonal antibodies against a target protein. Antibodies against similar targets are tested in pairs against all other antibodies in the library to determine whether the antibodies block each other's binding to the antigen epitope. After each antibody has a profile generated against all other antibodies in the library, a competitive blocking profile is generated for each antibody compared to the other antibodies in the library. Closely related binning profiles indicate that the antibodies have the same or closely related epitopes and are "binned" together.Similarly, conformational epitopes are easily identified by determining the spatial conformation of amino acids, for example, by hydrogen / deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. See, for example, the epitope mapping protocol described above. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using the Omiga version 1.0 software program available from Oxford Molecular Group. This computer program uses the Hopp / Woods method (Hopp et al., (1981) Proc. Natl. Acad. Sci. USA 78:3824-3828) to determine antigenic profiles, and the Kyte-Doolittle technique (Kyte et al., (1982) J. Mol. Biol. 157: 105-132) for hydropathy plots. To determine whether selected monoclonal antibodies against a target (e.g., CD19) bind to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using CD19 extracellular domain-coated ELISA plates. Biotinylated mAb binding can be detected with a streptavidin-alkaline phosphatase probe. Exemplary epitopes of the human CD20 antigen to which the scFvs, SARs, antibodies, and other immunotherapeutics of the present disclosure bind are set forth in SEQ ID NOS: 15149-15154 of patent application PCT / US18 / 53247, the entire contents of which are incorporated herein by reference. Exemplary epitopes of human BCMA to which the scFvs, SARs, antibodies, and other immunotherapeutics of the present disclosure bind are set forth in SEQ ID NOs: 15155-15159 of patent application PCT / US18 / 53247, the entire contents of which are incorporated herein by reference. Exemplary epitopes of human MPL antigen to which the scFvs, SARs, and antibodies of the present disclosure bind are set forth in SEQ ID NO: 15160 of patent application PCT / US18 / 53247, the entire contents of which are incorporated herein by reference.

[0139] As used herein, the terms "biological equivalent thereof," "variant," or "functional variant," when referring to a reference protein, antibody, or fragment thereof, polypeptide, or nucleic acid, are intended to be synonymous with "equivalent thereof," and refer to those that have minimal homology while maintaining the desired structure or function. Unless specifically stated herein, any of the above is considered to include its equivalent. For example, an equivalent is intended to be at least about 70% homologous or identical, or at least 80% homologous or identical, or at least about 85%, or at least about 90%, or at least about 95%, or even at least 98% homologous or identical, and exhibit substantially the same biological activity as the reference protein, polypeptide, antibody, or fragment thereof, or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent is a polynucleotide that hybridizes to the reference polynucleotide or its complement under stringent conditions. Alternatively, when referring to a polypeptide or protein, the equivalent is a polypeptide or protein expressed from a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding the reference polypeptide or protein, or its complement.

[0140] As used herein, the term "bispecific" refers to an agent (e.g., an antibody, antibody fragment, SAR, CAR, etc.) that can bind to two antigens.

[0141] As used herein, the term "biparatopic" refers to an agent (e.g., an antibody, antibody fragment, SAR, CAR, etc.) that can bind to two epitopes of a single antigen.

[0142] As used herein, the term "multispecific" refers to an agent (e.g., an antibody, antibody fragment, SAR, CAR, etc.) that can bind to three or more antigens.

[0143] 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 specific regions are described by Kabat et al., J. Bioi. Chern. 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. Bioi. 196:901-917 (1987); and MacCallum et al., J. Mol. Bioi. 25 262:732-745 (1996), and these definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. As used herein, different CDRs of an antibody can also be defined by a combination of different definitions.For example, VHCDR1 can be defined according to Kabat, and VHCDR2 can be defined according to Chothia.The amino acid residues encompassing the CDRs as defined by each of the above cited documents are as follows:

[0144] [Table 10]

[0145] The sequence numbers of the CDRs of the different vL and vH segments that may comprise the antigen-binding domains of the scFvs, CARs, AMRs, antibodies, and other immunotherapeutics of the present disclosure are set forth in SEQ ID NOS: 13204-14121 and 14122-15039, respectively, of PCT / US18 / 53247 (Table 6A, B) and Tables 5-6 of PCT / US2017 / 064379, which are incorporated herein by reference.

[0146] In some embodiments, when referring to an antigen binding module that specifically binds to a target antigen (e.g., a Fab-like or Fv-like antigen binding module), it refers to an antigen binding module that binds to the target antigen (a) with an affinity that is at least about 10-fold (e.g., about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more times) greater than its binding affinity for other molecules; or (b) with an affinity that is less than its K d At most about 1 / 10 (e.g., 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1175, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 750, 1 / 1000 or less) of K d Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, Malibu-Glo assay, Topanga assay, or radioimmunoprecipitation assay (RIA). d can be determined by methods known in the art, such as, for example, surface plasmon resonance (SPR) assays using a Biacore instrument, or equilibrium exclusion assays (KinExA) using, for example, a Sapidyne instrument.

[0147] "Cancer" and "cancerous" refer to or describe a physiological condition in mammals that is generally characterized by unregulated cell proliferation.Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and / or non-Hodgkin's lymphoma), T-cell lymphoma, myeloma, myelodysplastic syndrome, myeloproliferative disorder (for example, polycythemia vera, myelofibrosis, essential thrombocythemia, etc.), skin cancer, brain cancer, breast cancer, colon cancer, rectal cancer, esophageal cancer, anal cancer, cancer of unknown primary site, endocrine cancer, testicular cancer, lung cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, cancer of reproductive organs, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer (for example, glioblastoma multiforme), prostate cancer, including, but not limited to, androgen-dependent prostate cancer and androgen-independent prostate cancer, and leukemia. Other cancers and cell proliferative disorders will be readily recognized in the art. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms include solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors. The term "cancer" is meant to include any type of cancerous growth or oncogenic process, metastatic tissue, or malignantly transformed cell, tissue, or organ, regardless of histological type or invasive stage.

[0148] "Cell therapy" or "cell-based therapy" or "immune cell therapy" or "immune effector cell therapy" or "adoptive cell therapy" refers to therapy involving the use of cells for the prevention or treatment of disease. Non-limiting examples of cell therapy include CAR-T cell therapy, NK cell therapy, recombinant TCR-T cell therapy, and TIL (tumor-infiltrating lymphocytes). Biological agents such as antibodies (e.g., bispecific T cell engagers and DARTs) that mediate their effects by binding to and / or activating immune cells (e.g., T cells and NK cells) are other examples of cell therapy. Organ transplants and bone marrow transplants, including stem cells and autologous and allogeneic blood, are also examples of cell therapy.

[0149] The term "sentilin," as used herein, refers to a small, engineered protein derived from the human protein tenascin-C that can bind to a target antigen with high affinity and specificity. Sentilin has a highly stable fibronectin type III (FN3) domain. Exemplary centirins are shown in Table 7.

[0150] A "chemotherapeutic agent" is a compound known for use in cancer chemotherapy. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and Cytoxan® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; camptothecin (including the synthetic analog topotecan); bryostatin; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, and melphalan. nitrogen mustards such as novembichin, fenesterine, prednimustine, trophosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics such as calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxol bicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate and 5 -Antimetabolites such as fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; Antiadrenergic drugs such as noglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfomitine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol;Nitraerin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as Taxol® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Abraxane® Cremophor Free, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, Ill.), and Taxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)), and VEGF-A that reduce cell proliferation, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above, or combinations thereof.

[0151] A "chimeric antigen receptor" (CAR) is an artificial (non-naturally occurring) immune cell (e.g., T cell) receptor intended for use as a cancer therapy using a technique called adoptive cell transfer. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs are specifically engineered to stimulate T cell activation and proliferation in response to a specific antigen to which the CAR binds. Generally, a CAR refers to a set of polypeptides, typically two in the simplest embodiment, that, when expressed in an immune effector cell, provide that cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. In some embodiments, a CAR comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some aspects, the set of polypeptides is contiguous with one another. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-lBB (i.e., CD137), CD27, and / or CD28. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. In various embodiments, the CAR is a recombinant polypeptide comprising an antigen-specific domain (ASD), a hinge region (HR), a transmembrane domain (TMD), an optional costimulatory domain (CSD), and an intracellular signaling domain (ISD). The optional costimulatory domain is generally not present in first-generation CAR constructs.The nucleic acid sequences of several exemplary second-generation CARs comprising different antigen-binding domains (e.g., vL and vH fragments, vHH, ligand and receptor, etc.) and incorporating a 41BB costimulatory domain are set forth in SEQ ID NOs: 1455-1703 (Table 8) of PCT / US2020 / 014237. The corresponding amino acid sequences are set forth in SEQ ID NOs: 7341-7589 of PCT / US2020 / 014237. In certain embodiments, the present disclosure provides bispecific, biparatopic, and multispecific CARs.

[0152] The term SAR, as used herein, comprises CARs and also encompasses newer approaches to confer antigen specificity to cells, such as antibody-TCR chimeric molecules or Ab-TCRs (WO2017 / 070608A1, incorporated herein by reference), TCR receptor fusion proteins or TFPs (WO2016 / 187349A1, incorporated herein by reference), synthetic immunoreceptors (SIRs) (see WO2018 / 102795A1, incorporated herein by reference), trifunctional T cell antigen couplers (Tri-TACs or TACs) (see WO2015 / 117229A1, incorporated herein by reference), and zSIRs (see PCT / US2019 / 035096, incorporated herein by reference). The nucleic acid sequences of several exemplary TFPs comprising different antigen-binding domains (e.g., vL and vH fragments, vHH, ligands and receptors, etc.) based on the CD3ε, CD3δ, CD3γ, and CD3ζ chains and co-expressing the optional accessory module NEMO-K277A are set forth in SEQ ID NOs: 1900-2205, 2206-2511, 2512-2817, and 2818-3123, respectively, of PCT / US18 / 53247, the entire contents of which are incorporated herein by reference (Table 13). The order of antigen-binding domains included in the various CAR structural constructs and BiTEs listed in Table 13 of PCT / US18 / 53247, the entire contents of which are incorporated herein by reference, is the same as the order of the zCAR-K277A structural constructs shown in Table 12 of PCT / US18 / 53247, the entire contents of which are incorporated herein by reference. The term "SAR-T cells" is generally used to refer to T cells engineered to express a synthetic antigen receptor. Thus, T lymphocytes bearing such SARs are generally called SAR-T lymphocytes. If this SAR is a CAR, the T cells are called CAR-T cells. SARs can also be expressed in cells other than T cells, such as hematopoietic stem cells, induced pluripotent stem cells (iPSCs), NK cells, and macrophages.The present disclosure provides bispecific, biparatope, and multispecific SARs having scaffolds, such as CARs, SIRs, zSIRs, cTCRs, Ab-TCRs, AABD-TCRs, TFPs, and TCRs, comprising one or more AABDs. The AABD domains of the SARs in any of the above scaffolds can be fully human, humanized, or non-human. In some embodiments, the present disclosure provides SARs in any of the above scaffolds comprising one or more fully human vH domains. In some embodiments, the present disclosure provides SARs in any of the above scaffolds comprising one or more fully human vL domains.

[0153] "Codon optimization" or "control of species codon bias" refers to the preferred codon usage of a particular host cell. As one of skill in the art will appreciate, it may be advantageous to modify a coding sequence to enhance its expression in a particular host.

[0154] As used herein, "coexpression" refers to the expression of two or more polynucleotides or genes. A gene can be, for example, a nucleic acid encoding a single protein or a chimeric protein as a single polypeptide chain. The SAR or TCR described herein can be encoded by a single polynucleotide chain and expressed as a single polypeptide chain, which is then cleaved into different polypeptides, each representing a separate functional unit. In some embodiments, when a SAR or TCR consists of two or more functional polypeptide units, the different functional units are coexpressed using one or more polynucleotide chains. In one embodiment, costimulation is provided by an accessory module that is coexpressed with the SAR or TCR but is not an essential part of the SAR or TCR polypeptide. In another embodiment, these different polynucleotide chains are linked by a nucleic acid sequence encoding a cleavable linker (e.g., T2A, F2A, P2A, E2A, etc.) (Table 20). In another embodiment, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NOs: 1239 and 11929) is also added upstream of the cleavable linker sequence to improve cleavage efficiency. Polynucleotides encoding different units of SAR or TCR may be linked by an IRES (internal ribosome entry site) sequence. Alternatively, different functional units of SAR or TCR are not linked via a linker, but instead are encoded by two different polynucleotides, for example, encoded by two different vectors. The nucleic acid and amino acid sequences of exemplary cleavable linkers and furin cleavage sites are shown in Table 20.

[0155] "Conservative substitution" or "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics or function of the encoded protein. For example, "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics or function of the SAR construct of the present disclosure (e.g., conservative changes in a constant chain, antibody, antibody fragment, or non-immunoglobulin binding domain). Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Series of amino acid residues having similar side chains are defined in the art. These series include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a SAR of the present disclosure can be substituted with other amino acid residues from the same side chain series, and the altered SARs can be tested using the binding and / or functional assays described herein.

[0156] The term "TCR constant chain" or "constant region of a T cell receptor" is defined as the constant chains of TCRα / TCRa, TCRβ1 / TCRb1, TCRβ2 / TCRb2, TCRγ / TCRd, TCRδ / TCRd, and pre-TCRα. Exemplary TCR constant chains are listed in Table 12. TCR constant chains can be divided into several subdomains, such as the Ig-like C1 domain (e.g., SEQ ID NOS: 11848-11865; Table 13), the binding peptide (e.g., SEQ ID NOS: 11867-11875; Table 14), the transmembrane domain (SEQ ID NOS: 11877-11880; Table 15), and the cytosolic domain (e.g., SEQ ID NOS: 11883-11885; Table 16). The cytosolic domains of the TCRα, TCRβ1 / β2, TCRγ, and TCRδ chains are short and generally not thought to play a significant role in their signaling activity. The present disclosure also provides deletion mutants and variants of the TCR chains listed in Table 12, so long as they retain one or more functional and biological properties of the TCR chain, such as the ability to pair with a complementary TCR chain, the ability to assemble into a TCR / CD3 complex, and the ability to transduce a T cell signal (e.g., activate the NFAT pathway) upon engagement of a target antigen-expressing cell.

[0157] The term "T cell receptor-α constant region" or "T cell receptor-α constant chain" or "TCRα" or "Cα" is defined as the protein set forth in SEQ ID NO: 11735 or 11733 or equivalent residues (i.e., homologs or variants) from non-human species, e.g., mouse, rodent, monkey, ape, etc. The term also includes any deletion or point mutants and variants of TCRα that retain the biological and functional properties of TCRα, such as the ability to pair with a complementary TCRβ chain, assemble into a TCR / CD3 complex, and transduce T cell signals (e.g., activate the NFAT pathway) upon engagement of target antigen-expressing cells. The present disclosure also provides specific mutations to the TCRα polypeptide that can be used in constructing SIRs and Ab-TCRs (Table 12). For example, positions of Cα mutations that show enhanced expression and reduced mispairing are positions 91, 92, 93, and 94 of SEQ ID NO: 11735. TCR polypeptides (described in more detail elsewhere herein) with a Thr 48 Cys (T48C) mutation in Cα and a Ser-57-Cys (S57C) mutation in the Cβ1 or Cβ2 chain result in an additional disulfide bond between the two TCR constant chains (α and β). This, in turn, results in reduced mispairing with endogenous TCR chains and increased functionality in immune cells. Similarly, SIRs (described in more detail elsewhere herein) with a Ser 61 Arg (S61R) mutation in Cα and an Arg 79 Gly (R79G) mutation in the Cβ1 or Cβ2 chain result in reduced mispairing with endogenous TCR chains and increased functionality due to a "knob-and-hole" design for pairing. The present disclosure provides Cα polypeptides with one or more or all of the mutations shown in Tables A-8 and 12 below that can be used in the construction of SIRs and Ab-TCRs.

[0158] [Table 11]

[0159] The human genome encodes two highly homologous TCR β constant chains: TCR β1 (TCR β1 or TCR b1 or cβ1) and TCR β2 (TCR β2 or TCR b2 or cβ2). A SAR (e.g., SIR, Ab-TCR, or TFP) of the present disclosure can comprise either of these two chains. Similarly, either the TCR β1 or TCR β2 chains of other mammalian species can be used in the methods of the present disclosure.

[0160] The term "constant chain of T cell receptor-β1" or "constant region of T cell receptor-β1" (TCR-β1 or TCRβ1 or TCRb1 or hTCR-β1 or Cβ1) is defined as the protein set forth in SEQ ID NO: 11746 or equivalent residues (i.e., homologs) from a non-human species, e.g., mouse, rodent, monkey, ape, etc. The term also includes any deletion or point mutants and variants of TCRβ1 that retain the biological and functional properties of TCRβ1, such as the ability to pair with a complementary TCRα chain, assemble into a TCR / CD3 complex, and transduce T cell signals (e.g., activate the NFAT pathway) upon engagement of a target antigen-expressing cell. The present disclosure also provides specific mutations to the TCRβ1 polypeptide that can be used in constructing SARs (e.g., SIRs and Ab-TCRs) (Table 12). The present disclosure also provides several deletion mutants of TCRβ1 that can be used in constructing SARs (Table 12).

[0161] The term "constant chain of T cell receptor-β2" or "constant region of T cell receptor-β2" (TCR-β2 or TCRβ2 or TCRb2 or Cβ2) is defined as the protein set forth in SEQ ID NO: 11747 or equivalent residues (i.e., homologs) from non-human species, e.g., mouse, rodent, monkey, ape, etc. The term also includes any deletion or point mutants and variants of TCRβ2 that retain the biological and functional properties of TCRβ2, such as the ability to pair with a complementary TCRα chain, assemble into a TCR / CD3 complex, and transduce T cell signals (e.g., activate the NFAT pathway) upon engagement of target antigen-expressing cells. The present disclosure also provides specific mutations to the TCRβ2 polypeptide that can be used in constructing SARs (e.g., SIRs and Ab-TCRs) (Table 12). The present disclosure also provides several deletion mutants of TCRβ2 that can be used in constructing SARs (Table 12).

[0162] The protein sequences of both Cβ1 (SEQ ID NO: 11746) and Cβ2 (SEQ ID NO: 11747) are known (Table 6). Differences between the sequences of Cβ2 and Cβ1 are readily identified by sequence alignment using techniques typical and routine in the art. In general, the Cβ1 and Cβ2 chains are highly homologous. Thus, unless otherwise specified, the term TCRβ, TCR-β, or TCRb constant chain refers to either the TCRβ1 or TCRβ2 constant chain. Similarly, unless otherwise specified, the term Cβ applies to either Cβ1 or Cβ2. The present disclosure also provides specific mutations to the TCRβ chain that can be used in constructing SIRs and Ab-TCRs. For example, provided herein are Cβ mutation sites that exhibit enhanced expression and mispairing with the endogenous TCRα chain. These mutation sites in Cβ1 and Cβ2 are located at positions 18, 22, 57, 79, 133, 136, and 139 of SEQ ID NOs: 11746 and 11747, and are summarized in Tables A-9 and A10 below. The mutation sites in Cβ1 and Cβ2 are identical at these positions. The only difference between these two sequences is the mutation at position 136, where glutamic acid (E) is present in Cβ2 and valine is present in Cβ1.

[0163] [Table 12]

[0164] [Table 13]

[0165] The term "constant chain of TCR-γ" or "constant region of TCR-γ" (TCR-γ or TCRγ or TCRg or TCR-γ1 or TCRγ1 or TCRg1 or Cγ) is defined as the protein set forth as SEQ ID NO: 11771 or equivalent residues (i.e., homologs) from a non-human species, e.g., mouse, rodent, monkey, ape, etc. The term also includes any deletion or point mutants and variants of TCRγ that retain the biological and functional properties of TCRγ, such as the ability to pair with a complementary TCR (e.g., TCRδ) chain, assemble into a TCR / CD3 complex, and transduce T cell signals (e.g., activate the NFAT pathway) upon engagement of a target antigen-expressing cell. The present disclosure also provides specific mutations to the TCRγ polypeptide that can be used in the construction of SARs (e.g., SIRs and Ab-TCRs) (Table 12). The present disclosure also provides several deletion mutants of TCRβ that can be used in constructing SARs (Table 12).

[0166] The term "constant chain of TCR-δ" or "constant region of TCR-δ" (TCR-δ or TCRδ or TCRd or Cδ) is defined as the protein set forth in SEQ ID NO: 5982 or equivalent residues (i.e., homologs) from a non-human species, e.g., mouse, rodent, monkey, ape, etc. The term also includes any deletion or point mutants and variants of TCRδ that retain the biological and functional properties of TCRδ, such as the ability to pair with a complementary TCR (e.g., TCRγ) chain upon engagement of a target antigen-expressing cell, the ability to assemble into a TCR / CD3 complex, and the ability to transduce a T cell signal (e.g., activate the NFAT pathway). The present disclosure also provides specific mutations to the TCRδ polypeptide that can be used in constructing SARs (e.g., SIRs and Ab-TCRs) (Table 12). The present disclosure also provides several deletion mutants of TCRβ that can be used in constructing SARs (Table 12).

