POLYNUCLEOTIDES, POLYPEPTIDES, CELLS, AND COMPOSITIONS COMPRISING SYNTHETIC IMMUNE RECEPTORS WITH METHODS OF MANUFACTURE AND TREATMENT
The disclosures herein provide synthetic immune receptors (SIRs), nucleic acids encoding the SIRs, and methods of making and using the SIRs, in, for example, adoptive cell therapy.
1 . At least one recombinant polynucleotide encoding a synthetic immune receptor (SIR) wherein the SIR comprises: a heterodimer of T-cell receptor (TCR) constant chains or functional variants thereof selected from (a)(i) and (a)(ii), (a)(i) and (a)(iii), (a)(iv) and (a)(v), (a)(vi) and (a)(vi), (a)(i) and (a)(vi), a(ii) and a(vi), a(iii) and a(vi), a(iv) and a(vi), and a(v) and a(vi) below;
wherein the polynucleotide encoding the SIR is expressed in a T cell with impaired or abolished expression of one or more endogenous TCR chains, and/or
wherein the polynucleotide encoding the TCR constant chains or functional variants of the TCR constant chains comprises one or more mutations that:
enhance the expression and/or pairing of encoded TCR constant chains or functional variants thereof, and/or
reduce the pairing of the encoded TCR constant chains or functional variants thereof with the endogenous chains
wherein the SIR comprises:
(a) a T-cell receptor (TCR) constant chain or a functional variant thereof having an amino acid sequence selected from the group consisting of:
a T-cell receptor α (Cα) constant chain or region which may comprise an optional accessory module;
a T-cell receptor beta (Cβ) constant chain or region which may comprise an optional accessory module;
a pre-TCRα constant chain or region which may comprise an optional accessory module;
a T cell receptor γ (Cγ) constant chain or region which may comprise an optional accessory module;
a T cell receptor δ (Cδ) constant chain or region which may comprise an optional accessory module;
a T cell receptor constant chain region fused to a CD3z chain region and which may comprise an optional accessory module;
(b) an optional linker; and
(c) one or more non-natural TCR antigen binding domain(s) linked to (a) selected from the group consisting of:
(1) an antibody;
(2) an antibody fragment selected from the group consisting of a Fv, a Fab, and a (Fab′) 2;
(3) a heavy chain variable region of an antibody (vH domain) and a light chain variable region of an antibody (vL domain) specific for a target antigen, such that one of said vH and vL domains is attached to a first of said two TCR constant chains of (a) and the other of said vH and vL domains is attached to a second of said TCR constant chains of (a);
(4) a single chain variable fragment (scFv);
(5) a single domain antibody (SDAB);
(6) a camelid VHH domain;
(7) a monomeric variable region of an antibody;
(8) a non-immunoglobulin antigen binding scaffold optionally selected from the group consisting of a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obody, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a Kunitz domain, an armadillo repeat protein and a functional fragment thereof;
(9) a receptor or a fragment thereof; and
(10) a ligand or a fragment thereof.
2 . The recombinant polynucleotide of claim 1 , wherein the polynucleotide encoding the TCR constant chain(s) or functional variant(s) and/or the encoded TCR constant chain(s) or functional variant(s) comprise one or more mutations and/or features selected from the group consisting of:
a) are encoded by polynucleotide sequence(s) that are not wild-type;
b) are encoded by codon-optimized polynucleotide sequence(s);
c) result in additional cysteine residues in the encoded TCR constant chains or functional variants thereof;
d) create additional interchain disulfide bonds between the TCR constant chains or functional variants thereof;
e) engineer “knob-into-hole” and “hole-into-knob” configurations in the TCR constant chains or functional variants thereof;
f) remove of one or more N-glycosylation sites;
g) substitute one or more amino acid residues of one or both human TCR constant chains or functional variants thereof with the corresponding amino acids of a mouse TCR constant chain;
h) stabilize an interaction between the antigen binding domains; and
i) are of human, mouse or dog origin.
3 . The recombinant polynucleotide of claim 2 , wherein the TCR constant chain(s) or functional variants thereof of the encoded SIR comprise an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor α (Cα) constant chain or region that a) comprises one or more mutations at positions 10, 15, 45, 48, 61, 91, 92, 93, and/or 94 corresponding to a reference Cα chain represented by SEQ ID NO: 3010; and/or b) is encoded by a polynucleotide sequence that is not wild-type; and/or c) is encoded by a polynucleotide sequence that is codon-optimized;
(ii) a T-cell receptor beta (Cβ) constant chain or region that a) comprises one or more mutations at positions 15, 17, 18, 22, 57, 79, 133, 136 and/or 139 corresponding to a reference Cβ chain represented by SEQ ID NO:3024 or 3025; and/or b) is encoded by a polynucleotide sequence that is not wild-type; and/or c) is encoded by a polynucleotide sequence that is codon-optimized;
(iii) a pre-TCRα chain constant chain or region that is a) is encoded by a polynucleotide sequence that is not wild-type; and/or b) is encoded by a polynucleotide sequence that is codon-optimized;
(iv) a T cell receptor γ (Cγ) chain constant region that is encoded by a) a not wild-type polynucleotide sequence; and/or b) a codon-optimized polynucleotide sequence; (v) a T cell receptor δ (Cδ) chain constant region that is a) is encoded by a polynucleotide sequence that is not wild-type; and/or b) is encoded by a polynucleotide sequence that is codon-optimized; and
(vi) a T cell receptor constant chain region fused to a CD3z chain region.