[0167] The term "constant chain of pretc.R-α" or "constant region of pretc.R" (pretc.Rα or pretc.R-α or preCα) is defined as the protein set forth in SEQ ID NO: 11769 or equivalent residues (i.e., homologs) from non-human species, such as mouse, rodent, monkey, ape, etc. The term also includes any deletion or point mutants and variants of pretc.Rα that retain the biological and functional properties of pretc.Rα, such as the ability to pair with a complementary TCR (e.g., TCRβ) chain, assemble into a TCR / CD3 complex, and transduce T cell signals (e.g., activate the NFAT pathway) upon engagement of target antigen-expressing cells. The present disclosure also provides specific mutations to the pretc.Rα polypeptide that can be used in the construction of SARs (e.g., SIRs and Ab-TCRs) (Table 12). The present disclosure also provides several deletion mutants of pretc.Rα that can be used in the construction of SARs (Table 12).

[0168] It will be appreciated that proteins may have identity or homology to one another and retain similar or identical function. The present disclosure includes TCR constant regions having 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.

[0169] Thus, the present disclosure provides an amino acid sequence that is at least 70% identical to any of SEQ ID NOs: 11733-11742, 11744, 11745, 11793-11803, and optionally has one or more mutations at positions 61, 91, 92, 93, 94, 120, 127, and / or 129; or (b) an amino acid sequence that is at least 75% identical to any of SEQ ID NOs: 11746-11766, 11804-11817, and optionally has one or more mutations at positions 18, 22, 57, 79, 133, 136, and / or 139. (c) an amino acid sequence that may have one or more mutations in any of SEQ ID NOs: 11818 to 11823; (d) an amino acid sequence that is at least 70% identical to any of SEQ ID NOs: 11824 to 11830; and (e) an amino acid sequence that is at least 70% identical to any of SEQ ID NOs: 11769 to 11770. The T cell receptor constant chain of any of (a) to (d) retains at least one biological activity of the wild-type T cell receptor constant chain to which it has identity or homology.

[0170] In one embodiment, the present disclosure provides a modified TCR selected from the group consisting of a wild-type TCR, a high-affinity TCR, and a chimeric TCR. In another embodiment, the modified TCR comprises at least one additional disulfide bond. In yet another embodiment, the modified TCR comprises a TCR α chain and a TCR β chain.

[0171] The term "constitutively active" refers to a molecule, e.g., a protein, that has signaling activity without the need for stimulation. Exemplary constitutively active proteins are NEMO-K277A and vFLIP K13, because they can activate NF-κB signaling when expressed in suitable cells without the need for added stimulation.

[0172] The term "costimulatory molecule" or "costimulatory receptor" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory extracellular molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD28, 2B4, and 4-1BB (CD137). Costimulatory receptors can be expressed on cells other than T cells, such as NK cells or macrophages.

[0173] A "costimulatory intracellular signaling domain" or "costimulatory domain" (CSD) can be the intracellular portion of a costimulatory receptor. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, 2B4, CD40, ICOS, and the like. The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived, or a native intracellular signaling domain, or a functional fragment or derivative thereof. The SAR of the present disclosure can comprise one or more costimulatory domains.

[0174] The term "cTCR" refers to a wild-type TCR protein linked to a wild-type TCR nucleic acid coding sequence and an antigen-binding domain not derived from a TCR. The TCR chains of a cTCR are not codon-optimized and lack mutations that may enhance their expression or reduce pairing with endogenous TCR chains. In exemplary embodiments, the antigen-binding domain of a cTCR may comprise a vL, vH, or scFv fragment. cTCRs are described in (Gross, Waks & Eshhar, Proc. Natl. Acad. Sci. USA, 1989). In some embodiments, a cTCR is used as a reference control. For example, a cTCR having a CD19-binding domain and a CD19-SIR (comprising a mutant TCR chain or a codon-optimized TCR chain and a CD19-binding domain) has different expression and / or different binding affinity to a target antigen. The present disclosure provides bispecific, biparatopic, and multispecific SARs having a cTCR scaffold comprising one or more AABDs. The AABD domain of the SAR of the present disclosure having a cTCR framework can be fully human, humanized, or non-human. In some embodiments, the present disclosure provides a cTCR comprising one or more fully human vH domains. In some embodiments, the present disclosure provides a cTCR comprising one or more fully human vL domains.

[0175] The term "cytosolic" or "cytoplasmic" refers to an agent, e.g., a protein, that exists in its mature form in the cytosol of a cell. A cytosolic protein may translocate to the nucleus, but is not a transmembrane protein and is not secreted outside the cell. Exemplary cytosolic proteins are MC159 and K13.

[0176] Cytokine release syndrome (CRS) is a complication of cell-based therapies (e.g., SAR-T, bispecific T-cell engaging antibodies, etc.) that manifests with signs and symptoms such as fever, hypotension, shortness of breath, renal dysfunction, pulmonary dysfunction, and / or capillary leak syndrome.

[0177] DARPINs or DARPins (designed ankyrin repeat proteins) are generally engineered antibody mimetic proteins that generally exhibit high specificity and high affinity target protein binding. Exemplary DARPINs are shown in Table 7.

[0178] The term "degenerative disorder" refers to a disease that is based on degenerative cellular changes and is the result of an ongoing process affecting tissues or organs, which becomes increasingly worse over time, whether due to normal physical wear or lifestyle choices such as exercise or diet. Exemplary degenerative diseases include Alzheimer's disease, Creutzfeldt-Jakob disease, diabetes mellitus (type 2), and atherosclerosis.

[0179] As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. This term generally refers to the structural similarity between the first molecule and the second molecule and does not imply or imply any limitations on the process or source by which the first molecule is derived from the second molecule. For example, in the case of an antigen-binding domain derived from an antibody molecule, the antigen-binding domain retains sufficient antibody structure so that it has the required function, i.e., the ability to bind to an antigen. This does not imply or imply any limitations on the particular process by which the antibody is produced, and does not mean, for example, that one must start with an antibody sequence and delete unnecessary sequences or impose mutations to arrive at an antigen-binding domain.

[0180] A "dimerizing molecule," as the term is used herein, refers to a molecule that promotes the association of a first switch domain with a second switch domain.

[0181] "Diseases associated with expression of a target antigen" or "disease-associated antigen as described herein" include, but are not limited to, diseases associated with expression of a target antigen as described herein or conditions associated with cells expressing a target antigen as described herein, including, but not limited to, proliferative diseases, e.g., cancers or malignant or precancerous conditions, e.g., myelodysplasia, myelodysplastic syndromes, or myeloproliferative disorders or preleukemias; or non-cancer-related indications associated with cells expressing a target antigen as described herein. In one aspect, a cancer associated with expression of a tumor antigen as described herein is a hematological cancer. In one aspect, a cancer associated with expression of a tumor antigen as described herein is a solid cancer. Additional diseases associated with expression of a tumor antigen as described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer-related indications associated with expression of a target antigen as described herein include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the target antigen-expressing cells express, or have expressed at any time, mRNA encoding the target antigen. In other embodiments, the target antigen-expressing cells produce the target antigen protein (e.g., wild-type or mutant), and the target antigen protein may be present at normal or low levels. In other embodiments, the target antigen-expressing cells produce detectable levels of the target antigen protein at one time, and then produce substantially no detectable target antigen protein.

[0182] As used herein, "diseases targeted by genetically modified cells" encompasses the targeting of any cells involved in any disease in any manner by the genetically modified cells of the present disclosure, regardless of whether the genetically modified cells target diseased or healthy cells to produce a therapeutically beneficial result. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells, induced pluripotent stem cells (iPSCs), or embryonic stem cells. The genetically modified cells express conventional SARs and novel scaffolds containing conventional SARs with the accessory modules of the present disclosure, and these SARs can target any antigen expressed on the surface of the target cells. Examples of targetable antigens include, but are not limited to, antigens expressed on B cells; antigens expressed on carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas; antigens expressed on various immune cells; and antigens expressed on cells associated with various blood, autoimmune, and / or inflammatory diseases. Other targetable antigens will be apparent to those skilled in the art and may be targeted by the SAR of the present disclosure in connection with its alternative embodiments.

[0183] The term "dissociation constant (Kd)" is defined as the equilibrium constant for dissociation of a receptor-ligand (eg, binding domain-cognate) interaction.

[0184] As used herein, a "diverse non-naturally occurring immunoreceptor set" or a "diverse SAR set" or a "diverse SAR set" refers to a diverse set of signaling chains, or multiple non-naturally occurring immunoreceptors having the same or different binding domains linked to binding domains and "scaffolds," where each construct comprising a different signaling chain or "scaffold" provides a diverse range of binding to a target antigen and / or various expression levels. For example, depending on the mutation composition of the constant domain (e.g., mutant TCRa+TCRb), the binding affinity of the binding domain to its target varies. In some embodiments, bispecific and / or multispecific SARs having an SIR, Ab-TCR, or TFP scaffold of the present disclosure comprise a binding affinity lower than that of a second-generation CAR, but higher than that of a wild-type TCR (e.g., cTCR) having the same binding domain.

[0185] As used herein, an "epitope" is defined as a portion of an antigen that can elicit an immune response or that binds to an antibody or antibody fragment. An epitope can be a protein sequence or subsequence.

[0186] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or liposome-containing) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0187] As used in the art, "Fc receptor" and "FcR" refer to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR.

[0188] The term "functional portion," when used in reference to a SAR, refers to any portion or fragment of a SAR that retains the biological activity of the SAR of which it is a part (the parent SAR). A functional portion includes, for example, a SAR portion that retains the ability to recognize a target cell or detect, treat, or prevent a disease to a similar, identical, or 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.

[0189] "Genetically modified cells," "redirected cells," "genetically engineered cells," or "modified cells," as used herein, refer to cells that express the SARs of the present disclosure. In some embodiments, genetically modified cells comprise vectors encoding the SARs. In some embodiments, genetically modified cells comprise vectors encoding the SARs and one or more accessory molecules (e.g., PDL1, PDL2, crmA, MC159, etc.).

[0190] As used herein, "hinge region" (HR) refers to the hydrophilic region located between the antigen-binding domain and the transmembrane domain of a SAR. Hinge regions include, but are not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, a CD8a hinge, and an artificial spacer composed of a polypeptide that may be small, for example, Gly3 or similar to the CH1 and CH3 domains of IgG (e.g., human IgG4). Exemplary HRs are shown in Table 17 (SEQ ID NOs: 1198-1204).

[0191] "Immune cell," as used herein, refers to a cell of the mammalian immune system, including, but not limited to, antigen-presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells, and T cells.

[0192] The term "immune disorder" refers to a disease characterized by a malfunction of the immune system. Autoimmune diseases are conditions that result from an abnormal immune response against normal body parts. There are at least 80 types of autoimmune diseases.

[0193] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., 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, and bone marrow-derived phagocytes.

[0194] "Immune effector function" or "immune effector response," "effector function" refers to the specialized functions of various cells. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. For example, an immune effector function or response refers to a property of a T or NK cell that promotes killing of a target cell or inhibiting its growth or proliferation. In the case of a T cell, primary stimulation and costimulation are examples of immune effector functions or responses. In the case of an antigen-presenting cell (e.g., a dendritic cell), antigen presentation and cytokine secretion are examples of effector functions.

[0195] "Immune response," as used herein, refers to immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunological, inflammatory response, acquired (adaptive) immunity, autoimmunity, and / or hyperactive immunity.

[0196] "Intracellular signaling domain" (ISD) or "cytoplasmic domain" or "primary intracellular signaling domain" or "activation domain," as the term is used herein, refers to the intracellular signaling portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of the cell. Examples of immune effector function include cytolytic activity and helper activity, including cytokine secretion. The primary intracellular signaling domain may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), Fcγ RIIa, FcRβ (Fcε R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0197] The term "isolated," as used herein, refers to cellular material that is substantially free of molecules or biological agents or other materials. In one aspect, the term "isolated" refers to a nucleic acid, such as DNA or RNA, or a protein or polypeptide (e.g., an antibody or derivative thereof), or a cell or cell organelle, or a tissue or organ, that has been separated from other DNA or RNA, or proteins or polypeptides, or cells or cell organelles, or tissues or organs, respectively, that are present in their natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides that are isolated from other cellular proteins and is meant to include both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to include both cultured and engineered cells or tissues.

[0198] As used herein, the term "linker" (also "linker domain" or "linker region") refers to an oligo or polypeptide (or an oligo encoding a polypeptide) that interconnects two or more domains or regions of a SAR polynucleotide or polypeptide, respectively, disclosed herein. Linkers can be 1 to 500 amino acids in length or 3 to 1500 nucleotides in length. In certain embodiments, the linker is one or more amino acids in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 450, 500 amino acids and any number in between). In some embodiments, the linker is 1 to 100, 1 to 125, 1 to 150, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, or 3 to 500 amino acids in length. In some embodiments, the SAR of the present disclosure may comprise one or more linkers (e.g., 2, 3, 4 or more).

[0199] A "long linker" or "long linker domain" is a linker between 25 and 500 amino acids in length. In certain embodiments, a long linker is approximately 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 450, 500 amino acids in length, and any number in between. In certain embodiments, a long linker is 25 to 125 amino acids in length. In certain embodiments, a long linker is 50 to 150 amino acids in length. In some embodiments, the long linker is 75 to 175 amino acids in length. In some embodiments, the long linker is 100 to 200 amino acids in length. In some embodiments, the long linker is 120 to 220 amino acids in length. In some embodiments, the long linker is 100 to 300 amino acids in length.

[0200] In certain embodiments, the linker encodes or comprises an immunoglobulin (Ig) domain or an Ig-like domain, or a fragment thereof. The terms "Ig domain," "Ig linker domain," "Ig-like domain," or "Ig-like linker domain" are used interchangeably in this disclosure. An immunoglobulin domain is a type of protein domain consisting of a two-layer sandwich of seven to nine antiparallel β-strands arranged in two β-sheets with a Greek key topology, consisting of approximately 125 amino acids. Ig domains can be classified as IgV, IgC1, IgC2, or IgI. IgV domains, with nine β-strands, are generally longer than IgC domains, with seven β-strands. In certain embodiments, the linker comprises an IgV domain or a fragment thereof. In certain embodiments, the linker comprises an IgC domain or a fragment thereof. Ig domains are found in immunoglobulins, T cell receptor chains, class I MHC, class II MHC, β2-microglobulin, co-receptors (e.g., CD4, CD8, CD19, etc.), antigen receptor accessory molecules (e.g., CD3γ, CD3δ, CD3ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), NK cell receptors (e.g., KIR), leukocyte immunoglobulin-like receptors (LILR), IgSF CAMs (e.g., NCAM, ICAM, CD2, etc.), cytokine receptors (e.g., IL-1R, CSF-1R, etc.), growth factor receptors (e.g., PDGFR), receptor tyrosine kinases and phosphatases, Ig-binding receptors, cytoskeletal proteins (e.g., titin, palladin, etc.), and other proteins (e.g., CD147, CD90, etc.). Exemplary Ig linker domains are IgCL (SEQ ID NO: 1142) and IgG1-CH1 (SEQ ID NO: 1143). Additional exemplary Ig linkers are shown in Table 13 (SEQ ID NOs: (PRT): 11832-11865). In certain embodiments, the linker has an E-set domain.E-set domains are a family of "early" Ig-like folds that may be related to the immunoglobulin and / or type III fibronectin superfamilies. In one embodiment, the linker comprises a type III fibronectin domain.

[0201] The linker may be a flexible linker. The term "flexible polypeptide linker," as used herein, refers to a peptide linker consisting of or consisting mostly of amino acids such as glycine and / or serine residues, used alone or in combination to link polypeptide chains together (e.g., heavy and light chain variable regions together). In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, having the amino acid sequence (Gly-Gly-Gly-Ser). n (e.g., SEQ ID NO: 11715), where n is a positive integer greater than or equal to 1. For example, n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3.

[0202] In some embodiments, a "linker" is cleavable or non-cleavable. Unless otherwise specified, the term "linker" as used herein refers to a non-cleavable linker. The non-cleavable linker may be composed of flexible residues that allow free movement of adjacent protein domains. Non-limiting examples of such residues include glycine and serine. In some embodiments, the linker may be a non-flexible residue. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents and combinations thereof. In some embodiments, linkers include picornavirus 2A-like linkers, porcine teschovirus (P2A), the CHYSEL sequence of Thosea asigna virus (T2A), or combinations, variants, and functional equivalents thereof. In some embodiments, the linker sequence may comprise a motif that results in cleavage between 2A glycine and 2B proline (see, e.g., the T2A sequence). The nucleic acid sequences of several exemplary cleavable linkers are set forth in SEQ ID NOs: 1233 to 1238, and the amino acid sequences of several exemplary linkers are set forth in SEQ ID NOs: 11923 to 11928. Other cleavable linkers that can be used herein will be readily recognized by those of skill in the art.

[0203] In some embodiments, a Ser-Gly-Ser-Gly (SGSG) motif (SEQ ID NO: 11929) is also added upstream of the cleavable linker sequence to improve cleavage efficiency. A potential drawback of cleavable linkers is that a small 2A tag at the end of the N-terminal protein may affect protein function or contribute to the antigenicity of the protein. To overcome this limitation, in some embodiments, a furin cleavage site (RAKR) (SEQ ID NO: 11931) is added upstream of the SGSG motif to facilitate cleavage of the remaining 2A peptide after translation.

[0204] The linker can be a protease-cleavable linker. Exemplary protease-cleavable linkers are shown in Table 19.

[0205] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, and therefore, lentiviruses are one of the most effective gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0206] The term "lentiviral vector" refers specifically to vectors derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used in clinics include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX™ vector system from Lentigen. Non-clinical lentiviral vectors are also available and known to those skilled in the art. Other examples of lentiviral vectors include pLENTI-EF1α (SEQ ID NO: 1), pLENTI-EF1α-DWPRE (SEQ ID NO: 2), pCCLc-MNDU3-WPRE (SEQ ID NO: 4), and pCCLc-MNDU3-Eco-Nhe-Sal-WPRE (SEQ ID NO: 5). In an exemplary embodiment, a SAR, or a SAR and an accessory module, or a nucleic acid fragment encoding an accessory module, can be cloned between the Nhe I and Sal I sites present in the pLENTI-EF1α and pCCLc-MNDU3-Eco-Nhe-Sal-WPRE vectors using methods known in the art.

[0207] "Mammal," as used herein, refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees and other ape and monkey species; farm animals, e.g., cattle, sheep, pigs, goats, and horses; domestic mammals, e.g., dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs;

[0208] "Native" or "naturally occurring" or "endogenous," as used herein, 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 alpha chain polypeptide of a T cell consists of a variable domain (Vα) connected to a TCR alpha constant chain. A native or endogenous TCR alpha chain precursor polypeptide also consists of an amino-terminal signal peptide that is cleaved from the mature polypeptide.

[0209] The term "NIK agonist," as used herein, refers to an agent that activates the activity of NF-κB-inducing kinase. In certain embodiments, the NIK agonist is a SMAC mimetic.

[0210] The term "SMAC mimetic," as used herein, refers to an agent that mimics the activation of SMAC / DIABLO proteins.

[0211] The term "near the N-terminus," as used herein, means within the N-terminal 30 amino acids. For example, the term "AABD operably linked at or near the N-terminus of a vL and / or vH domain" means an AABD operably linked to the N-terminus of a vL or vH fragment, or within 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, the term "AABD operably linked to at or near the N-terminus of a Va and / or Vb domain" means an AABD operably linked to the N-terminus of a Va or Vb fragment, or 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 at the N-terminus comprising a Va or Vb domain. An AABD of the disclosure can be operably linked to at or near the N-terminus of another domain, either directly or by an intervening linker sequence.

[0212] As used herein, "non-naturally occurring factor" or "non-natural" or "exogenous" refers to a factor that is not naturally expressed in a cell. In other words, a non-naturally occurring factor is "engineered" to be expressed in a cell. A non-naturally occurring factor can be a cloned version of a naturally occurring factor. Exemplary non-naturally occurring factors include SARs (e.g., CARs, SIRs, Ab-TCRs, TFPs, recombinant TCRs, NEMO-K277A, vFLIP-K13, and K13-opt). A non-naturally occurring factor can be expressed in a cell using gene transfer techniques known in the art, such as lentivirus- or retrovirus-mediated gene transfer. A non-naturally occurring factor can be expressed in an immune cell using an exogenous promoter (e.g., EF1α promoter) or an endogenous promoter (e.g., TCRα promoter).

[0213] As used herein, a "non-naturally occurring immunoreceptor" or "exogenous immunoreceptor" refers to an immunoreceptor that is not naturally expressed in immune cells. In other words, a non-naturally occurring immunoreceptor has been "engineered" to be expressed in immune cells. A non-naturally occurring immunoreceptor can be a cloned version of a naturally occurring immunoreceptor. Alternatively, a non-naturally occurring immunoreceptor can be a chimeric receptor produced using recombinant molecular biology techniques. Exemplary non-naturally occurring immunoreceptors include SAR, SIR, Ab-TCR, TFP, and recombinant TCR. A non-naturally occurring immunoreceptor can be introduced into immune cells using gene transfer techniques known in the art, such as lentivirus- or retrovirus-mediated gene transfer. A non-naturally occurring immunoreceptor can be expressed in immune cells using an exogenous promoter (e.g., EF1α promoter) or an endogenous promoter (e.g., TCRα promoter).

[0214] As used herein, a "non-scFv antigen-binding domain" or "non-scFv-based antigen-binding domain" refers to an antigen-binding domain that is not composed of a single-chain variable fragment (i.e., vL-linker-vH or vH-linker-vL). Exemplary non-scFv-based antigen-binding domains include, but are not limited to, vHH, FHVH, SVL, non-immunoglobulin antigen-binding scaffolds (e.g., DARPIN, centrin, affibody, etc.), ligand-binding domains of receptors, receptor-binding domains of ligands, adaptor-binding domains (e.g., RZIP, EZIP, E4, K4, D domain, NKG2D-YA, NKG2D-AF, CD16A-V158, CD32, or CD64, etc.), and autoantigens. AABD is a non-scFv-based antigen-binding domain.

[0215] 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 TCR constant region that is chimeric and non-naturally occurring relative to a naturally occurring TCR. Stated differently, a non-naturally occurring TCR antigen-binding domain is "engineered" using recombinant molecular biology techniques to be operably linked to a TCR, and further, the antigen-binding domain is obtained or derived from a molecule different from the TCR found in nature. Antigen-binding domains that are different from the native TCR include antibody vH and vL fragments, humanized antibody fragments, chimeric antibody fragments, receptor ligands, and the like.

[0216] The term "NKT cells," as used herein, refers to a subset of T cells that not only co-express the αβ T cell receptor but also express various molecular markers commonly associated with NK cells, such as NK1.1. The best-known NKT cells differ from conventional αβ T cells in that the diversity of their T cell receptors is much more restricted ("non-mutant" or "type 1" NKT). These and other CD1d-restricted T cells ("type 2" NKT) recognize lipids and glycolipids presented by the CD1d molecule, a member of the CD1 family of antigen-presenting molecules, rather than by peptide-major histocompatibility complex (MHC).

[0217] The terms "operably linked" or "functionally linked" or "operably connected" or "operably attached" refer to a functional linkage or association between a first component and a second component such that each component is functional. For example, operably linked includes the association between a regulatory sequence and a heterologous nucleic acid sequence that confers expression. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. With respect to two different modules of a molecule, a first module is operably linked to a second module when their nucleotide sequences are connected in-frame. With respect to two operably linked polypeptides, the first polypeptide functions independently of any linkage, and the second polypeptide functions as if no linkage were present between them. The terms "operably linked" or "operably attached" are used interchangeably with the terms "linked," "attached," or "connected."

[0218] "Percent identity," with respect to 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 the same (e.g., 60% identity, or, optionally, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region, or, if not specified, the entire sequence), when compared and aligned for maximum correspondence over the comparison window or indicated region, as determined using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more usually over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0219] For sequence comparison, generally one sequence serves as a reference sequence, and test sequences are compared with it.Two examples of algorithms that can be used to determine percent sequence identity and sequence similarity are BLAST and BLAST2.0 algorithms, which are respectively described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Bioi. 215:403-410.Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information.

[0220] The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome. Examples of retroviral vectors include MSCVneo, MSCV-pac (or MSCV-puro), and MSCV-hygro, such as those available from Addgene or Clontech.

[0221] The term "Sleeping Beauty transposon" or "Sleeping Beauty transposon vector" refers to a vector derived from at least a portion of the Sleeping Beauty transposon genome.

[0222] The term "single-chain variable region" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked, for example, via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have the vL and vH variable regions in either order, e.g., with respect to the N- and C-termini of the polypeptide, and can comprise vL-linker-vH or vH-linker-vL. In the present disclosure, an scFv is also described as vL-Gly-Ser-linker-vH. Alternatively, an scFv is also described as (vL+vH) or (vH+vL).

[0223] The term "signaling domain" refers to a functional region of a protein that transmits information intracellularly to regulate cellular activity through a defined signaling pathway by generating second messengers or by responding to such messengers and thereby functioning as an effector.

[0224] The term "synthetic antigen receptor" or "SAR" refers to a non-naturally occurring polypeptide that, when expressed in an effector cell, provides the cell with specificity for a target cell, typically a cancer cell. SARs are engineered receptors that confer antigen specificity to a cell (e.g., T cells, NK cells, NKT cells, monocytes / macrophages, B lymphocytes, or a combination thereof), thus combining the antigen-binding properties of an antigen-binding domain with the effector function of the cell. Exemplary effector functions of T cells and NK cells may include target cell lysis, cytokine production, and self-renewal. In the case of monocytes / macrophages, the effector function may include phagocytosis of target cells. SARs, as this term is defined herein, encompass first-generation CARs, second-generation CARs, third-generation CARs, and next-generation CARs, such as synthetic immunoreceptors (SIRs), cTCRs, Ab-TCRs, AABD-TCRs, TFPs (e.g., TFPε, TFPγ, TFPδ), TACs, recombinant TCRs, etc. SARs may be single-chain, double-chain, or 1.5-chain. SARs can be monospecific, bispecific, or multispecific. SARs can have one or more antigen-binding domains. SARs can have the backbone of a CAR (e.g., a second-generation CAR), SIR, cTCR, Ab-TCR, AABD-TCR, TFP, TAC, or TCR. As used herein, SARs include artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. However, the term SAR is not limited to T cell receptors or immunoreceptors. SARs may or may not have their own signaling domains. Exemplary SARs that lack their own signaling domains are SARs with the backbone of an SIR or TCR, which transmit signals through the recruitment of other signaling proteins with signaling domains. SARs can also be any non-natural antigen-binding receptor (ABR). The terms "antigen-binding domain" or "antigen-specific targeting domain," as used herein, refer to the region of a SAR that targets and binds to a specific antigen.The antigen-binding domain of a SAR can consist of an antibody or antibody fragment (e.g., vL, vH, Fv, Fab, scFv, vHH, single-domain antibody, etc.), a T cell receptor (TCR) or a fragment of a TCR (e.g., Va, Vb, Vg, Vd, or single variable domain TCR, etc.). The antigen-binding domain of a SAR can comprise one or more autonomous antigen-binding domains (AABDs), such as a non-immunoglobulin antigen-binding scaffold (e.g., DARPIN, centilin, affibody, D domain, etc.), a receptor-binding domain of a ligand, a ligand-binding domain of a receptor, an autoantigen, an adaptor-binding domain (e.g., RZIP, EZIP, E4, K4, NKG2D-AF, etc.), an Fc-binding domain of a receptor (e.g., Fc-binding region of CD16A-V158, CD32, CD64, etc.), or a combination thereof. A SAR can have one or more antigen-binding domains. Various modules and domains of SAR can be connected by one or more linkers. SAR can also comprise one or more epitope tags or mimotopes that can be used to detect SAR expression, isolate and purify SAR-expressing cells, monitor the persistence of SAR-expressing cells, and deplete SAR-expressing cells. In some embodiments, epitopes and / or mimotopes are targeted by antibodies, antibody fragments, or antibody derivatives (e.g., antibody-drug conjugates) to function as "suicide switches" to reduce or eliminate SAR-expressing cells in the event of toxicity. When SAR is expressed in a host cell, this domain forms an extracellular domain (ectodomain).

[0225] The term SVH domain as used herein refers to a single human V H Domain antibodies (V HsdAb). Thus, these terms are used interchangeably. The term SVH is also used interchangeably, independent of the vH domain. SVH is an example of an autonomous antigen-binding domain (AABD). An exemplary SVH is a fully human vH domain (FHVH) set forth in SEQ ID NOs: (DNA) 827-828 and SEQ ID NOs: (PRT) 11517-11518. Another exemplary SVH is a chVH domain set forth in SEQ ID NOs: (DNA) 830-831 and SEQ ID NOs: (PRT) 11520-11521. Another exemplary SVH is a VH domain set forth in SEQ ID NOs: (DNA) 850-851 and SEQ ID NOs: (PRT) 11540-11541. SEQ ID NOs for other exemplary SVH domains are shown in Table 5. Additional SVH domains that can be used to construct the SARs of the present disclosure are provided in WO2016062988, WO2016113556, WO2017191476, WO2018039180, WO2019006072, WO2018237037, WO2018119215, WO2019126756, WO2019055689, and WO2020018922, the entire contents of which are incorporated herein by reference.

[0226] The term SVL domain, as used herein, refers to a single human vL domain antibody (vL sdAb). Thus, these terms are used interchangeably. The term SVL is also used interchangeably independent of the vL domain. SVL is an example of an autonomous antigen-binding domain (AABD).

[0227] As used herein, a "single variable domain T cell receptor" or "svd-TCR" refers to a variable domain of a T cell receptor that can specifically bind to an epitope in the absence of a second TCR variable domain. For example, an svd-TCR comprising a Vb variable domain can bind to an epitope independent of and / or in the absence of a Va variable domain, and an svd-TCR comprising a Va variable domain can bind to an epitope independent of and / or in the absence of an nb variable domain. Exemplary epitopes recognized by svd-TCRs include peptide:MHC complexes (pMHC complexes).

[0228] The term "synthetic immunoreceptor" or alternatively "SIR" refers to a set, in some embodiments, typically two polypeptides, that, when expressed in an effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and the generation of an intracellular signal. SIRs represent the next-generation CAR platform described in WO 2018 / 102795 A1, incorporated herein by reference. In an exemplary embodiment, 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 connected to one or more T cell receptor constant chains or regions via optional linkers. 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 SIRs are contiguous with each other (functional polypeptide unit 1) but not with the polypeptides of the other sets (functional polypeptide unit 2). In some embodiments, the T cell receptor constant chain (or region) of the SIR is selected from the group consisting of human T cell receptor-α (TCR-α or TCRα or TCRa or hTCR-α or hTCRα or hTCRa ​​or Cα), human T cell receptor-β1 (TCR-β1 or TCRβ1 or TCRb1 or hTCR-β1 or hTCRβ1 or hTCRb1 or Cβ1), human T cell receptor-β2 (TCR-β2 or TCRβ2 or TCRb2 or hTCR, also referred to as TCR-β, TCRβ or TCRb or Cβ), human T cell receptor-β3 (TCR-β4 or TCRβ5 or TCRβ6 or TCRβ7), human T cell receptor-β5 (TCR-β6 or TCRβ7 or TCRβ8 or TCRβ9), human T cell receptor-β6 (TCR-β7 or TCRβ8 or TCRβ9, also referred to as TCR-β1 or TCRβ10), human T cell receptor-β7 (TCR-β8 or TCRβ9 or TCRβ11), human T cell receptor-β9 (TCR-β9 or TCRβ12 or TCRβ13), human T cell receptor-β10 (TCR-β10 or TCRβ11 or TCRβ12, also referred to as TCR-β10 or TCRβ1 ... The constant chain is selected from the constant chains of human precursor T-cell receptor alpha (pretc.R-alpha or pretc.Ralpha or pretc.Ra or preCalpha), human T-cell receptor gamma (TCR-gamma or TCRγ or TCRg or hTCR-gamma or hTCRγl or hTCRγl or Cγ), or human T-cell receptor delta (TCR-δ or TCRd or TCRδ or hTCR-δ or hTCRd or hTCRδ or Cδ).In some embodiments, the TCR constant chains of the SIRs are encoded by their wild-type nucleotide sequences, while in other aspects, the TCR constant chains of the SIRs are encoded by non-wild-type nucleotide sequences. In some embodiments, the TCR constant chains of the SIRs are encoded by their codon-optimized sequences. In some embodiments, the TCR constant chains of the SIRs encode wild-type polypeptide sequences, while in other embodiments, the TCR constant chains of the SIRs encode polypeptides with one or more mutations. In some embodiments, the TCR constant chains of the SIRs are encoded by their codon-optimized sequences with one or more mutations. The present disclosure also includes deletion mutants of TCR constant chains that retain at least one of the biological and functional properties of the corresponding full-length TCR chain. An SIR comprising an antigen-binding domain (e.g., scFv or vHH) targeting a specific tumor marker "X," such as those described herein, is also referred to as an X-SIR or XSIR. For example, an SIR comprising an antigen-binding domain targeting CD19 is referred to as a CD19-SIR or CD19SIR. The TCR constant chain / domain of the SIR can be derived from the same species as the SIR will ultimately be used in. For example, for use in humans, it may be advantageous for the TCR constant chain of the SIR to be derived from or consist of a human TCR constant chain. However, in some cases, it may be advantageous for the SIR to be derived from the same species as the SIR will ultimately be used in, but for the TCR constant chain to be modified to have amino acid substitutions that enhance TCR constant chain expression. For example, for use in humans, it may be advantageous for the TCR constant chain of the SIR to be derived from or consist of a human TCR constant chain, but for certain amino acids to be substituted with corresponding amino acids from a mouse TCR constant chain. Such a murine TCR constant chain provides enhanced SIR expression. The SIR or a functional portion thereof can contain additional amino acids at the amino or carboxy terminus, or both, that are not found in the amino acid sequence of the TCR or antigen-binding domain that constitutes the SIR.Desirably, these additional amino acids do not interfere with the biological function of the SIR or functional portion, such as, for example, recognizing target cells, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance biological activity compared to the biological activity of the parent SIR. The present disclosure provides bispecific, biparatopic, and multispecific SARs having an SIR scaffold comprising one or more AABDs. The AABD domains of the SARs of the present disclosure having an SIR scaffold can be fully human, humanized, or non-human. In one embodiment, the present disclosure provides an SIR comprising one or more fully human vH domains. In one embodiment, the present disclosure provides an Ab-TCR comprising one or more fully human vL domains.

[0229] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand (or target antigen), thereby mediating a signal transduction event, such as, but not limited to, TCR / CD3-mediated signaling. Stimulation can mediate altered expression of specific molecules.

[0230] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides cytoplasmic signaling sequences that stimulatoryly regulate immune cell activation with respect to at least some aspect of an immune cell signaling pathway.

[0231] The term "subject" is intended to include organisms in which an immune response can be elicited (e.g., any domestic mammal or human). "Subject" or "individual" or "animal" or "patient" are used interchangeably to refer to any subject, particularly a mammalian subject, to whom administration of a composition or pharmaceutical composition of the present disclosure is desired. Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc., with humans being preferred.

[0232] A "switch domain" or "dimerization domain," as used herein, generally refers to a polypeptide-based entity that associates with another switch domain in the presence of a dimerization molecule. This association results in functional coupling of, e.g., a first entity linked to, e.g., fused to, a first switch domain, with a second entity linked to, e.g., fused to, a second switch domain. The first switch domain and the second switch domain are collectively referred to as a dimerization switch.

[0233] The term "target antigen" refers to an antigen to which an antigen-binding agent (e.g., an antibody, antibody fragment, SAR-T cell, SAR adapter, etc.) binds. The term "target antigen-expressing cell" refers to a cell that expresses the antigen to which an antigen-binding agent binds, i.e., a target cell. In certain embodiments, a cell expressing a SAR of the present disclosure undergoes activation, proliferation, and induces effector functions (e.g., cytokine production, cytotoxicity, etc.) when it binds to the target antigen-expressing cell directly or indirectly via a SAR adapter. Non-limiting examples of target antigens are shown in Table 8. A SAR of the present disclosure may bind to one or more (e.g., two, three, four, five, or more) target antigens listed in Table 8 directly or via a SAR adapter described herein.

[0234] [Table 14] JPEG2023518049000016.jpg219146JPEG2023518049000017.jpg75146

[0235] The terms "T cells" and "T lymphocytes" are interchangeable and are used synonymously herein. Examples include, but are not limited to, naive T cells ("lymphocyte progenitor cells"), central memory T cells, effector memory T cells, stem memory T cells (T scm ), iPSC-derived T cells, synthetic T cells, or a combination thereof.

[0236] The term "T / NK cell activating antibody therapy," as used herein, refers to an antibody therapy that activates T cells and / or NK cells. Examples of T / NK cell activating antibody therapies include bispecific T cell engaging antibodies (e.g., blinatumomab) or bispecific NK cell engaging antibodies.

[0237] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and are linked to the IMGT public database of TCR sequences. Naturally occurring α-β heterodimeric TCRs have an α chain (TCRα or TCRα) and a β chain (TCRβ or TCRβ). γ-δ heterodimeric TCRs have a γ (TCRγ) chain and a TCR-δ (TCRγ) chain. Broadly speaking, each chain comprises a variable region, a connecting region, and a constant region; β chains also typically contain a short diversity region between the variable and connecting regions, which is often considered part of the connecting region. Each variable region comprises three CDRs (complementarity-determining regions) embedded in framework sequences, one of which is a hypervariable region designated CDR3. There are several α chain variable (Vα or Va) regions and several β chain variable (Vβ or Vb) regions, distinguished by framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. Va / Va types are referenced by unique TRAV numbers in the IMGT nomenclature. Thus, "TRAV21" defines a TCR Va / Va region with unique framework and CDR1 and CDR2 sequences and a CDR3 sequence that is partially defined by conserved amino acid sequences among TCRs but also contains amino acid sequences that vary among TCRs. Similarly, "TRBV5-1" defines a TCR Vβ / Vb region with unique framework and CDR1 and CDR2 sequences but only partially defined CDR3 sequences. The variable regions of TCR gamma (TCRg) and TCR delta (TCRd) are designated Vg and Vd, respectively. The variable regions of TCRs (e.g., Va, Vβ, Vγ, Vδ) can bind antigen in an MHC (or HLA)-dependent manner. The variable regions of a TCR (e.g., Vα, Vβ, Vγ, Vδ) can bind to antigens in an MHC (or HLA)-independent manner. In an exemplary embodiment, the variable regions of a TCR (e.g., Vα, Vβ, Vγ, Vδ) can bind to extracellular antigens (e.g., CD19, CD20, CD22, mesothelin, etc.) in an MHC (or HLA)-independent manner. Such TCRs are referred to as HLA-independent TCRs.In some embodiments, the SAR of the present disclosure may comprise an HLA-dependent and / or HLA-independent TCR variable region. In another exemplary embodiment, the SAR of the present disclosure may comprise one variable region of a TCR and one variable region of an antibody. Thus, a dual-chain SAR may comprise a Va fragment attached to a TCR alpha constant chain and a vH (or vL) fragment attached to a TCR beta constant chain. The AABD may be attached to one or both of these chains.

[0238] The α and β chains of the αβ TCR are generally considered to have two "domains," respectively: a variable domain and a constant domain. The variable domain consists of the connection of the variable region and the connecting region. Thus, in this specification and claims, the term "TCRα variable domain or Va or Vα" refers to the connection of the TRAV and TRAJ regions, and the term TCRα constant domain (Cα) refers to the extracellular TRAC region or a C-terminal truncated TRAC sequence. Similarly, the term "TCRβ variable domain or Vβ or Vb" refers to the connection of the TRBV region and the TRBD / TRBJ regions, and the term TCRβ constant domain (Cβ) refers to the extracellular TRBC region or a C-terminal truncated TRBC sequence.

[0239] The TCRs of the present disclosure may be non-naturally occurring and / or purified and / or engineered. The TCRs of the present disclosure may have two or more mutations present in the α chain variable domain and / or the β chain variable domain compared to a parent TCR. "Engineered TCR" and "mutant TCR" are used interchangeably herein and generally refer to a TCR that has one or more mutations introduced relative to a parent TCR, particularly in its Va and / or Vb or Vg and / or Vd domains. Engineered TCRs may bind antigen in an HLA-dependent or HLA-independent manner.

[0240] An "HLA-independent TCR" or "MHC-independent TCR," as defined herein, is a TCR that can recognize an antigen without relying on MHC restriction. In an exemplary embodiment, an HLA-independent TCR can bind to a cell surface antigen that is not presented by an MHC complex. In an exemplary embodiment, an HLA-independent TCR can bind to an antigen expressed on a cell surface without relying on presentation by an MHC complex. An HLA-independent TCR can be a naturally occurring TCR. In an exemplary embodiment, an HLA-independent TCR is MC.7.G5 (MC7G5), which recognizes MR1, a ubiquitously expressed monomorphic antigen-presenting molecule. An HLA-independent TCR can be an engineered or recombinant TCR. In an exemplary embodiment, an HLA-dependent TCR is an engineered TCR that can bind to a cell surface-expressed protein such as CD19, CD20, mesothelin, PSMS, or BCMA. Methods for engineering TCR variable domains (e.g., CDR grafting, etc.) are known in the art and can be used to create HLA-independent TCRs that can bind to extracellularly expressed proteins (e.g., CD19, MSLN, PSMA, etc.) or protein epitopes independent of MHC complexes. The present disclosure provides bispecific, biparatope, and multispecific SARs having a TCR framework, including HLA-independent TCRs comprising one or more AABDs. The AABD domains of the SARs of the present disclosure having a TCR (e.g., HLA-independent TCR) framework can be fully human, humanized, or non-human. In certain embodiments, the present disclosure provides TCRs (e.g., HLA-independent TCRs) comprising one or more fully human vH domains. In certain embodiments, the present disclosure provides TCRs (e.g., HLA-independent TCRs) comprising one or more fully human vL domains.

[0241] An "HLA-independent TCR variable domain," as defined herein, is a variable domain of a TCR that can bind to an antigen in an HLA-independent manner. The HLA-independent variable domain can be the variable domain of an HLA-independent TCRα, TCRβ, TCRγ, TCRδ, or pre-TCRα. The HLA-independent TCR variable domain can be a single variable domain TCR (i.e., svd-TCR). The HLA-independent TCR variable domain can be a naturally occurring HLA-independent variable domain or an engineered HLA-independent variable domain. In an exemplary embodiment, an engineered HLA-independent variable domain can be generated against the extracellular domain of a protein (e.g., CD19, CD22, BCMA, MSLN, PSMA) using techniques known in the art (e.g., CDR grafting, phage display library screening, etc.).

[0242] 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 binding peptide and / or all or part of the intracellular domain.

[0243] The term "TCR-associated signaling module" refers to a molecule containing a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) that is part of the TCR-CD3 complex. TCR-associated signaling modules include CDγε, CDδε, and CD3ζζ.