4 . The recombinant polynucleotide of claim 3 , wherein the TCR constant chain(s) or functional variants thereof of the encoded SIR comprise an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor a (Cα) constant chain or region with an amino acid sequence that a) comprises one or more of the following position-amino acids 10C, 15C, 45C, 48C, 61R, 91S, 92D, 93V, and/or 94P corresponding to a reference Cα chain represented by SEQ ID NO: 3010; and/or b) is at least 85% identical to SEQ ID NO: 3010 and comprises one or more of the following position-amino acids 10C, 15C, 45C, 48C, 61R, 91S, 92D, 93V, and/or 94P, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; or c) at least 85% identical to SEQ ID NO: 3022 and comprises one or more of the amino acid positions 10C, 15C, 45C and/or 48C corresponding to a reference Cα chain represented by SEQ ID NO: 3010, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(ii) a T-cell receptor β (Cβ) constant chain or region with an amino acid sequence that a) comprises one or more of the following position-amino acids 15C, 17C, 18K or R, 22A, 57C, 59C, 77C, 79G, 133I, 136A and/or 139H corresponding to a reference Cβ chain represented by SEQ ID NO: 3024 or 3025; and/or b) is at least 85% identical to SEQ ID NOs: 3024 or 3025 and comprises one or more of the following position-amino acids 15C, 17C, 18K or R, 22A, 57C, 59C, 77C, 79G, 133I, 136A and/or 139H, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; or c) at least 85% identical to SEQ ID NO: 3043 and comprises one or more of the following position-amino acids 15C, 17C, 57C and/or 77C, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iii) a pre-TCRα chain constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3046, 3047 or 3048, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iv) a T-cell receptor γ (Cγ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NO: 3049, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(v) a T-cell receptor δ (Cδ) constant chain or region an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3051 or 3052, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(vi) a T cell receptor α (Cα) constant chain region fused to a CD3z chain region and which may comprise an optional costimulatory domain having an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3021, or 12421 to 12428; and
(vii) a T cell receptor β (Cβ) constant chain region fused to a CD3z chain region and which may comprise an optional costimulatory domain having an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3045, or 12401 to 12409.
5 . At least one recombinant polynucleotide encoding the synthetic immune receptor (SIR) of claim 2 , wherein the SIR comprises a heterodimer of T-cell receptor (TCR) constant chains or variants thereof selected from (a)(i) and (a)(ii), (a)(i) and (a)(iii), (a)(iv) and (a)(v), (a)(vi) and (a)(vi), (a)(i) and (a)(vi), a(ii) and a(vi), a(iii) and a(vi), a(iv) and a(vi), and a(v) and a(vi) below, wherein the TCR constant chains of the encoded SIR comprise:
(a) a T-cell receptor (TCR) constant chain or a variant thereof having an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor α (Cα) constant chain or region that comprises one or more mutations at positions 10, 15, 45, 48, 61, 91, 92, 93, and/or 94 corresponding to SEQ ID NO: 3010, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(ii) a T-cell receptor beta (Cβ) constant chain or region that comprises one or more mutations at positions 15, 17, 18, 22, 57, 79, 133, 136 and/or 139 corresponding to SEQ ID NO: 3024 or 3025, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iii) a pre-TCRα constant chain or region that is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iv) a T cell receptor γ (Cγ) constant chain or region that is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(v) a T cell receptor δ (Cδ) constant chain or region that is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; and
(vi) a T cell receptor constant chain region fused to a CD3z chain region, and which may comprise an optional accessory module;
(b) an optional linker; and
(c) one or more non-natural TCR antigen binding domain(s) linked to (a) selected from the group consisting of:
(1) an antibody;
(2) an antibody fragment selected from the group consisting of a Fv, a Fab, and a (Fab′)2;
(3) a heavy chain variable region of an antibody (vH domain) and a light chain variable region of an antibody (vL domain) specific for a target antigen wherein one of the vH and vL domains is attached to a first of the two TCR constant chains of (a) and the other of the vH and vL domains is attached to a second of the TCR constant chains of (a);
(4) a single chain variable fragment (scFv);
(5) a single domain antibody (SDAB);
(6) a camelid VHH domain;
(7) a monomeric variable region of an antibody;
(8) a non-immunoglobulin antigen binding scaffold optionally selected from the group consisting of a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obody, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a Kunitz domain, an armadillo repeat protein, and a functional fragment thereof;
(9) a receptor or a fragment thereof, and
(10) a ligand or a fragment thereof.