[0244] "Therapeutic agent," as used herein, refers to an agent used to, for example, treat, suppress, prevent, alleviate the effects of, reduce the severity of, reduce the likelihood of onset of, slow the progression of, and / or cure a disease. Diseases targeted by therapeutic agents include, but are not limited to, infectious diseases, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, antigens expressed on various immune cells and cells associated with various blood disorders, and / or inflammatory diseases.

[0245] "Therapeutic control," as used herein, refers to an element used to control the activity of SAR (including next-generation CAR)-expressing cells. In some embodiments, the therapeutic control for controlling the activity of the SAR-expressing cells of the present disclosure is a truncated epidermal growth factor receptor (tEGFR), a truncated epidermal growth factor receptor viii (tEGFRviii), a truncated CD30 (tCD30), a truncated BCMA (tBCMA), a truncated CD19 (tCD19), a thymidine kinase, a cytosine deaminase, a nitroreductase, a xanthine guanine phosphoribosyltransferase, a human caspase 8, a human caspase 9, an inducible caspase 9, a purine nucleotide The gene encoding the nucleoside phosphorylase comprises any one or more of: nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), selection marker, and combinations thereof.

[0246] 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 the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, a reduction in cancer cell survival, a reduction in the titer of an infectious pathogen, a reduction in the number of colonies of an infectious pathogen, and an improvement in various physiological symptoms associated with a pathological condition. A "therapeutic effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the onset of disease in the first place or to prevent the recurrence of disease.

[0247] The term "therapeutically effective amount," as used herein, refers to an amount of a pharmaceutical composition comprising one or more peptides or mutants, variants, analogs or derivatives thereof as disclosed herein to alleviate at least one or more symptoms of a disease or disorder, and relates to the amount of a pharmacological composition sufficient to provide the desired effect. The phrase "therapeutically effective amount," as used herein, means the amount of the composition sufficient to treat a disorder at a reasonable benefit / risk ratio applicable to any medical treatment.

[0248] A therapeutically or prophylactically significant alleviation of symptoms is, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more of a measured parameter compared to a control or untreated subject or the condition of the subject prior to administration of an oligopeptide described herein.

[0249] The term "TCR receptor fusion protein" or "TFP" refers to a next-generation SAR platform such as that described in WO2016 / 187349A1, which is incorporated herein by reference. In one embodiment, the TFP comprises an antibody moiety that specifically binds to a target antigen fused to a TCR chain, such as CD3ε, CD3γ, CD3δ, TCRα, or TCRβ. Exemplary TCR chains that can be used in constructing TFPs are represented by SEQ ID NOs: 11903-11906 of the present disclosure and are provided in WO2017 / 070608A1, which is incorporated herein by reference. A TFP incorporating a CD3ε chain is referred to as CD3ε TFP or TFPε. A TFP incorporating a CD3γ chain is referred to as CD3γ TFP or TFPγ. A TFP incorporating a CD3δ chain is referred to as CD3δ TFP or TFPδ. TFPs incorporating CD3ε, CD3γ, or CD3δ chains are collectively referred to as CD3ε / γ / δ TFPs or TFPε / γ / δ. The present disclosure provides bispecific, biparatopic, and multispecific SARs having a TFP (e.g., TFPε) backbone comprising one or more AABDs. The AABD domains of the SARs of the present disclosure having a TFP backbone can be fully human, humanized, or non-human. In some embodiments, the present disclosure provides a TFP comprising one or more fully human vH domains. In some embodiments, the present disclosure provides a TFP comprising one or more fully human vL domains. In some embodiments, the present disclosure provides a TFP comprising a non-immunoglobulin antigen-binding scaffold (e.g., centilin or DARPIN). In some embodiments, the present disclosure provides a TFP comprising an adaptor-binding domain (e.g., a leucine zipper domain, e.g., RZip, Ezip, E4, K4, etc.).

[0250] The term "transfer vector" refers to a subject composition comprising an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0251] "Transmembrane domain" (TMD), as used herein, refers to the region of a SAR that crosses the plasma membrane. The transmembrane domain of a SAR of the present disclosure is a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Transmembrane domains will be apparent to those skilled in the art and can be used in connection with other embodiments of the present disclosure. In some embodiments, the SAR encoded by the TMD comprises a transmembrane domain selected from the transmembrane domains of the TCR alpha, TCR beta, TCR gamma, TCR delta chains of the T cell receptor, CD3 gamma, CD3 epsilon, CD3 delta, CD3 zeta, FcR gamma, CD28, CD45, CD4, CD5, CD8, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0252] As used herein, "trifunctional T cell antigen coupler" or "tri-TAC" or "TAC" refers to the next-generation SAR platform described in WO2015 / 117229A1, which is incorporated herein by reference. Tri-TACs targeting different antigens can be constructed using techniques known in the art and antigen-binding domains (e.g., vL and vH fragments, scFv, vHH, ligands and receptors, etc.) described in this disclosure.

[0253] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment whose purpose is to reverse, alleviate, improve, inhibit, slow or halt the progression or severity of pathology associated with a disease or disorder.

[0254] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0255] "Vector," "cloning vector," and "expression vector," as used herein, refer to a vehicle by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

[0256] The term "viral vector" refers to a vector obtained or derived from a virus. Generally, the virus is a retrovirus, including but not limited to a lentivirus and a gamma retrovirus. The viral vector of the present disclosure may be a retroviral vector, such as a gamma retroviral vector. The viral vector of the present disclosure may be a lentiviral vector.

[0257] The terms "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" are defined as the protein provided as GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species.

[0258] The binding domain of the SAR binds to a desired epitope or antigen. For example, the epitope recognized by the SAR is determined from the epitope recognized by the scFv used as the binding domain of the SAR. For example, the antigen-specific domain of this SAR is CD8SP-hu-mROO5-1-vL-Gly-Ser-linker-hu-mROO5-1-vH-Myc-CD8TM-BBz-T2A-PAC (SEQ ID NO: 7340) targeting CD19 is composed of a humanized scFv (SEQ ID NO: 11323), so the SAR is likely to target the same epitope as the scFv and / or the parent antibody from which the scFv is derived. The epitopes recognized by several scFvs and / or their parent antibodies used in the construction of the SARs and scaffolds of the present disclosure are known in the art. Alternatively, the epitope targeted by AMR or SAR can be determined by creating a series of mutants of the target antigen and testing the ability of the mutants to bind to SAR-expressing cells using techniques known in the art, for example, using the Topanga assay. For example, the epitope recognized by SARCD8SP-hu-mROO5-1-vL-Gly-Ser-linker-hu-mROO5-1-vH-Myc-CD8TM-BBz-T2A-PAC (SEQ ID NO: 7340) targeting CD19 can be determined by creating a panel of deletion and point mutants of CD19-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac (DNA SEQ ID NO: 1282 and PRT SEQ ID NO: 11972). These mutant constructs are transfected into suitable cell lines (for example, 293FT cells), and the supernatant containing fusion protein is collected and assayed for NLuc activity, confirming that various mutant CD19-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis fusion proteins are secreted into the supernatant.Then, these fusion proteins are tested for their ability to bind to cells (for example, Jurkat cells or T cells) that express SAR (SEQ ID NO: 7340).The mutants that cannot bind to SAR-expressing cells are candidates that contain the epitope targeted by CD19-specific SAR.Another approach to determining the epitope recognized by a particular SAR can involve functional competition assays with various test antibodies. For example, T cells expressing SAR (SEQ ID NO: 7340) can be co-cultured with a cell line expressing CD19 (e.g., RAJI cells) in the absence and presence of increasing concentrations of various test CD19 antibodies. If the epitope recognized by a test CD19 antibody overlaps with the epitope recognized by SAR (SEQ ID NO: 7340), the test antibody will dose-dependently block target cell death and cytokine production induced by T cells expressing SAR (SEQ ID NO: 7340). A non-specific antibody of the same isotype as the test antibody is included as a control and will have no effect on target cell death and cytokine production induced by T cells expressing SAR. Similarly, a specific SAR can be expressed in Jurkat-NFAT-EGFP cells, and the ability of the test antibody to block EGFP induction by SAR-expressing Jurkat-NFAT-GFP cells when co-cultured with a target cell line can be used to determine whether the epitope recognized by the test antibody overlaps with the epitope recognized by the SAR.

[0259] [Table 15]

[0260] [Table 16]

[0261] [Table 17] JPEG2023518049000021.jpg181133JPEG2023518049000022.jpg172133JPEG202 3518049000023.jpg181134JPEG2023518049000024.jpg180133JPEG20235180490 00025.jpg180133JPEG2023518049000026.jpg180134JPEG2023518049000027.j pg175133JPEG2023518049000028.jpg172134JPEG2023518049000029.jpg164134

[0262] Table 18

[0263] Table 19 JPEG2023518049000032.jpg226146JPEG2023518049000033.jpg144146

[0264] Table 20

[0265] Table 21

[0266] Table 22

[0267] Table 23

[0268] Table 24

[0269] Table 25

[0270] Table 26 JPEG2023518049000041.jpg132146JPEG2023518049000042.jpg110145JPEG2023518049000043.jpg213146

[0271] Table 27

[0272] Table 28

[0273] Table 29

[0274] Table 30

[0275] Table 31

[0276] Table 32

[0277] Table 33

[0278] Table 34

[0279] Table 35

[0280] Table 36

[0281] Table 37

[0282] Table 38

[0283] Table 39 JPEG2023518049000057.jpg173146JPEG2023518049000058.jpg177146JPEG2023518049000059.jpg162146JPEG2023518049000060.jpg169146 JPEG2023518049000061.jpg182146JPEG2023518049000062.jpg168146JPEG2023518049000063.jpg183146JPEG2023518049000064.jpg149146

[0284] Table 40

[0285] Table 41 JPEG2023518049000067.jpg177145JPEG2023518049000068.jpg77145

[0286]

Table 42

[0287]

Table 43

[0288]

Table 44

[0289]

Table 45

[0290]

Table 46

[0291]

Table 47

[0292] Table 48

[0293] Table 49 TIFF2023518049000122.tif212146TIFF2023518049000123.tif190146TIFF2023518049000124.tif179146TIFF2023518049000125.tif50146

[0294] Table 50 TIFF2023518049000127.tif187140TIFF2023518049000128.tif112140

[0295] Table 51

[0296] Table 52

[0297] Table 53 TIFF2023518049000132.tif112144

[0298] Table 54 JPEG2023518049000134.jpg203143JPEG2023518049000135.jpg203143JPEG2023518049000136.jpg195144JPEG2023518049000137.jpg49144

[0299] In one aspect, the present disclosure provides novel compositions of synthetic antigen receptors (SARs). In another aspect, the present disclosure provides novel configurations / structures of SARs. In another aspect, the present disclosure provides SARs with useful biological properties (e.g., expression, binding affinity, effector function, etc.). In another aspect, the present disclosure provides SARs capable of binding to one or more antigens. In another aspect, the present disclosure provides SARs capable of binding to one or more epitopes of an antigen.

[0300] In one aspect, the present disclosure provides a synthetic antigen receptor (SAR) comprising two or more (i.e., 2, 3, 4, 5, or more) antigen-binding domains. In another aspect, the present disclosure provides a SAR capable of binding to and / or responding to two or more antigens or two or more epitopes of antigens. In another aspect, the present disclosure provides a bispecific and / or multispecific SAR capable of binding to and / or responding to two or more antigens or two or more epitopes of antigens. In another aspect, the present disclosure provides antigen-binding domains useful for constructing bispecific and / or multispecific SARs. In another aspect, the present disclosure provides configurations (i.e., locations of different domains) useful for bispecific and / or multispecific SARs. The disclosed bispecific and multispecific SARs, when expressed in an immune effector cell (e.g., a T cell, an NKT cell, or an NK cell), confer upon it the ability to bind to and / or respond to two or more antigens or two or more epitopes of antigens with approximately equal or greater efficacy than two or more monospecific SARs targeting those same antigens or the same epitopes of those antigens.

[0301] The presence of two or more antigen-binding domains in bispecific or multispecific SARs can result in steric hindrance, nonspecific aggregation, insufficient expression, protein unfolding, and / or interference with antigen binding. Furthermore, to optimize signal transduction by the resulting receptor, it is necessary to optimize the position of the antigen-binding domain relative to the transmembrane domain of the SAR. Bispecific and multispecific CARs incorporating two or more scFvs have been described in the art. However, the present disclosure identifies that the presence of two or more scFvs (i.e., 2, 3, 4, or more) in SARs (e.g., second-generation CARs, SIRs, Ab-TCRs, zSIRs, TFPs, or rTCRs) often results in steric hindrance, nonspecific aggregation, persistent signal transduction, insufficient expression, protein unfolding, and / or interference with antigen binding, resulting in insufficient signal transduction and effector function (e.g., cytokine production, cytotoxicity, etc.). Therefore, a major challenge in generating bispecific and multispecific SARs comprising two or more antigen-binding domains is determining useful antigen-binding domains (e.g., scFv, Fv, Fab, vHH, FHVH, centilin, affibody, cytokine, receptor, svd-TCR, etc.) to incorporate into such SARs to mitigate steric hindrance, nonspecific aggregation, persistent signaling, poor expression, protein unfolding, and / or interference with antigen binding, which can result in poor signaling and effector function (e.g., cytokine production, cytotoxicity, etc.).

[0302] The second challenge is determining useful configurations of the various antigen-binding domains comprising bispecific and multispecific SARs. For example, the optimal order of the various antigen-binding domains relative to each other and to other components of the SAR (e.g., hinge domain, transmembrane domain, etc.) must be determined to mitigate nonspecific aggregation, persistent signal transduction, insufficient expression, protein unfolding, and / or interference with antigen binding, resulting in insufficient signal transduction and effector function (e.g., cytokine production, cytotoxicity, etc.). This is an important challenge for all SARs, especially for multi-chain SARs such as SIRs, Ab-TCRs, zSIRs, αβTFPs, or γδTFPs, whose antigen-binding domains are composed of two different fragments (e.g., vL and vH, Va and Vb, or Vg and Vd, etc.). For example, attachment of a second antigen-binding domain (e.g., an scFv or vHH domain) to a dual-chain SIR (e.g., CD8SP-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-SP-hu-mROO5-1-vH-[hTCRa-T48C]; SEQ ID NO: 7348) that binds CD19 via vL and vH fragments operably linked to two separate TCR constant chains and is connected to form an Fv that binds CD19 may interfere with the interaction between the vL and vH fragments, preventing the formation of a functional Fv that can bind CD19.

[0303] The length of the hinge domain, which determines the distance between the antigen-binding domain and the cell membrane, may affect signal transduction via chimeric antigen receptors. Therefore, another challenge in this field is that it is currently unclear whether the attachment of multiple antigen-binding domains may adversely affect the formation of an effective immune synapse and SAR-mediated signal transduction by increasing the distance between the target antigen and the cell membrane.

[0304] Fusion of multiple antigen-binding domains in SARs may result in steric hindrance and improper folding. Another challenge in this field is that it is currently unclear whether a linker domain is required between the different antigen-binding domains of bispecific / multispecific SARs. The length and nature of the linker domain are also unknown. This is particularly important in the case of double-chain SARs (e.g., double-chain SIRs, double-chain zSIRs, Ab-TCRs, αβTFP, or γδTFP), because adding an inappropriate linker may prevent the interaction between the two chains or the formation of a functional Fv. Furthermore, linkers may negatively affect the formation of an effective immune synapse and SAR-mediated signaling by increasing the distance between the target antigen and the cell membrane.

[0305] In one aspect, the present disclosure provides a solution to the above problem.

[0306] In one aspect, the present disclosure provides SARs having one or more antigen-binding domains and one or more transmembrane domains. In some embodiments, the present disclosure provides antigen-binding domains useful for constructing bispecific and multispecific SARs.

[0307] The present disclosure provides several exemplary SARs, including different antigen-binding domains, hinge domains, linker domains, binding peptides, transmembrane domains, activation domains, costimulatory domains, accessory modules, and therapeutic controls. The names and SEQ ID NOS (DNA) and (PRT) of exemplary components that can be used in constructing SARs are shown in Tables 1-20 and 24. The names and SEQ ID NOS (DNA) and (PRT) of exemplary SARs are shown in Tables 25-36 and 41-50. The target antigens, organization, and composition of SARs can be deduced from their nucleic acid and amino acid sequences shown in the present disclosure by performing a sequence homology search of their component modules using a program such as BLAST. Alternatively, software such as ApE (https: / / jorgensen.biology.utah.edu / wayned / ape / ) can be used to determine the composition of different SAR constructs whose nucleic acid sequences are shown in the present disclosure. Finally, the organization and composition of different SARs of the present disclosure can be deduced from their names by those skilled in the art.

[0308] In certain embodiments, the present disclosure provides novel SARs having a structure and / or composition represented by any of the exemplary SARs shown in Tables 25-36 and 41-50. In certain embodiments, the present disclosure provides novel SARs or functional variants thereof having the composition of any of the exemplary SARs shown in Tables 25-36 and 41-50. In certain embodiments, the present disclosure provides novel SARs having at least 70% homology (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% homology) to the amino acid sequence of any of the exemplary SARs shown in Tables 25-36 and 41-50. In certain embodiments, the present disclosure provides novel SARs that have at least 70% homology (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% homology) to the amino acid sequence of any of the exemplary SARs shown in Tables 25-36 and 41-50, excluding optional accessory modules. The nucleic acid and amino acid sequences of the SARs in Tables 25-36 and 41-50 include their signal peptides. However, if the signal peptide is removed from the mature SAR polypeptide chain, the sequence encoding the signal peptide is excluded for purposes of determining homology. Similarly, sequences encoding accessory modules and / or any cleavage linkers (e.g., P2A, F2A, etc.) or furin cleavage sites are excluded for purposes of determining sequence homology. Thus, in one embodiment, the present disclosure provides novel SARs having at least 70% homology (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% homology) in the region comprising the antigen binding domain and signaling chain (e.g., TCR constant chain) to the amino acid sequence of any of the exemplary SARs shown in Tables 25-36 and 41-50.

[0309] In one aspect, the present disclosure provides a method for the preparation of SARs having advantageous antigen-binding domains, comprising the steps of: H domains, generally multiple human V H The present invention relates to the use of autonomous antigen binding domains (AABDs) comprising domains.

[0310] In one aspect, the present disclosure relates to autonomous antigen-binding domains (AABDs), methods for making them, and the use of such domains for the construction of synthetic antigen receptors and potentially antibody therapeutics. In one embodiment, the AABD domain has improved stability. In another embodiment, the AABD domain has improved thermal stability. In another embodiment, the AABD domain has improved solubility. In another embodiment, the AABD domain has a reduced tendency toward self-aggregation. In another embodiment, the AABD domain has an improved ability to be secreted into the extracellular space when expressed in mammalian cells using an N-terminal signal peptide.

[0311] In one aspect, the AABD is a single domain antibody or antibody fragment. In one aspect, the AABD is a single heavy chain variable chain (VH or vH) domain (SVH domain) or a fragment thereof that can bind to an antigen in the absence of a light chain variable chain (VL or vL) domain. In another aspect, the AABD is a single heavy chain variable chain (VH) domain (or SVH domain) of a fragment thereof that can be expressed as a soluble protein in the absence of a vL domain. In another aspect, the AABD is a single heavy chain variable chain (VH) domain (or SVH domain) or a fragment thereof that can be expressed as a secreted protein in the absence of a vL domain when connected to an N-terminal secretion signal. Certain embodiments of the present disclosure relate to SARs comprising a first AABD that specifically binds to an antigen in the absence of a second domain.

[0312] In one aspect, the AABD is a single light chain variable chain (VL or vL) domain or SVL domain, or a fragment thereof, that can bind to an antigen in the absence of a heavy chain variable chain (VH or vH) domain. In another aspect, the AABD is a single light chain variable chain (VL) domain (or SVL domain), or a fragment thereof, that can be expressed as a soluble protein in the absence of a vH domain. In another aspect, the AABD is a single light chain variable chain (VL) domain (or SVL domain), or a fragment thereof, that can be expressed as a secreted protein in the absence of a vH domain when connected to an N-terminal secretion signal.

[0313] In certain embodiments, the AABD is a non-scFv-based antigen binding domain or fragment thereof.

[0314] In one embodiment, the AABD is a camelid vHH domain or a humanized vHH domain or a fragment thereof.

[0315] In certain embodiments, the AABD is a non-immunoglobulin antigen-binding scaffold or fragment thereof.

[0316] In certain embodiments, the AABD is a cytokine or ligand or a fragment thereof.

[0317] In certain embodiments, the AABD is the extracellular domain of a receptor or a fragment thereof.

[0318] In one embodiment, the AABD is a single variable domain T-cell receptor (TCR) or a fragment thereof.

[0319] In certain embodiments, the AABD is an autoantigen or a fragment thereof.

[0320] In one embodiment, the AABD is an adaptor domain, an adaptor binding domain, or a fragment thereof. Exemplary adaptor and adaptor binding domains include, but are not limited to, RZIP, EZIP, E4, K4, NKG2D-YA, NKG2D-AF, D domains, and the like.

[0321] The terms "single domain antibody, variable single domain, or immunoglobulin single variable domain (ISV)" are all well known in the art and refer to a single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. As explained below, preferred embodiments of various aspects of the present disclosure relate to SARs comprising a single heavy chain variable domain antibody / immunoglobulin heavy chain single variable domain, referred to as an SVH domain, that binds to different antigens, such as CD19, CD20, CD22, BCMA, CD38, MPL, CD123, CD33, mesothelin, Her2, CS1 / SLAMF7, CD30, GD2, GD3, FLT3, ROR1, CD79b, Lym1, Lym2, PSCA, PSMA, ALK, CD138, CEA, FAP, TAJ, CD229, IL13Ra2, CD32b, GPC3, Mucl6, and KIR3DL2, in the absence of a light chain. Human heavy chain single variable domain antibodies are particularly preferred.

[0322] Thus, in some preferred embodiments, the SARs of the present disclosure comprise a binding domain that comprises or consists of a single domain antibody, said domain being a single human heavy chain variable domain (SVH). L The polypeptide comprises one or more binding domains lacking a domain.

[0323] Thus, in some preferred embodiments, the SAR of the present disclosure comprises a binding domain comprising or consisting of a single domain antibody, said domain being a camelid vHH (or VHH) domain or a humanized vHH domain.

[0324] As used herein, VH Domain: Human V H domain or non-human V H Domain. H The term "domain" includes the human V H Domain V derived from or based on amino acid or nucleic acid sequences H As used herein, the term includes heavy chain variable domains derived from human germline immunoglobulin sequences. H The term domain particularly includes V domains isolated from transgenic mice that express human immunoglobulin V genes in response to immunization with an antigen of interest, e.g., as described in WO2016 / 062990. H Such domains are generally fully human. In one embodiment, human V H The domain is V like this H Human V encoding domain H Domain V derived from or based on amino acid or nucleic acid sequences H The term may also include heavy chain variable domains derived from or encoded by human germline immunoglobulin sequences. H Substantially human V derived from or based on the domain H Domain or V H A domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g., by random or site-directed mutagenesis, or by somatic mutation in vivo). Thus, a "human V" domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g., by random or site-directed mutagenesis, or by somatic mutation in vivo). H The term "domain" also includes a substantially human V domain in which one or more amino acid residues have been modified. H domains, such as substantially human V H Domain V H A domain may contain up to 10, eg, 1, 2, 3, 4 or 5 amino acid modifications relative to the fully human sequence.

[0325] As used herein, VH, VH Alternatively, the term "variable domain" refers to an immunoglobulin variable domain as defined by Kabat et al., Sequences of Immunological Interest, 5th ed., US Dept. Health & Human Services, Washington, DC (1991). The numbering and positions of CDR amino acid residues within a variable domain are according to the well-known Kabat numbering convention.

[0326] In one aspect, the novel AABD of SAR is a single variable heavy chain (VH) domain or SVH domain. The term SVH domain as used herein refers to a single human VH domain antibody (VH sdAb). These terms are used interchangeably. The term SVH is also used interchangeably with independent vH domain or autonomous vH domain. The present disclosure relates to the use of human, generally multiple human SVH domains as building blocks for creating SAR.

[0327] SVH domains are small molecules of 12-14 kDa that can be combined in various formats to obtain multivalent or multispecific antigen-binding domains for SAR. SVH domains are robust and characterized by high affinity and stability in serum. SVH domains are also characterized by high solubility in serum and lack of aggregation.

[0328] Each single V H A domain (SVH) antibody comprises three CDRs and four FRs arranged from amino to carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, in one embodiment of the invention, the domain is a human heavy chain variable (VH) having the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. H ) domain.

[0329] In some embodiments, the present disclosure provides single-chain and multi-chain SARs (e.g., SIRs, cTCR Ab-TCRs, TCRs, αβTFPs, or γδTFPs, etc.) that can be constructed using an SVH domain with a W103R substitution according to the Kabat system. An exemplary SVH-targeted CD19 with a W103R substitution is CD19-FHVH-354, represented by SEQ ID NO: (DNA): 836 and SEQ ID NO: (PRT): 11526 (Table 5). Additional exemplary AABDs comprising an SVH with a W103R substitution are listed in Table 5.

[0330] In another embodiment, the present disclosure provides a multi-chain SAR (e.g., SIR, cTCR Ab-TCR, αβTFP, γδTFP, or recombinant TCR) having a bispecific, bivalent, or biparatopic antigen-binding moiety comprising an SVH domain with a W103R substitution according to the Kabat system. Exemplary SARs based on the SIR framework and comprising CD19-FHVH-354 as one of the antigen-binding domains targeting different antigens are shown in Table 25.

[0331] In another embodiment, the present disclosure provides a multi-chain SAR (e.g., SIR, cTCR Ab-TCR, αβTFP, γδTFP, or recombinant TCR) having a multispecific, multivalent, or multiparatope antigen-binding moiety comprising an SVH domain with a W103R substitution according to the Kabat system. Exemplary bispecific and trispecific SARs targeting different antigens based on the SIR, zSIR, and Ab-TCR scaffolds and comprising CD19-FHVH-354 as one of the antigen-binding domains are shown in Table 26. Because AABDs are modular in nature, CD19-FHVH-354 can be replaced with other AABDs targeting different antigens to develop SARs targeting those antigens.

[0332] In another embodiment, the present disclosure provides a single-chain SAR (e.g., CAR, TFP, etc.) having a bispecific, bivalent, or biparatopic antigen-binding moiety comprising an SVH with a W103R substitution according to the Kabat system. An exemplary single-chain bispecific SAR targeting CD22 and CD19, comprising CD22-FHVH-24 as one of the antigen-binding domains, is CD8SP-CD22-FHVH-24-G4Sx3-R1-hu-mROO5-1-vL-Gly-Ser-linker-hu-mROO5-1-vH-Myc-28z (SEQ ID NO: (DNA): 4118 and SEQ ID NO: (PRT): 14808). In this SAR construct, CD22-FHVH-24 is operably linked to the N-terminus of hu-mROO5-1 scFV via a glycine-serine (G4Sx3) linker. The scFV is fused in-frame to the human CD28 hinge and transmembrane domain via a Myc linker. The SAR also has a CD3z activation domain at its C-terminus. SARs with similar modular structures are also within the scope of the present disclosure.

[0333] In another embodiment, the present disclosure provides a single-chain SAR (e.g., CAR, TFP, etc.) having a multispecific, multivalent, or multiparatopic antigen-binding moiety comprising an SVH having a W103R substitution according to the Kabat system.

[0334] In some embodiments, the present disclosure demonstrates that single-chain and multi-chain SARs can be constructed using SVHs that can form disulfide bonds under suitable conditions and / or are stabilized by the introduction of non-canonical cysteines that form disulfide bridges.An exemplary SVH comprising non-canonical cysteines is CEA-300-aVH, and is shown in SEQ ID NO: (DNA): 954 and SEQ ID NO: (PRT): 11644.Further exemplary such SVHs are shown in WO2019149715, the entire contents of which are incorporated herein by reference.An exemplary SAR incorporating CEA-300-aVH is represented by SEQ ID NO: 21956.

[0335] In one embodiment, the present disclosure aims to alleviate the shortcomings of existing adoptive cell therapies by providing single-chain SARs (e.g., CARs, TFPs, etc.) comprising an SVH, wherein these SVH domains contain substituted cysteines at positions (i) 52a and 71 or (ii) 33 and 52 according to the Kabat numbering, wherein said cysteines are capable of forming disulfide bonds and / or disulfide bridges under suitable conditions.

[0336] In certain embodiments of the present disclosure, the SVH domain used to generate the SAR comprises a substitution selected from the group consisting of 44E, 45E, 45R, (101-1)Y, and 101D according to Kabat numbering. In particular, the SVH comprises a 44E, 45E or 45R, (101-1)Y, and 101D substitution according to Kabat numbering. In certain embodiments, the SVH domain comprises a substitution selected from the group consisting of G44E, T45E, T45R, F(101-1)Y, and A101D according to Kabat numbering. In certain embodiments, the SVH domain comprises a G44E, T45E, T45R, F(101-1)Y, and A101D substitution according to Kabat numbering.

[0337] In certain embodiments of the present disclosure, the SAR comprises an SVH domain having a substitution selected from the group consisting of 44E, 45E, and (101-1)Y according to Kabat numbering. In certain embodiments, the SAR comprises an SVH domain having a 44E, 45E, and (101-1)Y substitution according to Kabat numbering. In certain embodiments, the SVH domain comprises a substitution, when present in the SVH domain, selected from the group consisting of G44E, T45E, and F(101-1)Y according to Kabat numbering. In certain embodiments, the SAR comprises an SVH domain comprising a G44E, T45E, and F(101-1)Y substitution according to Kabat numbering.

[0338] In one embodiment, the SVH domain of the SAR comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21411, 21412, 21413 and 21414, respectively.

[0339] In one embodiment, the SVH domain of the SAR comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21415, 21416, 21417, and 21418, respectively.

[0340] In one embodiment, the SVH domain of the SAR comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21419, 21420, 21421, and 21422, respectively.

[0341] In certain embodiments of the present disclosure, the SVH domain of the SAR comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21423, 21424, 21425, and 21426, respectively.

[0342] In certain embodiments of the present disclosure, the SVH domain comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21427, 21428, 21429, and 21430, respectively.

[0343] In certain embodiments of the present disclosure, the SVH domain comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21431, 21432, 21433, and 21434, respectively.

[0344] In one embodiment, the SVH domain comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21435, 21436, 21437, and 21438, respectively.

[0345] In one embodiment, the SVH domain comprises a vH framework comprising FR1, FR2, FR3 and FR4 having at least 85% sequence identity to the amino acid sequences of SEQ ID NOs: 21439, 21440, 21441, and 21442, respectively.

[0346] SVH domains are particularly useful for constructing SARs because FR1-4 of SEQ ID NOs: 21411-21442 are not immunogenic in humans.

[0347] The present disclosure also relates to the use of human, generally multiple human SVH domains, as building blocks for creating SARs. The term SVH domain, as used herein, refers to a single human VH domain antibody (VH sdAb). Thus, these terms are used interchangeably. The term SVH is also used interchangeably with independent vH domain or autonomous vH domain. SVH is a type of AABD.

[0348] SVH domains are small molecules of 12-14 kDa that can be combined in various formats to obtain multivalent or multispecific antigen-binding domains for SAR. SVH domains are robust and characterized by high affinity and stability in serum. SVH domains are also characterized by high solubility in serum and lack of aggregation.

[0349] In some embodiments, the SAR constructs described herein include a human SVH domain (generally multiple human SVH domains) that recognizes a target protein of interest, e.g., a protein expressed on tumor cells, such as an antigen.

[0350] In some embodiments, the SAR constructs described herein include a human SVL domain (generally multiple human SVL domains) that recognizes a target protein of interest, for example, a protein expressed on tumor cells, such as an antigen. The term SVL domain, as used herein, refers to a single human VL domain antibody (VL sdAb). Thus, these terms are used interchangeably. The term SVL is also used interchangeably with independent vL domain or autonomous vL domain. SVL is a type of AABD.

[0351] In one aspect, the AABD of the SAR is a camelid vHH domain. The present disclosure also relates to a SAR comprising multiple vHH domains. The present disclosure also relates to a SAR comprising a humanized vHH domain. Exemplary vHH domains that can be used to construct the SAR of the present disclosure and their target antigens are shown in Table 5.

[0352] In one aspect, the AABD of the SAR is a non-immunoglobulin antigen-binding scaffold such as a DARPIN, affibody, affilin, adnectin, afftin, obody, lipibody, finomer, alphabody, avimer, atrimer, centilin, pronectin, anticalin, Kunitz domain, armadillo repeat protein, D domain, or fragment thereof. The present disclosure also relates to SARs comprising multiple non-immunoglobulin antigen-binding scaffolds. Exemplary non-immunoglobulin antigen-binding scaffolds and their target antigens that can be used to construct the SARs of the present disclosure are shown in Tables 7-9.

[0353] In one aspect, the AABD of the SAR is an adaptor-binding domain (e.g., RZIP, EZIP, E4, K4, NKG2D-AF, NKG2D-YA, or D domain, etc.). The present disclosure also includes SARs that bind to multiple adaptors. In one embodiment, the adaptor-binding domain is a leucine zipper domain. In one aspect, the AABD of the SAR binds to an adaptor (e.g., RZIP, EZIP, E4, K4, D domain, Streptag, FITC, biotin, ULBP2R, ULBP2-S3, etc.). It will be understood by those skilled in the art that adaptors and adaptor-binding proteins can be substituted for each other. Thus, a SAR can comprise an RZIP module that binds to a SAR adaptor comprising an EZIP module. Alternatively, a SAR can comprise an EZIP module, and a SAR adaptor can comprise an RZIP module. The present disclosure also includes SARs that bind to multiple adaptors. Exemplary adaptors and adaptor-binding proteins are shown in Table 10.

[0354] In one aspect, the AABD of the SAR is an extracellular ligand-binding domain of a receptor or a fragment thereof. The present disclosure also includes SARs comprising multiple extracellular ligand-binding domains of a receptor. Exemplary extracellular ligand-binding domains of receptors that can be used to construct SARs are shown in Table 8.

[0355] In one aspect, the AABD of the SAR is an extracellular receptor binding domain of a ligand or cytokine, or a fragment thereof. The present disclosure also includes SARs comprising multiple extracellular receptor binding domains of a ligand or cytokine. Exemplary extracellular receptor binding domains of ligands or cytokines that can be used to construct SARs are shown in Table 9.

[0356] In one aspect, the AABD of the SAR is an autoantigen. The present disclosure also includes SARs comprising multiple autoantigens. An exemplary autoantigen that can be used to construct a SAR is Dsg3 (SEQ ID NO: 11687) or a fragment thereof.

[0357] In one aspect, the AABD of the SAR is a single variable domain of a T cell receptor (svd-TCR). The present disclosure also includes SARs comprising multiple single variable domains of a T cell receptor. Exemplary polynucleotides comprising svd-TCR domains are set forth in SEQ ID NOs: (DNA): 21563-21564 and WO2021030182, the entire contents of which are incorporated herein by reference.

[0358] In one aspect, the AABD of the SAR is any protein that can bind to an antigen expressed on the surface of a cell.

[0359] The multiple AABDs in a SAR can be present in various combinations (eg, two centirins, one centirin and one vHH domain, a vHH domain, an SVH domain and centirin, etc.).

[0360] In one aspect, the AABD of the SAR is centirin. The present disclosure also relates to a SAR comprising multiple centirins. In one aspect, the AABD of the SAR is DARPINS. The present disclosure also relates to a SAR comprising multiple DARPINs. Similarly, the present disclosure relates to a SAR containing multiple non-immunoglobulin antigen-binding domains, such as affibodies, affilins, adnectins, affitins, obodies, lipibodies, fynomers, alphabodies, avimers, atrimers, pronectins, anticalins, Kunitz domains, and armadillo repeat proteins.

[0361] In some embodiments, the SAR comprises multiple AABDs. In certain embodiments, a first AABD is linked to a second AABD, and the first AABD and the second AABD specifically bind to an antigen. In certain embodiments, the antigen recognized by the SAR is a peptide antigen bound to an MHC complex. In some embodiments, two or more AABDs of the SAR recognize the same antigen. In other embodiments, two or more AABDs of the SAR recognize different antigens.

[0362] While scFvs, commonly used in SARs such as second-generation CARs, have the potential for unwanted aggregation, clustering, and immunogenicity, the use of autonomous antigen-binding domains (AABDs) provides a stable format with substantially reduced potential for immunogenicity, nonspecific aggregation, or unfolding. This is particularly useful when designing SARs with bispecific, bivalent, or biparatopic antigen-binding moieties. As demonstrated herein by the inventors, multiple AABD domains can be readily used in such multimeric formats, facilitating the creation of multispecific SARs that enable simultaneous targeting of two or more target antigens or two or more epitopes of an antigen.

[0363] In some embodiments, the present disclosure provides SARs that can target one or more antigens (e.g., 1, 2, 3, 4, 5, 6, or more antigens). In some embodiments, the present disclosure provides SARs that can target one or more epitopes (e.g., 1, 2, 3, 4, 5, 6, or more epitopes). In some embodiments, the present disclosure provides SARs that comprise one or more antigen-binding domains (e.g., 1, 2, 3, 4, 5, 6, or more antigen-binding domains).

[0364] In one embodiment, the present disclosure provides a SAR comprising one or more chains, each chain comprising zero, one, or more antigen-binding domains operably linked to a transmembrane domain, an optional activation domain, and an optional costimulatory domain. In one embodiment, the activation domain encodes one or more ITAM motifs.

[0365] In one embodiment, the present disclosure provides a single-chain SAR comprising one or more antigen-binding domains operably linked to a transmembrane domain, an activation domain, and an optional costimulatory domain. In one embodiment, the activation domain encodes one or more ITAM motifs. Exemplary such SARs are first-generation CARs, second-generation CARs, third-generation CARs, or SARs expressed in the backbone of εTFP, γTFP, δTFP, or ζTFP. Exemplary such SARs are represented by SEQ ID NOs: (DNA): 4087, 4110-4113, 4118-4120, SEQ ID NOs: (PRT): 14777, 14800-14803, and 14808-14810.

[0366] The present disclosure also provides second-generation CARs, K13-CARs, and single-chain SARs based on TFP (e.g., TFPε, TFPγ, TFPδ) backbones (Tables 33 and 34). The present disclosure provides single-chain SARs with modular domain structures and the structure of an exemplary SAR on a second-generation CAR backbone that targets BCMA and CD19, represented by CD8SP-BCMA-FHVH-93-SG4S-CD19-FHVH-354-Myc-28z (SEQ ID NO: 17667). Another exemplary SAR on a second-generation CAR backbone that targets BCMA and CD19 is CD8SP-CD19-FHVH-354-SG4S-BCMA-FHVH-93-Myc-CD8TM-BBz (SEQ ID NO: 17669). Exemplary SARs on a BCMA- and CD19-targeting TFP scaffold are CD8SP-BCMA-FHVH-93-SG4S-CD19-FHVH-354-CD3e-ECDTMCP-opt2 (SEQ ID NO: 17696), CD8SP-BCMA-FHVH-93-SG4S-CD19-FHVH-354-CD3d-ECDTMCP-opt2 (SEQ ID NO: 17697), and CD8SP-BCMA-FHVH-93-SG4S-CD19-FHVH-354-CD3g-ECDTMCP-opt2 (SEQ ID NO: 17698). The present disclosure provides single-chain SARs with modular domain structures and additional exemplary SAR structures on a BCMA- and CD19-targeting TFP scaffold, such as those represented by SEQ ID NOs: 17700-17703.

[0367] The present disclosure provides single-chain SARs with modular domain structures and exemplary SAR structures in the backbone of second-generation CARs targeting CD20 and CD22, represented by CD8SP-CD20-vHH-USC1-2HC2D6-SG4S-CD22-USC1-FHVH-160-Myc-28z and CD8SP-CD22-USC1-FHVH-160-SG4S--CD20-vHH-USC1-2HC2D6-Myc-CD8TM-BBz, represented by SEQ ID NOs: 17813 and 17815. The present disclosure provides SARs with modular domain structures and exemplary SAR structures in a first generation CAR backbone that co-expresses K13 and targets CD20 and CD22, represented by CD8SP-CD20-vHH-USC1-2HC2D6-SG4S-CD22-USC1-FHVH-160-Myc-CD8TM-z-P2A-K13-FLAG-T2A-PAC (SEQ ID NO: 17816). The present disclosure provides SARs with modular domain structures and exemplary SAR structures in a TFP backbone that targets CD20 and CD22, as represented by SEQ ID NOs: 17842-17849.

[0368] Exemplary SARs on second generation CAR scaffolds targeting BCMA and CD38 are CD8SP-BCMA-FHVH-74-SG4S-CD38-FHVH-USC1-32184-Myc-28z and CD8SP-CD38-FHVH-USC1-32184-SG4S-BCMA-FHVH-74-Myc-CD8TM-BBz, represented by SEQ ID NOs: 17886 and 17888. An exemplary SAR on the scaffold of a first generation CAR that co-expresses K13 and targets BCMA and CD38 is CD8SP-BCMA-FHVH-74-[hTCRb-S57C]-F-P2A-SP-CD38-FHVH-USC1-32184-[hTCRa-T48C]-F-F2A-K13-opt (SEQ ID NO: 17893). Exemplary SAR on the scaffold of a TFP that targets BCMA and CD38 are represented by SEQ ID NOs: 17915-17922.

[0369] Exemplary SARs on the backbone of second-generation CARs targeting CD20 and CD19 are CD8SP-CD20-vHH-USC1-2HC2D6-SG4S-CD19-FHVH-354-Myc-28z and CD8SP-CD20-vHH-USC1-2HC2D6-SG4S-CD19-FHVH-354-Myc-CD8TM-BBz, represented by SEQ ID NOs: 17959 to 17960. An exemplary SAR on the backbone of a first-generation CAR co-expressing K13 and targeting CD20 and CD19 is CD8SP-CD20-vHH-USC1-2HC2D6-SG4S-CD19-FHVH-354-Myc-CD8TM-z-P2A-K13-FLAG-T2A-PAC (SEQ ID NO: 17962). Exemplary SARs on TFP scaffolds targeting BCMA and CD38 are represented by SEQ ID NOs: 17988-17995.

[0370] Exemplary SARs on the backbone of second-generation CARs targeting CD22 and CEA are CD8SP-CD22-FHVH-24-SG4S-CEA-aVH-3001-Myc-28z and CD8SP-CD22-FHVH-24-SG4S-CEA-aVH-3001-Myc-CD8TM-BBz, represented by SEQ ID NOs: 17521 to 17522. An exemplary first-generation CAR co-expressing K13 and an optional PAC module and targeting CD22 and CEA is CD8SP-CD22-FHVH-24-SG4S-CEA-aVH-3001-Myc-CD8TM-z-P2A-K13-FLAG-T2A-PAC (SEQ ID NO: 17524). Exemplary SARs on TFP scaffolds targeting CD22 and CEA are represented by SEQ ID NOs: 17550-17557.

[0371] An exemplary double-chain SAR on an AABD-TCR scaffold targeting CD20 and CD22 is CD8SP-CD20-vHH-USC1-2HC2D6-IgG1-hinge-[TCRg-6MD]-F-P2A-SP-CD22-USC1-FHVH-160-IgG1-hinge-v2-[TCRd-6MD], represented by SEQ ID NO: 17883. Exemplary SARs on the AABD-TCR scaffold targeting CD20 and CD22 are CD8SP-CD20-vHH-USC1-2HC2D6-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-SP-CD22-USC1-FHVH-160-[IgG1-CH1-TCRa-wt-opt2-6MD] (SEQ ID NO: 17868) and CD8SP-CD20-vHH-USC1-2HC2D6-[IgCL-TCRg-6MD]-F-P2A-SP-CD22-USC1-FHVH-160-[IgG1-CH1-TCRd-6MD] (SEQ ID NO: 17877). Exemplary SARs on AABD-TCR scaffolds targeting BCMA and CD38 are CD8SP-BCMA-FHVH-74-[IgCL-TCRg-6MD]-F-P2A-SP-CD38-FHVH-USC1-32184-[IgG1-CH1-TCRd-6MD] and CD8SP-BCMA-FHVH-74-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-SP-CD38-FHVH-USC1-32184-[IgG1-CH1-TCRa-wt-op2-6MD], represented by SEQ ID NOs: 17912 and 17914. Exemplary SARs on AABD-TCR scaffolds targeting CD20 and CD19 are CD8SP-CD20-vHH-USC1-2HC2D6-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-SP-CD19-FHVH-354-[IgG1-CH1-TCRa-wt-opt2-6MD] and CD8SP-CD20-vHH-USC1-2HC2D6-[IgCL-TCRg-6MD]-F-P2A-SP-CD19-FHVH-354-[IgG1-CH1-TCRd-6MD], represented by SEQ ID NOs: 18016 and 18023, respectively.Because SARs are modular in nature, the AABDs of the above SARs can be replaced with AABDs targeting different antigens to develop SARs targeting those antigens. Table 33 shows the names, module compositions, and SEQ ID NOs of SARs targeting CD22 and PSMA. Table 34 shows SEQ ID NOs of SARs created from the SARSs listed in Table 33 by replacing the AABD targeting CD22 (i.e., CD22-FHVH-24) and the AABD targeting PSMA (i.e., PSMA-chVH-71v2) with AABDs targeting different antigens. Otherwise, the order of the constructs in Table 34 is the same as the order of the constructs in Table 33.

[0372] In one embodiment, the present disclosure provides a synthetic antigen receptor comprising (a) one or more antigen-specific targeting regions, (b) at least one extracellular linker domain, (c) at least one transmembrane domain, (d) an optional costimulatory domain, and (e) an optional intracellular signaling domain, wherein one antigen-specific targeting region comprises an antigen-specific single-chain Fv (scFv) fragment, and the second antigen-specific targeting domain comprises an AABD. In an exemplary embodiment, the AABD is a non-scFv antigen binding module (e.g., SVH, vHH, FHVH, SVL, svd-TCR, centrin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, D domain, etc.).

[0373] In one embodiment, the present disclosure provides a bispecific or multispecific synthetic antigen receptor comprising (a) at least two antigen-specific targeting regions, (b) at least one extracellular linker domain, (c) at least one transmembrane domain, (d) an optional costimulatory domain, and (e) an optional intracellular signaling domain, wherein one antigen-specific targeting region comprises an antigen-specific single-chain Fv (scFv) fragment and the second antigen-specific targeting domain comprises an AABD. In one exemplary embodiment, the AABD is a non-scFv antigen binding module (e.g., SVH, vHH, FHVH, SVL, svd-TCR, centrin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, D domain, etc.). In one embodiment, the present disclosure provides a bispecific or multispecific synthetic antigen receptor having the general formula: (AABD)n-optional linker domain-scFv-hinge domain-transmembrane domain-optional one or more costimulatory domains-activation domain; n=0, 1, 2, 3, 4, 5, or more, where the activation domain may contain one or more ITAM motifs. An exemplary such SAR is represented by IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-hu-mROO5-1-vH-Gly-Ser-linker-vL-Myc-CD8TM-BBz and has SEQ ID NO: (DNA): 4087. This SAR has one antigen-binding domain represented by humanized hu-mROO5-1 targeting CD19 and a second antigen-binding domain represented by CD20-vHH-2HCD25 targeting CD20. These two antigen-binding domains are linked via a Gly-Ser (G4Sx3v2) flexible linker. This SAR construct also comprises a Myc tag, a human CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3z activation domain. Other exemplary such constructs are represented by first-generation CARs, second-generation CARs, third-generation CARs, or SARs with εTFP, γTFP, δTFP, or ζTFP backbones.Exemplary such SARs are represented by SEQ ID NOs: (DNA): 4087, 4110-4113, 4118-4120, SEQ ID NOs: (PRT): 14777, 14800-14803 and 14808-14810.

[0374] In some embodiments, the present disclosure provides SARs comprising one or more chains, each chain comprising zero, one, two, or more antigen-binding domains operably linked to a transmembrane domain, but lacking an activation domain. Such SARs lack their own activation domains but are capable of signal transduction through the recruitment of a signaling module comprising a protein encoding the activation domain. Exemplary such SARs are SARs based on the scaffold of SIR, cTCR, Ab-TCR, TCR, αβTFP, or γδTFP. In some exemplary embodiments, the present disclosure provides SARs in which one or more AABDs are attached to or near the N-terminus of one or both chains of SIR, cTCR, Ab-TCR, TCR, αβTFP, or γδTFP. In some exemplary embodiments, the present disclosure provides SARs in which one or more AABDs are attached to or near the N-terminus of a vL or vH fragment comprising one or both chains of SIR, cTCR, Ab-TCR, αβTFP, or γδTFP. In certain exemplary embodiments, the present disclosure provides SARs in which one or more AABDs are attached at or near the N-terminus of a Va, Vb, Vg, or Vd fragment comprising one or both chains of a TCR. Exemplary such SARs are represented by SEQ ID NOs: (DNA): 4088-4098, 4107-4109, 4166, and 3500-3510 and SEQ ID NOs: (PRT): 14778-14788, 14797-14799, 14856, and 14190-14200. Further exemplary such SARs are set forth in Tables 25-32. In certain exemplary embodiments, the AABD is a non-scFv antigen binding module (e.g., an SVH, vHH, FHVH, SVL, svd-TCR, centirin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, D domain, etc.).

[0375] In one embodiment, the present disclosure provides a single-chain, 1.5-chain, or dual-chain SAR having the general formula: (AABD)n-optional linker domain-scFv-optional linker-TCR constant chain; n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain comprises at least one chain representing a constant chain of TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, pre-TCRα, or a mutant or variant thereof. Exemplary such SARs are represented by SEQ ID NOs: (DNA): 4127 and 4128 and SEQ ID NOs: (PRT): 14817-14818. In one embodiment, the scFv is replaced with a single-chain soluble TCR comprising the Va and Vb fragments of an αβTCR connected by a flexible linker or the Vg and Vd fragments from a γδTCR connected by a flexible linker.

[0376] In certain embodiments, the present disclosure provides a 1.5-chain SAR or a dual-chain SAR comprising one chain having the general formula: (AABD)n-optional linker domain-scFv-optional linker-TCR constant chain; and a second chain having the general formula (AABD)n-optional linker domain-TCR constant chain, where n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain represents the constant chain of TCR alpha (Cα), TCR beta 1 (Cβ1), TCR beta 2 (Cβ2), TCR gamma (Cγ), TCR delta (Cδ), or pre-TCR alpha (pre-Cα), or a mutant or variant thereof. In certain embodiments, the two chains of the SAR comprise complementary TCR constant chains; i.e., Cα and Cβ, pretc. Rα and Cβ, or Cγ and Cδ. The Cβ chain can represent either Cβ1 or Cβ2. Exemplary such SARs are represented by SEQ ID NOs: (DNA): 4127 and 4128 and SEQ ID NOs: (PRT): 14817-14818. In one embodiment, the scFv is replaced with a single-chain soluble TCR.

[0377] In one embodiment, the present disclosure provides a 1.5-chain synthetic antigen receptor comprising two TCR constant chains or fragments thereof, one of which comprises an AABD connected via an optional linker to an antigen-specific single-chain Fv (scFv) fragment, which is in turn connected to a TCR constant chain or fragment thereof, and the second chain comprises the complementary TCR constant chain of the fragment. In an exemplary embodiment, the AABD is a non-scFv antigen-binding module (e.g., SVH, vHH, FHVH, SVL, svd-TCR, centrin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, D domain, etc.). An exemplary such SAR is represented by CD8SP-MYC-[hTCRa-T48C-opt1]-F-F2A-SP-CD22-FHVH-24-G4Sx3-R1-hu-mROO5-1-vL-Gly-Ser-linker-hu-mROO5-1-vH-V5-[hTCRb-S57C-opt1]-F-P2A-PAC, and has SEQ ID NO: (DNA): 4128 and SEQ ID NO: (PRT): 14818. This SAR has a backbone of a 1.5-strand SIR (see WO2018102795) and comprises one strand encoding a MYC tag joined in-frame to a mutant human TCR alpha constant chain (hTCRa-T48C-opt1; SEQ ID NO: 11738). The second chain of this SAR comprises one antigen-binding domain represented by the hu-mROO5-1 scFv (SEQ ID NO:633 and SEQ ID NO:11323) targeting CD19 and a second AABD represented by the fully human vH domain CD22-FHVH-24 (SEQ ID NO:843 and SEQ ID NO:11533) targeting CD22. These two antigen-binding domains are linked via a Gly-Ser (G4Sx3) flexible linker. The hu-mROO5-1 scFv is in-frame connected to a mutant human TCR β constant chain with an S57C mutation (hTCRb-S57C-opt1; SEQ ID NO:11749) module via a V5 tag.These two chains are encoded by a single polynucleotide and are separated by a P2A cleavable linker. The SAR also optionally encodes a puromycin resistance gene (PAC). Another exemplary SAR construct is represented by CD8SP-V5-[hTCRb-KACIAH]-F-P2A-CD8SP-CD22-FHVH-24-G4Sx3-R1-hu-mROO5-1-vL-Gly-Ser-linker-hu-mROO5-1-vH-Myc-[hTCRa-CSDVP]-F-F2A-PAC, and is represented by SEQ ID NO: (DNA): 4127 and SEQ ID NO: (PRT): 14817. This SAR also has a framework of 1.5 chain SIR (see WO2018102795) and comprises one chain encoding a V5 tag linked in-frame to a mutant human TCR beta constant chain (hTCRb-KACIAH; SEQ ID NO: 11750). The second chain of this SAR comprises one antigen-binding domain represented by hu-mROO5-1 scFv targeting CD19 and a second antigen-binding domain represented by CD22-FHVH-24 targeting CD22. These two antigen-binding domains are linked via a Gly-Ser (G4Sx3) flexible linker. The humanized hu-mROO5-1 scFv is linked in-frame to a mutant human TCR alpha constant chain (hTCRa-CSDVP; SEQ ID NO: 11734) module via a Myc epitope tag.

[0378] In one embodiment, the present disclosure provides a bispecific synthetic antigen receptor comprising (a) at least two antigen-specific targeting regions, (b) at least one extracellular linker domain, (c) at least one transmembrane domain, (d) an optional costimulatory domain, and (e) an optional intracellular signaling domain, wherein the antigen-specific targeting domain comprises an AABD. In one exemplary embodiment, both AABDs are non-scFv antigen binding modules (e.g., SVH, vHH, FHVH, SVL, svd-TCR, centilin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, and D domains, etc.). An exemplary such SAR is CD8SP-CD22-FHVH-24-SG4S-PSMA-chVH-71v2-Myc-CD8TM-BBz-T2A-PAC (SEQ ID NO: 6464), which comprises a CD8 signal peptide, a CD22-targeting fully human vH domain (FVHV), a Ser-Glyx4-Ser linker, a PSMA-targeting single vH domain, a Myc tag, a CD8 hinge and transmembrane region, a 4-1BB costimulatory domain, and a CD3 activation domain.

[0379] In certain embodiments, the disclosure provides single-chain, 1.5-chain, or double-chain SARs having the general formula: (AABD)n-optional linker domain-vL or vH fragment-optional linker-TCR constant chain; n=0, 1, 2, 3, 4, 5, or more, wherein the TCR constant chain comprises at least one chain representing a constant chain of TCR alpha, TCR beta 1, TCR beta 2, TCR gamma, TCR delta, or pre-TCR alpha, or a mutant or variant thereof.

[0380] In one embodiment, the present disclosure provides a double-chain SAR comprising one chain having the general formula: (AABD)n-optional linker domain-vL fragment-optional linker-TCR constant chain; and a second chain having the general formula: (AABD)n-optional linker domain-vH fragment-optional linker-TCR constant chain, where n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain represents the constant chain of TCR alpha (Cα), TCR beta 1 (Cβ1), TCR beta 2 (Cβ2), TCR gamma (Cγ), TCR delta (Cδ), or pre-TCR alpha (pre-Cα), or a mutant or variant thereof. In one embodiment, the two chains of the SAR comprise complementary TCR constant chains; i.e., Cα and Cβ, pretc. Rα and Cβ, or Cγ and Cδ. The Cβ chain can represent either Cβ1 or Cβ2.

[0381] In one embodiment, the present disclosure provides a dual-chain bispecific synthetic antigen receptor comprising two chains, each chain comprising (a) one or more antigen-specific targeting regions, (b) at least one extracellular linker domain, (c) at least one transmembrane domain, (d) an optional costimulatory domain, and (e) an optional intracellular signaling domain, wherein one antigen-specific targeting region comprises a variable region fragment (vL and / or vH fragment) that can be combined with a vH and / or vL fragment present in a second chain to form an Fv, and the second antigen-specific targeting domain comprises an AABD (e.g., a vHH, SVH, centilin, affibody, etc.). In one embodiment, the Fv binds to the antigen. In another embodiment, the Fv does not bind to the antigen. In one embodiment, the Fv serves as a scaffold for attachment of a second antigen-specific targeting domain comprising an AABD. In one embodiment, the AABD is a non-scFv antigen-binding domain.

[0382] An exemplary dual-chain bispecific SAR comprising two chains is CD8SP-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-USC1-vHH-2HCD26-G4Sx3v2-hu-mROO5-1-vH-[hTCRa-T48C], represented by SEQ ID NO:(DNA):7406 and SEQ ID NO:(PRT):18096. This SAR construct has the backbone of the SIR described in WO2018102795, the entire contents of which are incorporated herein by reference. One chain of the SAR construct comprises a humanized hu-mROO5-1 vL fragment fused to the constant chain of human TCRb with an S57C mutation (hTCRb-S57C), and the other chain of the SAR comprises a humanized hu-mROO5-1 vH fragment fused to the constant chain of human TCRa with a T48C mutation (hTCRa-T48C). The hu-mROO5-1 vL fragment and hu-mROO5-1 vH fragment of the SAR together form an Fv targeting CD19. A vHH fragment targeting CD20 (CD20-USC1-vHH-2HCD26; SEQ ID NO: 841) is fused to the N-terminus of the hu-mROO5-1 vH fragment via a glycine-serine linker. Thus, the SAR targets CD19 via the hu-mROO5-1 Fv and CD20 via CD20-USC1-vHH-2HCD26. Note that the SAR is modular. Therefore, one module of the SAR can be replaced with another module. For example, the hu-mROO5-1 vL and hu-mROO5-1 vH fragments can be replaced with vL / vH fragments targeting different antigens. Table 25 shows the target antigens, names, nucleic acid sequence numbers, and amino acid sequence numbers of several exemplary bispecific SARs on an SIR scaffold comprising vL / vH fragments targeting different antigens and also comprising an SVH (CD19-FHVH-354) targeting CD19. In the SARs listed in Table 25, the vL fragment is operably linked to the hTCRb-S57C chain, and the vH fragment is operably linked to the hTCRa-T48C chain.A SAR can be constructed in which the vL fragment is operably linked to the hTCRa-T48C chain or the hTCRb-S57C chain, and the vH fragment is operably linked to the hTCRb-S57C chain. Exemplary such SAR constructs targeting different antigens are represented by SEQ ID NOs: (DNA): 2014-2177 and (PRT): 12704-12868. The order of the vL / vH fragments in these constructs is the same as the order of the vL / vH fragments in the constructs shown in Table 25. Thus, the SAR construct with the BCMA-J6M0 vL / vH fragment is the eighth construct in Table 25. Thus, the nucleic acid and amino acid sequences of the SAR construct CD8SP-CD19-FHVH-354-G4S3-BCMA-J6M0-vL-[hTCRa-T48C]-F-F2A-SP-BCMA-J6M0-vH-[hTCRb-S57C] carrying the BCMA-J6M0 vL / vH fragment can be calculated to be 2021 (i.e., 2014+8) and 12711 (i.e., 12704+8), respectively. Note that due to the modular nature of the SAR, the hTCRb-S57C chain can be substituted with deletion mutants and / or variants as long as it retains its biological activity (e.g., the ability to associate with the TCRa chain and / or the TCR / CD3 complex and / or the ability to induce antigen-dependent T cell signaling). Nucleic acid and amino acid SEQ ID NOs for exemplary deletion mutants and variants of the TCRb chain are listed in Table 12 (e.g., SEQ ID NOs:(DNA):1056-1076 and SEQ ID NOs:(DNA):1114-1127). Similarly, the hTCRa-T48C chain can be substituted with deletion mutants and / or variants so long as it retains its biological activity (e.g., the ability to associate with the TCRb chain and / or the TCR / CD3 complex and / or the ability to induce antigen-dependent T cell signaling). Nucleic acid and amino acid SEQ ID NOs for exemplary deletion mutants and variants of the TCRa chain are listed in Table 12 (e.g., SEQ ID NOs:(DNA):10421052 and SEQ ID NOs:(DNA):1103-1113).

[0383] The present disclosure also describes novel deletion mutants of the TCRa and TCRb chains that can be used to construct SIRs and bispecific / multispecific SARs. An exemplary SIR comprising deletion mutants of the TCRa and TCRb constant chains is CD8SP-hu-mROO5-1-vL-[hTCRb-S57C-opt-d17]-F-P2A-SP-hu-mROO5-1-vH-[hTCRa-T48C-opt-d17], represented by nucleic acid SEQ ID NO: 1331 and amino acid SEQ ID NO: 12021. SIR / SAR constructs containing deletion mutants of the TCRa and TCRb chains have the advantage of being small in size and therefore increasing the titer of the lentiviral / retroviral vectors encoding them, since the titer of a lentiviral / retroviral vector is inversely proportional to the insert size.

[0384] The present disclosure provides, in Table 26, the target antigens, SAR names, module compositions, DNA SEQ ID NOs, and amino acid SEQ ID NOs for several exemplary bispecific and trispecific SARs comprising vL / vH fragments targeting different antigens on different backbones (i.e., with different TCR constant chains). These SAR constructs comprise a fully human single vH domain targeting CD19 (CD19-FHVH-354) and / or a fully human single vH domain targeting CD22 (CD22-FHVH-24). The names and compositions of the first constructs in each series are shown in Table 26 and can be used to determine the TCR constant chain modules present in that series. For example, it can be determined from Table 26 that in the constructs represented by nucleic acid SEQ ID NOs: 2178-2343, the vL fragment is operably linked to the [hTCRbECD-CD3zECDTMCP-opt] module and the vH fragment is operably linked to the [hTCRaECD-CD3zECDTMCP-opt2] module. The order of the different constructs in this series is the same as the order of the constructs in the series shown in Table 25, and the vL / vH fragments of the different constructs in this series can be determined from the constructs with the same vL / vH fragments in Table 25. A similar approach can be used to determine the SEQ ID NOs of other constructs with different vL / vH fragments in the series listed in Table 26 with reference to Table 25. Other SARs based on the modular structure of the SARs described in Tables 25-26 are also within the scope of the present disclosure.

[0385] The nucleic acid and amino acid SEQ ID NOs for exemplary bispecific and trispecific SARs based on vL and vH fragments derived from FMC63 scFv and with different TCR constant chains are shown in Table 26. The nucleic acid and amino acid SEQ ID NOs for exemplary bispecific and trispecific SARs based on vL and vH fragments derived from other scFv fragments can be derived by replacing the vL and vH fragments of FMC63 scFv with the vL and vH fragments comprising the SARs listed in Table 25. The order of the SAR constructs comprising different vL / vH fragments in Table 26 is the same as the order of the SARs shown in Table 25 for the SARs.

[0386] Another exemplary dual-chain bispecific SAR comprising two chains is CD8SP-hu-mROO5-1-vL-[IgCL-TCRg-6MD]-F-P2A-IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-hu-mROO5-1-vH-[IgG1-CH1-TCRd-6MD], represented by SEQ ID NO: (DNA): 5037 and SEQ ID NO: (PRT): 15727. In this SAR construct, one chain of the SAR comprises the hu-mROO5-1 vL fragment fused to a truncated constant chain of human TCRg (TCRg-6MD) via an IgCL linker, and the other chain of the SAR comprises the hu-mROO5-1 vH fragment fused to a truncated constant chain of human TCRd (TCRd-6MD) via an IgG1-CH1 linker. The hu-mROO5-1 vL fragment and hu-mROO5-1 vH fragment of the SAR assemble to form an Fv targeting CD19. A vHH fragment targeting CD20 (CD20-vHH-2HCD25) is fused to the N-terminus of the hu-mROO5-1 vH fragment via a glycine-serine linker. Thus, the SAR targets CD19 via the hu-mROO5-1 Fv and CD20 via CD20-USC1-vHH-2HCD25. Table 26 shows the nucleic acid SEQ ID NOs (2839-3003) and amino acid SEQ ID NOs (13529-13693) of several exemplary bispecific SARs in which the vL fragment is operably linked to an [IgCL-TCRg-6MD] module and the vH fragment is operably linked to an [IgG1-CH1-TCRd-6MD] module, and which comprise vL / vH fragments targeting different antigens. These SAR constructs also comprise an SVH (CD19-FHVH-354) targeting CD19. The order of the different constructs in this series is the same as the order of the constructs in the series shown in Table 25, so the vL / vH fragments of the different constructs in this series can be determined from the constructs with the same vL / vH fragments listed in Table 25.Because SARs are modular, it is possible to replace one module with another module or a variant thereof, as long as the functional activity of the SAR (i.e., its ability to bind to its target antigen, its ability to induce T cell signaling upon antigen binding, its ability to assemble into a TCR / CD3 complex, etc.) is maintained. Thus, in an exemplary embodiment, the vL fragment of the SAR is operably linked to an [IgG1-CH1-TCRd-6MD] module or a variant thereof, and the vH fragment is operably linked to an [IgCL-TCRg-6MD] module or a variant thereof. The IgCL and IgG1-CH1 linkers can be replaced with other linkers, as long as the functional activity of the SAR (i.e., its ability to bind to its target antigen, its ability to induce T cell signaling upon antigen binding, its ability to assemble into a TCR / CD3 complex, etc.) is maintained. Exemplary such linkers are shown in Table 13. In a preferred embodiment, one chain of the bispecific SAR comprises IgCL (SEQ ID NO: 11832) or a variant thereof, and the complementary strand comprises a linker represented by SEQ ID NOs: 11833-11847. Table 13 also lists components of TCR constant chains that can function as linkers in the construction of SARs. Exemplary linkers based on the TCRa / TCRα constant chain are represented by SEQ ID NOs: 11848-11849, 11857-11859. Exemplary linkers based on the TCRb / TCRβ constant chain are represented by SEQ ID NOs: 11850-11851, 11856, 11860-11861. Exemplary linkers based on the TCRg / TCRγ constant chain are represented by SEQ ID NOs: 11852-11853, 11862, 11863. Exemplary linkers based on the TCRd / TCRδ constant chain are represented by SEQ ID NOs: 11854-11855, 11862, 11864-11865. In one embodiment, one chain of the double-chain SAR comprises a linker based on the TCRa chain, and the other chain comprises a linker based on the TCRb chain. In another embodiment, one chain of the double-chain SAR comprises a linker based on the TCRg / TCRγ chain, and the other chain comprises a linker based on the TCRd / TCRδ chain.In one embodiment, one chain of the double-chain SAR comprises a linker based on a TCRa chain, and the other chain comprises a linker based on an IgCL (SEQ ID NO: 11832). Other linkers based on immunoglobulin-like modules are known in the art and can be used in other embodiments of the present disclosure. Finally, in addition to TCR chains and modules, the CD20-vHH-2HCD25 fragment can be replaced with another AABD that targets a different antigen or a different epitope of CD20. Exemplary AABDs are shown in Tables 5, 7-10. Additional AABDs (e.g., vHH, SVH, centilin, etc.) that target different antigens are also known in the art.

[0387] An exemplary double-chain bispecific SAR comprising two chains is CD8SP-hu-mROO5-1-vL-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-hu-mROO5-1-vH-[IgG1-CH1-TCRa-wt-op2-6MD], represented by DNA SEQ ID NO: 5039 and PRT SEQ ID NO: 15729. This SAR is similar to the SAR construct CD8SP-hu-mROO5-1-vL-[IgCL-TCRg-6MD]-F-P2A-IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-hu-mROO5-1-vH-[IgG1-CH1-TCRd-6MD] represented by SEQ ID NOs:(DNA):5037 and (PRT):15727, except that the TCRg-MD and TCRd-MD modules are replaced with the (TCRb-wt-opt2-6MD; SEQ ID NO:1126) and (TCRa-wt-op2-6MD; SEQ ID NO:1112) modules, respectively. Thus, the SAR targets CD19 via the hu-mROO5-1 Fv and CD20 via CD20-USC1-vHH-2HCD25. Table 26 shows the DNA SEQ ID NOs (3169-3333) and amino acid SEQ ID NOs (13859-14023) of several exemplary bispecific SARs comprising vL / vH fragments targeting different antigens, in which the vL fragment is operably linked to a [TCRb-wt-opt2-6MD] module and the vH fragment is operably linked to a [TCRa-wt-op2-6MD] module. These SARs also comprise a CD19-targeting SVH (CD19-FHVH-354; SEQ ID NO: 836). Because the order of the different constructs in this series is the same as the order of the constructs in the series shown in Table 25, the vL / vH fragments of the different constructs in this series can be determined from the constructs with the same vL / vH fragments listed in Table 25. Because the SAR is modular, it is possible to replace one module with another module or a variant thereof, as long as the functional activity of the SAR is maintained.Thus, in an exemplary embodiment, the vL fragment of the SAR is operably linked to the [TCRa-wt-op2-6MD] module or a variant thereof, and the vH fragment is operably linked to the [TCRb-wt-opt2-6MD] module or a variant thereof. Similarly, the CD20-vHH-2HCD25 fragment can be replaced with another AABD that targets a different antigen or a different epitope of CD20. Exemplary AABDs are shown in Tables 5, 7-10. Additional AABDs (e.g., vHH, SVH, centilin, etc.) that target different antigens are also known in the art.

[0388] Another exemplary double-chain bispecific SAR comprising two chains is CD8SP-CD38-USC1-FHVH-32184-G4Sx3-R1-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-SP-hu-mROO5-1-vH-[hTCRa-T48C], represented by DNA SEQ ID NO: 7405 and PRT SEQ ID NO: 18095. This SAR construct has a SIR backbone. The hu-mROO5-1 vL fragment and hu-mROO5-1 vH fragment of the SAR dimerize to form an Fv targeting CD19. The CD38-targeting SVH fragment (CD38-USC1-FHVH-32184) is fused to the N-terminus of the hu-mROO5-1 vL fragment via a glycine-serine linker. Thus, SAR targets CD19 via hu-mROO5-1 Fv and CD38 via CD38-USC1-FHVH-32184.

[0389] Another exemplary dual-chain trispecific SAR comprising two chains is CD8SP-CD38-USC1-FHVH-32184-G4Sx3-R1-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-USC1-vHH-2HCD26-G4Sx3v2-hu-mROO5-1-vH-[hTCRa-T48C], represented by DNA SEQ ID NO: 7408 and PRT SEQ ID NO: 18098. This SAR construct has the framework of an SIR. The hu-mROO5-1 vL fragment and hu-mROO5-1 vH fragment of the SIR dimerize to form an Fv that targets CD19. The CD38-targeting SVH fragment (CD38-USC1-FHVH-32184) is fused to the N-terminus of the hu-mROO5-1 vL fragment via a glycine-serine linker. The CD20-targeting vHH fragment (CD20-USC1-vHH-2HCD26) is fused to the N-terminus of the hu-mROO5-1 vH fragment via a glycine-serine linker. Thus, the SAR targets CD19 via the hu-mROO5-1 Fv, CD38 via CD38-USC1-FHVH-32184, and CD20 via CD20-USC1-vHH-2HCD26. Another exemplary trispecific SAR construct having a similar format is CD8SP-CD19-FHVH-354-G4S3-FMC63-vL-[hTCRb-S57C-opt]-F-P2A-SP-CD22-FHVH-24-FMC63-vH-[hTCRa-T48C-opt], ​​represented by SEQ ID NO:(DNA):2344 and SEQ ID NO:(PRT):13034, respectively. Additional SARs of this format in which the vL / vH of FMC63 is replaced with vL / vH fragments derived from other scFvs and targeting different antigens are shown in Table 26 (SEQ ID NOs:2344-2508 and 13034-13198). Since the order of the vL / vH fragments in these constructs is the same as the order of the vL / vH fragments in the constructs in Table 25, the vL / vH fragments in these constructs can be determined from their sequence numbers by consulting Table 25.As noted above, the TCR constant chains of these constructs can be replaced with other TCR constant chains listed in Table 12 or their deletion mutants and functional variants, so long as they retain the functional activity of the SAR (i.e., the ability to bind to its target antigen, the ability to induce T cell signaling upon antigen binding, the ability to assemble into a TCR / CD3 complex, etc.). An exemplary SAR comprising deletion mutants of the TCRa and TCRb constant chains is CD8SP-CD38-USC1-FHVH-32184-G4Sx3-R1-hu-mROO5-1-vL-[hTCRb-S57C-opt-del17]-F-P2A-IgSP-Apa-CD20-USC1-vHH-2HCD26-G4Sx3v2-hu-mROO5-1-vH-[hTCRa-T48C-opt-del17], represented by nucleic acid SEQ ID NO: 1332 and amino acid SEQ ID NO: 12022. SAR constructs with deletion mutants of the TCRa and TCRb chains have the advantage of being small in size and therefore increasing the titer of the lentiviral / retroviral vectors encoding them, as the titer of a lentiviral / retroviral vector is inversely proportional to the insert size. Finally, in addition to the TCR chains and modules, the CD38-USC1-FHVH-32184 and CD20-USC1-vHH-2HCD26 fragments can be replaced with other AABDs that target different antigens or epitopes. Exemplary AABDs are shown in Tables 5, 7-10. Additional AABDs (e.g., vHHs, SVHs, centilins, etc.) that target different antigens are known in the art.

[0390] An exemplary dual-chain trispecific SAR comprising two chains is CD8SP-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-IgSP-CD22-USC1-FHVH-160-G4Sx2-CD20-USC1-vHH-2HCD26-G4Sx3v2-hu-mROO5-1-vH-[hTCRa-T48C], represented by DNA SEQ ID NO: 7407 and PRT SEQ ID NO: 18097. This SAR construct has the framework of an SIR. The hu-mROO5-1 vL fragment and hu-mROO5-1 vH fragment of the SIR dimerize to form an Fv that targets CD19. The CD20-targeting vHH fragment (CD20-USC1-vHH-2HCD26) is fused to the N-terminus of the hu-mROO5-1 vH fragment via a glycine-serine linker. Furthermore, the CD22-targeting SVH fragment (CD22-USC1-FHVH-160) is fused to the N-terminus of the CD20 vHH fragment via another glycine-serine linker. Thus, the SAR targets CD19 via the hu-mROO5-1 Fv, CD20 via CD22-USC1-FHVH-160, and CD20 via CD20-USC1-vHH-2HCD26. Because the SAR is modular, the TCR chains can be replaced with other TCR chains or their variants, the AABDs can be replaced with other AABDs or their variants, and the vL / vH fragments can be replaced with other vL / vH fragments or their variants.

[0391] An exemplary dual-chain tetraspecific SAR comprising two chains is CD8SP-BCMA-FHVH-33-G3Sx2-CD38-FHVH-309021-G4Sx3-R1-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-hu-mROO5-1-vH-[hTCRa-T48C], represented by DNA SEQ ID NO: 5362 and PRT SEQ ID NO: 16052. This SAR construct has a SIR backbone. The hu-mROO5-1 vL fragment and hu-mROO5-1 vH fragment of the SIR dimerize to form an Fv that targets CD19. The CD20-targeting vHH fragment (CD20-vHH-2HCD25; SEQ ID NO: 835) is fused to the N-terminus of the hu-mROO5-1 vH fragment via a glycine-serine linker. The CD38-targeting SVH fragment (CD38-FHVH-309021; SEQ ID NO: 846) is fused to the N-terminus of the hu-mROO5-1-vL fragment via a glycine-serine linker. The BCMA-targeting SVH (BCMA-FHVH-33; SEQ ID NO: 856) is fused to the N-terminus of the CD38-FHVH-309021 fragment via another glycine-serine linker. Thus, the SAR targets CD19 via hu-mROO5-1 Fv, CD20 via CD20-vHH-2HCD25, CD38 via CD38-FHVH-309021, and BCMA via BCMA-FHVH-33. Because the SAR is modular, the TCR chains can be replaced with other TCR chains or their variants, the AABDs can be replaced with other AABDs or their variants, and the vL / vH fragments can be replaced with other vL / vH fragments or their variants.

[0392] An exemplary dual-chain pentaspecific SAR comprising two chains is CD8SP-BCMA-FHVH-33-G3Sx2-CD38-FHVH-309021-G4Sx3-R1-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-IgSP-CD22-FHVH-158-G4Sx2-CD20-vHH-2HCD25-G4Sx3v2-hu-mROO5-1-vH-[hTCRa-T48C], represented by DNA SEQ ID NO: 5437 and PRT SEQ ID NO: 16127. This SAR construct resembles the construct represented by DNA SEQ ID NO:5362 and PRT SEQ ID NO:16052, but further includes a CD22-targeting SVH fragment (CD22-FHVH-158; SEQ ID NO:848) fused to the N-terminus of the CD20-vHH-2HCD25 fragment via a glycine-serine linker. Thus, the SAR targets CD19 via the hu-mROO5-1 Fv, CD20 via CD20-vHH-2HCD25, CD38 via CD38-FHVH-309021, and BCMA via BCMA-FHVH-33. Because the SAR is modular, the TCR chains can be replaced with other TCR chains or their variants, the AABDs can be replaced with other AABDs or their variants, and the vL / vH fragments can be replaced with other vL / vH fragments or their variants. The vL / vH fragment comprising the SAR can be fully human, humanized, chimeric or of non-human origin.

[0393] In the above examples, the bispecific and multispecific SAR scaffolds are composed of vL and vH fragments to which one or more AABDs are operably linked. The present disclosure also describes SARs in which the scaffolds are composed of variable fragments (Va / Vb or Vg / Vd) from TCRs.

[0394] In certain embodiments, the present disclosure provides single-, 1.5-chain, or double-chain SARs having the general formula: (AABD)n-optional linker domain-TCR variable fragment-optional linker-TCR constant chain; n=0, 1, 2, 3, 4, 5, or more, where the TCR variable fragment represents Va, Vb, Vg, or Vd, and the TCR constant chain comprises at least one chain representing the constant chain of TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, or pre-TCRα, or a mutant or variant thereof. In certain exemplary embodiments, the AABD is a non-scFv antigen-binding module (e.g., SVH, vHH, FHVH, SVL, svd-TCR, centrin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, D domain, etc.).

[0395] In one embodiment, the present disclosure provides a double-chain SAR comprising one chain having the general formula: (AABD)n-optional linker domain-Va fragment-optional linker-TCR constant chain; and a second chain having the general formula: (AABD)n-optional linker domain-Vb fragment-optional linker-TCR constant chain, where n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain represents the constant chain of TCR alpha (Cα), TCR beta 1 (Cβ1), TCR beta 2 (Cβ2), TCR gamma (Cγ), TCR delta (Cδ), or pre-TCR alpha (pre-Cα), or a mutant or variant thereof. In one embodiment, the two chains of the SAR comprise complementary TCR constant chains; i.e., Cα and Cβ, pretc. Rα and Cβ, or Cγ and Cδ. The Cβ chain can represent either Cβ1 or Cβ2.

[0396] In one embodiment, the present disclosure provides a double-chain SAR comprising one chain having the general formula: (AABD)n-optional linker domain-Vg fragment-optional linker-TCR constant chain; and a second chain having the general formula: (AABD)n-optional linker domain-Vd fragment-optional linker-TCR constant chain, where n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain represents the constant chain of TCR alpha (Cα), TCR beta 1 (Cβ1), TCR beta 2 (Cβ2), TCR gamma (Cγ), TCR delta (Cδ), or pre-TCR alpha (pre-Cα), or a mutant or variant thereof. In one embodiment, the two chains of the SAR comprise complementary TCR constant chains; i.e., Cα and Cβ, pretc. Rα and Cβ, or Cγ and Cδ. The Cβ chain can represent either Cβ1 or Cβ2.

[0397] An exemplary SAR in which AABD is operably linked to the Va and / or Vb chain is CD8SP-MC7G5-Vb-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-MC7G5-Va-[hTCRa-T48C]-F-F2A-PAC, represented by nucleic acid SEQ ID NO: 3502 and amino acid SEQ ID NO: 14192. This SAR construct has the MC7G5-Vb fragment fused to the constant chain of human TCRb with an S57C mutation (hTCRb-S57C) and the MC7G5-Va fragment fused to the constant chain of human TCRa with a T48C mutation (hTCRa-T48C). The MC7G5-Va and MC7G5-Vb fragments of the SAR bind to MR1. The CD20-targeting vHH fragment (CD20-vHH-2HCD25) was fused to the N-terminus of the MC7G5-Va fragment via a glycine-serine linker (G4Sx3v2). Thus, the SAR targets MR1 ​​via the MC7G5 Va / Vb fragment and CD20 via CD20-vHH-2HCD25.

[0398] An exemplary SAR based on the gamma delta TCR backbone is CD8SP-BCMA-FHVH-93-G4S-TCR-Vg9-F-P2A-IgHSP-CD20-vHH2-HCD25-G4S-TCR-Vd2-F-F2A-PAC (SEQ ID NO: 22201). This SAR has the Vgamma / Vdelta TCR backbone. Vgamma / Vdelta T cells represent a minor and unconventional component (0.5-5%) of the peripheral blood leukocyte population, but are thought to play an essential early role in sensing "danger" from invading pathogens. They recognize the same small microbial compound, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a natural intermediate in the non-mevalonate pathway of isopentenyl pyrophosphate (IPP) biosynthesis. The above-described SAR on a Vy9 / V52 backbone can also recognize BCMA via BCMA-FHVH-93, which is attached to the N-terminus of the Vg9 variable domain via a G4S linker, and CD20 via CD20-vHH2-HCD25, which is attached to the N-terminus of the Vd2 variable domain. Additional Vy9 / V52-based SARs can be constructed by replacing BCMA-FHVH-93 and CD20-vHH2-HCD25 with antigen-binding domains (e.g., AABDs) that bind other antigens. Exemplary Vy9 / V52-based SARs can also be constructed by replacing the MC.7.G5 (MC7G5) Va and Vb domains, having SAR SEQ ID NOs: 3500-3595, with a Vy9 / V52 variable domain.

[0399] The present disclosure also describes SARs in which the vL and / or vH fragments of the SARs described in the previous section are replaced with Va and / or Vb fragments derived from a T cell receptor (TCR). Based on the Va and Vb fragments of the TCR (MC7G5) directed against MIR1, a monomorphic MHC class I-related protein (MR1), as a framework, several exemplary bispecific and trispecific SARs with AABDs targeting CD19 and / or CD20 are shown in Table 27. The composition of these SARs can be determined from their names. Additional TCR-based SARs are listed in Table 28. Other SARs based on the modular structures of the SARs described in Tables 27-28 are also within the scope of the present disclosure.

[0400] The present disclosure also provides exemplary SARs in which one or more AABDs are operably linked to or near the N-terminus of a variable fragment (e.g., Va, Vb, Vg, or Vd) derived from a TCR. An exemplary such SAR construct is CD8SP-Sph-CD19-vHH-048-G4Sx3-R1-MC7G5-Vb-[hTCRb-S57C]-F-P2A-SP-MC7G5-Va-[hTCRa-T48C], which is represented by nucleic acid SEQ ID NO: 3535 and amino acid SEQ ID NO: 14225. An exemplary trispecific SAR based on the MC7G5 backbone is CD8SP-Sph-CD19-vHH-048-G4Sx3-R1-MC7G5-Vb-[hTCRb-S57C]-F-P2A-IgSP-Apa-CD20-vHH-2HCD25-G4Sx3v2-MC7G5-Va-[hTCRa-T48C], represented by nucleic acid sequence number 3578 and amino acid sequence number 14268.

[0401] Table 27 shows several exemplary bispecific and trispecific SARs based on the Va and Vb fragments of the TCR (MC7G5) directed against the monomorphic MHC class I-related protein (MR1), MIR1, as a framework, with AABDs targeting CD19 and / or CD20. The composition of these SARs can be determined from their names. It is possible to replace one module of these SARs with another. Modules that can be replaced include the Va / Vb fragment, the TCR constant chain (i.e., hTCRb-S57C and hTCRa-T48C), and the AABD (i.e., CD20-vHH-2HCD25). Additional SARs based on different TCR variable domains, TCR constant chains, and AABDs are listed in Table 28. SARs in which the Va / Vb fragment of an MC7G5-based SAR listed in Table 27 is replaced with a Va / Vb derived from a TCR-targeted CMV-pp65-derived peptide (SEQ ID NO:21452) complexed with HLA-A2 are represented by nucleic acid SEQ ID NOs:3891-3988 and amino acid SEQ ID NOs:14581-14678, respectively (Table 28). Similarly, exemplary SARs in which the Va / Vb fragment of an MC7G5-based SAR listed in Table 27 is replaced with a Va / Vb derived from a TCR-targeted NYESO-1-derived peptide (SEQ ID NO:21461) complexed with HLA-A2 are represented by nucleic acid SEQ ID NOs:3989-4086 and amino acid SEQ ID NOs:14679-14776, respectively (Table 28). The order of these constructs is the same as the order of the MC7G5-based constructs listed in Table 27.

[0402] The present disclosure also provides a SAR polypeptide encoding a SAR, wherein one chain comprises a variable fragment derived from an antibody (e.g., vL or vH), and the second chain comprises a variable fragment derived from a TCR (e.g., Va, Vb, Vg, or Vd). Exemplary such SAR polypeptides are represented by SEQ ID NOs: (PRT): 23020 and 23021. The variable fragments derived from the antibody and TCR in such SARs can serve as a scaffold for attachment of one or more AABDs.

[0403] In some embodiments, the present disclosure provides single-, 1.5-, or double-chain SARs having the general formula: (AABD)n-optional linker-TCR constant chain; n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain comprises at least one chain representing a constant chain of TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, or pre-TCRα, or a mutant or variant thereof. In some embodiments, the linker is an Ig linker domain (e.g., IgCL, IgG1-CH1, etc.). Exemplary Ig (or Ig-like) linker domains are shown in Table 13. In certain exemplary embodiments, the AABD is a non-scFv antigen binding module (e.g., an SVH, vHH, FHVH, SVL, svd-TCR, centirin, DARPIN, CD16A, CD64, CD32, NKG2D, NKG2D-AF, NKG2D-YA, RZIP, EZIP, E4, K4, D domain, etc.). In certain exemplary embodiments, the AABD is an adaptor binding domain.

[0404] In one embodiment, the present disclosure provides a double-chain SAR comprising one chain having the general formula: (AABD)n-Ig linker domain-TCR constant chain; and a second chain having the general formula: (AABD)n-Ig linker domain-TCR constant chain, where n=0, 1, 2, 3, 4, 5, or more, where the TCR constant chain represents the constant chain of TCR alp...

Claims

1. At least one recombinant polynucleotide encoding at least one synthetic antigen receptor (SAR), wherein the at least one SAR comprises a heterodimer of a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain and / or the second polypeptide chain are i) Single vH domain (SVH); ii) Single vL domain (SVL); iii) vHH domain; iv) Single-domain antibody; v) Single variable domain of the T cell receptor (svd-TCR); vi) DARPIN, afibody, adonectin, afitin, lipibody, finomer, alphabody, avimer, atrimer, centinrin, pronectin, antikalin, Knitz domain, armadillo repeat protein and D domain, or any non-immunoglobulin antigen-binding scaffolds optionally selected from any of the above fragments; vii) The ligand-binding domain or fragment of a receptor; viiii) Receptor-binding domain of the ligand; ix) Autoantigen or its fragment; x) Adapter binding domain; xi) Fc binding domain A first module comprising one or more non-scFv autonomous antigen-binding domains (AABDs) or fragments thereof selected from the group consisting of the following: (i) The first polypeptide chain further a) A second module comprising a heavy chain variable domain (vH), a light chain variable domain (vL), or an Ig linker domain, b) A third module (if any) including an extracellular domain, a linked peptide, or a hinge domain, c) A fourth module including a transmembrane domain, and d) comprising a fifth module (if any) containing one or more intracellular signaling domains, (ii) The second polypeptide chain further a) A second module comprising a light chain variable domain (vL), a heavy chain variable domain (vH), or an Ig linker domain, b) A third module (if any) including an extracellular domain, a linked peptide, or a hinge domain, c) A fourth module including a transmembrane domain and d) comprising a fifth module (if any) containing one or more intracellular signaling domains, A first module is operably linked to or near the N-terminus of a second module, and the first, second, third (if any), fourth and fifth (if any) modules are operably linked via one or more linkers (if any), and at least one recombinant polynucleotide encoding at least one SAR polypeptide, wherein the first polypeptide chain and the second polypeptide chain each contain an antibody variable domain in the second module, the variable domain of one chain being vH and the variable domain of the other chain being vL.

2. The one or more non-scFvAABDs are linked via an optional linker to the N-terminus or near the N-terminus of a second module of a first polypeptide chain, the second module of the first polypeptide chain is operably linked via an optional linker to a first T cell receptor constant chain fragment or CD3 chain fragment containing a first linked peptide operably linked to a first transmembrane domain, and / or The second module of the second polypeptide chain comprises at least one recombinant polynucleotide according to claim 1, wherein the second module comprises a vL, vH, or Ig linker domain fragment operably linked to a second T cell receptor constant chain fragment or CD3 chain fragment containing a second linked peptide operably linked to a second transmembrane domain via an optional linker.

3. The at least one recombinant polynucleotide according to claim 2, wherein the vH of the encoded first peptide chain and the vL of the second peptide chain form an antigen-binding module that specifically binds to a target antigen.

4. The at least one recombinant polynucleotide according to claim 1 or 2, wherein the AABD, vL, vH, and / or Ig linker domains are fully human, humanized, chimeric, or non-human domains.

5. The at least one recombinant polynucleotide according to claim 1 or 2, comprising a polypeptide having sequence numbers 11832 to 11865 or a fragment or variant thereof having at least 95% sequence homology to a polypeptide having the sequence numbers 11832 to 11865, wherein the encoded Ig linker domain comprises

6. The at least one recombinant polynucleotide according to claim 1 or 2, wherein the optionally encoded linker domain comprises a polypeptide having sequence numbers 11832-11865, 11714-11730, or a fragment or variant thereof having at least 95% sequence homology to a polypeptide having sequence numbers 11832-11865, 11714-11730, or a domain of 25-500 amino acid length.

7. The at least one recombinant polynucleotide according to claim 2, wherein the encoded T cell receptor constant chain fragment comprises a polypeptide having any one sequence of SEQ ID NOs: 11732-11742, 11744-11766, 11768-11777, or 11793-11830, or a polypeptide having at least 95% sequence homology with any one sequence of SEQ ID NOs: 11732-11742, 11744-11766, 11768-11777, or 11793-11830.

8. The at least one recombinant polynucleotide according to claim 2, wherein the encoded T cell receptor constant chain comprises a binding peptide having the sequence of any one of SEQ ID NOs. 11867 to 11875 or a polypeptide having at least 95% sequence homology with any one of SEQ ID NOs. 11867 to 11875.

9. The at least one recombinant polynucleotide according to claim 1 or 2, comprising a transmembrane domain having any one sequence of SEQ ID NOs: 11877-11881 or 23332-23334, or a polypeptide having at least 95% sequence homology with any one sequence of SEQ ID NOs: 11877-11881 or 23332-23334.

10. The at least one recombinant polynucleotide according to claim 1, wherein the early intracellular signaling domain comprises a cytoplasmic domain having one of the sequences of SEQ ID NOs: 11883-11886, 11785, or 23335-23337, or a polypeptide having at least 95% sequence homology with one of the sequences of SEQ ID NOs: 11883-11886, 11785, or 23335-23337.

11. At least one recombinant polynucleotide according to claim 1, having a skeleton of 1.5-chain SIR, double-chain SIR, zSIR, 1.5-chain cTCR, double-chain cTCR, Ab-TCR, AABD-TCR, or HLA-independent TCR.

12. The aforementioned SAR, i) at least one antigen; and / or ii) At least one epitope of one or more antigens At least one recombinant polynucleotide according to claim 1 or 2, which can bind to.

13. One or more antigen-binding domains are encoded by CD5; CD19; CD123; CD22; CD30; CD171; CS1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); type C lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviiii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); 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 in acute leukemia or lymphoma but not in hematopoietic progenitor cells, expressed in non-hematopoietic malignancies. Glycosylated CD43 epitope, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor α (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS2) 1) Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific fetal antigen-4 (SSEA-4); CD20; Folate receptor α (FRa or FR1); Folate receptor β (FRb); Receptor tyrosine protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface-related (MUC1); Epidermal growth factor receptor (E) GFR); neuronal adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); prolongation factor 2 mutation (ELF2M); ephrin B2; fibroblast-activating protein α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CALX); proteasome (prosome, macropain) subunit, β-type, 9 (LMP2); glycoprotein 100 (gpl00);Breakpoint cluster region (BCR); Oncogene fusion protein (bcr-abl) consisting of Abelson mouse leukemia virus oncogene homolog 1 (Abl); Tyrosinase; Ephrin type A receptor 2 (EphA2); Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDClarp(l-4)bDClcp(l-1)Cer); Transglutaminase 5 (TGS5); High molecular weight melanoma-associated antigen (HM WMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK) ); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globeH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor β3 (ADRB3); panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); auditory receptor 51E2 (OR51E2) ;TCR gamma-selective reading frame protein (TARP); Bilmus tumor protein (WT1); cancer / testicular antigen 1 (NY-ESO-1); cancer / testicular antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member lA (XAGEL); angiopoietin-binding cell surface receptor 2 (Tie 2) Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 variant; Prostain; Survivin; Telomerase; Prostate cancer tumor antigen-1 (PCT A-1 or Galectin 8), Melanoma antigen 1 recognized by T cells (Melan A or MARTI); Rat sarcoma (Ras) variant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint;Apoptotic melanoma inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin Bl; v-myc avian myelomatosis virus oncogene neuroblastoma homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 LB 1 (CYPLB 1); CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-section 2 (SSX2); advanced glycation end product receptor (RAGE-1); renal ubiquitous protein 1 (RUl); renal ubiquitous protein 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); enteric carboxylesterase; heat shock protein 70-2 variant (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLLl); MPL; Biotin; c-MYC epitope tag; CD34; LAMP1 TROP2; GFRα4; CDH17; CDH6; NYBR1; CDH19; CD200R; Slea (CA19.9; sialyl Lewis antigen); Fucosyl-GM1;PTK7; gpNMB; CDH1-CD324; DLL3; CD276 / B7H3; IL11Ra; IL13Ra2; CD179b-IGLl1; TCRγδ; NKG2D; CD32 (FCGR2A),; Tn ag; Tim1- / HVCR1; CSF2RA (GM-CSFR-α); TGFβR2; Lews Ag; TCR-β1 chain; TCR-β2 chain; TCR-γ chain; TCR-δ chain; FITC; Luteinizing hormone receptor (LHR); Follicle-stimulating hormone receptor (FSHR); Gonadotropin receptor (CGHR or GR); CCR4; GD3; SLAMF6; SLAMF4; HIV-1 envelope glycoprotein; HTLV1-Tax; CMV pp65; EBV-EBNA3c; KSHV K8.1; KSHV-gH; influenza A hemagglutinin (HA); GAD; PDL1; guanylyl cyclase C (GCC); autoantibody against desmoglein 3 (Dsg3); autoantibody against desmoglein 1 (Dsg1); HLA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DP; HLA-DM; HLA-DOA; HLA-DOB; HLA-DQ; HLA-DR; HLA-G; IgE, CD99; Ras At least one recombinant polynucleotide according to claim 1 or 2, which binds to at least one antigen selected from the group consisting of G12V; tissue factor 1 (TF1); AFP; GPRC5D; claudin 18.2 (CLD18A2 or CLDN18A.2); P-glycoprotein; STEAP1; Liv1; nectin-4; Cripto; gpA33; BST1 / CD157; low-conductance chloride ion channel (LCCC); TAJ / TROY; MPL (TPO-R); KIR3DL2; CD32b; CD229; Toso; GPC3; BAFF-R; MR1; CMV-pp65 / MHC complex; and NYESO-1 / MHC complex.

14. The coded SAR polypeptide is, (i) A heavy chain variable region (vH) comprising any one sequence of sequence numbers 10978-11214 and 23148-23161, or a sequence containing the three complementarity-determining regions (CDRs) described above; and A light chain variable region (vL) comprising one of the sequences 10736-10972 and 23136-23147, or a sequence containing the three complementarity-determining regions (CDRs) mentioned above; (ii) Camelid VHH domain comprising one of sequence numbers 11524-11525, 11530-11531, 11549-11575, 11576-11592 and 23163-23173, or a sequence containing the three complementarity-determining regions (CDRs) mentioned above; (iii) Non-immunoglobulin scaffolds encoded by polynucleotides of any one sequence of sequence numbers 11662-11673, or sequences having at least 95% identity with any one sequence of sequence numbers 11662-11673; (iv) A ligand-binding domain of a receptor comprising any one sequence of sequence numbers 11674 to 11691, or a sequence having at least 95% identity thereto and encoding a polypeptide that binds to that cognitive; (v) Receptor-binding domain of a ligand comprising one of sequence numbers 11692-11702, 22391-22392, 22402-22404, or a sequence having at least 95% identity thereto and encoding a polypeptide that binds to that cognitive; (vi) A single vH domain comprising one of sequence numbers 11519-11523, 11526-11529, 11532-11548, 11644-11645 and 23174, or a sequence containing the three complementarity-determining regions (CDRs) mentioned above; (vii) an adapter-binding domain comprising one of sequence numbers 11704-11712 or 22383, or a sequence having at least 95% identity thereto, and encoding a polypeptide that binds to the adapter; (viiii) an autoantigen comprising any one sequence of sequence numbers 11687, 22406-22407 to 11712, or 22383, or a sequence having at least 95% identity thereto and encoding a polypeptide that binds to the autoantibody or autoantibody-producing cell; and (ix) A single variable TCR domain (svd-TCR) comprising any one sequence of sequence numbers 22399 to 22400, or a sequence containing the three complementarity-determining regions (CDRs) described above. At least one recombinant polynucleotide encoding at least one SAR according to claim 1 or 2, comprising one or more antigen-binding domains selected from the group consisting of the following.

15. (1) at least one recombinant polynucleotide as described in claim 1, and (2) a therapeutic control operably linked to the at least one recombinant polynucleotide, wherein the therapeutic control is a truncated epidermal growth factor receptor (tEGFR), truncated epidermal growth factor receptor viiii (tEGFRviiii), truncated CD30 (tCD30), truncated BCMA (tBCMA), truncated CD19 (tCD19), CD34, thymidine kinase, cytosine deaminase, nitroreductase, xanthine guanine phosphoribosyltransferase, human caspase 8, human caspase 9 Inducible caspase 9 (i-caspase 9), purine nucleoside phosphorylase, linamarase / linamarin / glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP) / indole-3-acetic acid (IAA), γ-glutamylcysteine ​​synthetase, CD20 / αCD20, CD34 / thymidine kinase chimeric, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903 / Fas system, chimeric cytokine receptor (CCR), 41BBL, CD40L, K13, MC159, cFLIP-L / MRITα, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2 Tax-RS mutant, FKBPx2-K13, FKBPx2-HTLV2-Tax, FKBPx2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4 A recombinant expression system selected from the group consisting of scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-ipilimumab-scFv, CTLA4-ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, CD3z, CD3z-GGGS-41BB, CD3-BBz, CD3-CD28z, CD3-CD28-Lck fusion protein, shRNA targeting Brd4, chimeric antigen receptor (CAR), hTERT, heparinase, CAR, inhibitory CAR, and combinations thereof.

16. At least one vector comprising a recombinant polynucleotide as described in claim 1, selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, retrovirus vectors, baculovirus vectors, sleeping beauty transposon vectors, and piggybac transposon vectors.

17. The at least one SAR polypeptide encoded by at least one recombinant polynucleotide as described in claim 1.

18. Use of at least one recombinant polynucleotide according to claim 1, or the vector according to claim 16, in the manufacture of a pharmaceutical product comprising recombinant cells or a population of cells expressing at least one recombinant polynucleotide, for the treatment of a disease associated with the expression of a disease-related antigen, wherein the recombinant cells express a SAR polypeptide that binds to the disease-related antigen.

19. CD5, CD19; CD123; CD22; CD23, CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; Epidermal growth factor receptor variant III (EGFRviii); Ganglioside G2 (GD2); Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); 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 in acute leukemia or lymphoma but not in hematopoietic progenitor cells, expressed in non-hematopoietic malignancies. Glycosylated CD43 epitope, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor α (IL-llRa); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS2) 1) Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific fetal antigen-4 (SSEA-4); CD20; Folate receptor α (FRa or FR1); Folate receptor β (FRb); Receptor tyrosine protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface-related (MUC1); Epidermal growth factor receptor (E) GFR); neuronal cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); prolongation factor 2 mutation (ELF2M); ephrin B2; fibroblast-activating protein α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CALX); proteasome (prosome, macropain) subunit, β-type, 9 (LMP2); glycoprotein 100 (gpl00);Oncogene fusion protein (bcr-abl) consisting of a cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClarp(l-4)bDClcp(l-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma Related antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma Kiner Ze (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globeH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor β3 (ADRB3); panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); auditory receptor 51E2 (OR51) E2); TCR gamma-selective reading frame protein (TARP); Bilmus tumor protein (WT1); cancer / testicular antigen 1 (NY-ES0-1); cancer / testicular antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member lA (XAGEL); angiopoietin-binding cell surface receptor 2 (Tie 2) Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 variant; Prostain; Survivin; Telomerase; Prostate cancer tumor antigen-1 (PCT A-1 or Galectin 8), Melanoma antigen 1 recognized by T cells (Melan A or MARTI); Rat sarcoma (Ras) variant; Human telomerase reverse transcriptase (hTERT);Sarcoma translocation breakpoint; apoptotic melanoma inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin Bl; v-myc avian myelomatosis virus oncogene neuroblastoma homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 lb 1 (CYPlb 1); CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-section 2 (SSX2); advanced glycation end product receptor (RAGE-1); renal ubiquitous protein 1 (RUl); renal ubiquitous protein 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); enteric carboxylesterase; heat shock protein 70-2 variant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin λ-like polypeptide 1 (IGLLl), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFRα4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen);Fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL11Ra, IL13Ra2, CD179b-I GLl1, TCRγδ, NKG2D, CD32 (FCGR2A), Tim1- / HVCR1, CSF2RA (GM-CSFR-α), TGFβR2, Lews Ag, TCR-β1 chain, TCR-β2 chain, TCR-γ chain, TCR-δ chain, FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin receptor (CGHR), CCR4, SLAMF6, SLAMF4, HIV-1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV Autoantibodies against K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IGE, CD99, RAS The use according to claim 18 in the manufacture of a pharmaceutical product for treating a disease associated with one or more disease-related antigens selected from the group consisting of G12V, tissue factor 1 (TF1), AFP, GPRC5D, claudin 18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, LIV1, nectin-4, CRIPTO, GPA33, BST1 / CD157, low-conductance chloride ion channel, TAJ / TROY, MPL (TPO-R), KIR3DL2, CD32b, CD229, Toso, GPC3, MR1, NYESO-1 / MHC complex, and CMVpp65 / MHC complex.

20. The use according to claim 18 or 19, wherein the disease is selected from the group consisting of proliferative disorders, precancerous conditions, cancer, infectious diseases, autoimmune diseases, allergic diseases, and degenerative diseases.

21. Sequence numbers 12539-14188, 14778-14799, 14811-14835, 14853-14899, 14905-14926, 14938-14962, 14978-15026, 15032-15053, 15065-15090, 15105-15153, 15159-15180, 15192-15216, 15234-15280, 15286-15307, 15319-15343, 15361-15407, 15413-15434, 15446-15470, 15488-15534 , 15540-15561, 15573-15597, 15615-15661, 15669-15693, 15708-15729, 15738-15759, 15763-15787, 15801-15842, 15846-15867, 15876-15911, 15669-15759, 15763-15911, 15917-15941, 15956-15977, 15986-16007, 16011-16035, 16049-16091, 16094-16115, 16124-16159, 16 165-16189, 16204-16225, 16234-16255, 16259-16283, 16297-16338, 16342-16363, 16372-16407, 16413-16437, 16452-16473, 16507-16531, 16545-16586, 16590-16611, 16661-16685, 16700-16721, 16730-16751, 16755-16779, 16793-16834, 16838-16859, 16909-16933, 16948 A recombinant polynucleotide according to any one of claims 1 to 14, encoding a SAR polypeptide consisting of the amino acid sequences of two polypeptide chains of SAR selected from the group consisting of ~16969, 16978~16999, 17003~17027, 17041~17083, 17086~17107, 17116~17151, 17160~17184, 17234~17257, 17306~17330, 17379~17403, 17452~17476, 17525~17549, and 22445~22460.

22. Recombinant cells comprising or expressing at least one recombinant polynucleotide according to any one of claims 1 to 14, wherein the recombinant cells are immune effector cells or stem cells capable of giving rise to immune effector cells, provided that the cells are not human embryonic stem cells.

23. Recombinant cells according to claim 22, wherein the cells are selected from one or more of the following: a) autologous T cells, allogeneic T cells, autologous natural killer T (NKT) cells, allogeneic NKT cells, autologous hematopoietic stem cells, allogeneic hematopoietic stem cells, autologous induced pluripotent stem cells (iPSCs), or allogeneic iPSCs that can give rise to immune effector cells; and / or b) Alpha / beta T cells, gamma / delta T cells, regulatory T cells (TREG), CD8+ T cells, CD4+ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, naive T cells, central memory T cells, effector memory T cells, stem memory T cells (Tscm), iPSC-derived T cells, synthetic T cells, bone marrow-derived phagocytes, or stem cell-derived immune effector cells; or c) Hematopoietic stem cells, peripheral blood stem cells, bone marrow-derived stem cells, immune stem cells, induced pluripotent stem cells (iPSCs).

24. A composition comprising at least one recombinant polynucleotide according to claim 1, a SAR polypeptide according to claim 17, a recombinant cell according to claim 22 or 23 and / or a vector according to claim 16, and a pharmaceutically acceptable excipient.

25. A kit comprising at least one recombinant polynucleotide according to claim 1, a SAR polypeptide according to claim 17, a recombinant cell according to claim 22 or 23, and / or a vector according to claim 16.