6 . The recombinant polynucleotide of claim 1 , wherein the TCR constant chain(s) or functional variants thereof of the encoded SIR comprise an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor α (Cα) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3010, 3012, 3015 to 3020, 3022, 3023, 3053, 12384 to 12386, or 18234 to 18235, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(ii) a T-cell receptor β (Cβ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3024, 3025, 3027, 3028, 3030, 3033 to 3044, 3054 to 3055, 12387 to 12390, or 18236 to 18237, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iii) a pre-TCRα chain constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3046, 3047 or 3048, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iv) a T-cell receptor γ (Cγ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NO: 3049, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(v) a T-cell receptor δ (Cδ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3051 or 3052, and/or is encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(vi) a T cell receptor α (Cα) constant chain region fused to a CD3z chain region with an optional costimulatory domain with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3021, or 12421 to 12428; and
(vii) a T cell receptor β (Cβ) constant chain region fused to a CD3z chain region with an optional costimulatory domain with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3045, or 12401 to 12409.
7 . The recombinant polynucleotide of claim 1 , wherein the SIR comprises a heterodimer of T-cell receptor (TCR) constant chains or functional variants thereof having mutations according to reference SEQ ID NOs: 3010 and 3024 respectively located at any one or more of the pairs of T-cell receptor α (Cα) constant chain or region and the T-cell receptor β (Cβ) constant chain or region at the amino acid positions consisting of:
a) the T-cell receptor α (Cα) constant chain or region with 48C and the T-cell receptor β (Cβ) constant chain or region with 57C;
b) the T-cell receptor a (Cα) constant chain or region with 15C and the T-cell receptor β (Cβ) constant chain or region or region with 15C;
c) the T-cell receptor α (Cα) constant chain or region with 45C and the T-cell receptor β (Cβ) constant chain or region with 59C;
d) the T-cell receptor a (Cα) constant chain or region with 45C and the T-cell receptor β (Cβ) constant chain or region with 77C; and
e) the T-cell receptor α (Cα) constant chain or region with 10C and the T-cell receptor β (Cβ) constant chain or region with 17C.
8 . The recombinant polynucleotide of claim 7 , wherein
(i) the T-cell receptor a (Cα) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3022, and the T-cell receptor β (Cβ) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3043; or
(ii) the T-cell receptor α (Cα) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3022 and has a C at the position corresponding to residue 48 of SEQ ID NO: 3010, and the T-cell receptor β (Cβ) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3043 and has a C at the position corresponding to residue 57 of SEQ ID NO: 3024; or
(iii) the T-cell receptor α (Cα) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3023, and the T-cell receptor β (Cβ) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3044; or
(iv) the T-cell receptor α (Cα) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3023 and has a C at the position corresponding to residue 48 of SEQ ID NO: 3010, and the T-cell receptor β (Cβ) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3044 and has a C at the position corresponding to residue 57 of SEQ ID NO: 3024; or
(v) the T-cell receptor α (Cα) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3010, 3012, 3015 to 3020, 3053, 12384 to 12386, or 18234 to 18235 and the T-cell receptor β (Cβ) constant chain or region comprises an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3024, 3025, 3027, 3028, 3030, 3033 to 3042, 3054 to 3055, 12387 to 12390, or 18236 to 18237.
9 . The recombinant polynucleotide of claim 1 , wherein the one or more non-natural TCR antigen binding domains are selected from the group consisting of:
(a) two single chain variable fragments (scFv) specific for one or more target antigens wherein expression of one of the scFv is attached to one of the two T-cell constant chains and the other of the scFv is attached to the other of the T-cell constant chains;
(b) two antibody fragments specific for one or more target antigens wherein upon expression, one of the antibody fragments is attached to one of the two T-cell constant chains and the other of the antibody fragments is attached to the other of the T-cell constant chains;
(c) two single domain antibody (SDAB) fragments specific for one or more target antigens wherein upon expression, one of the SDAB fragments is attached to one of the two T-cell constant chains and the other of the SDAB fragments is attached to the other of the T-cell constant chains;
(d) two camelid vHH domains specific for one or more target antigens, such that, when expressed, one of said vHH domains is attached to one of said two chains of said T-cell constant chains and the other of vHH domains is attached to the other of said two chains of said T-cell constant chains;
(e) two non-immunoglobulin antigen binding scaffolds specific for one or more target antigens, such that, when expressed, one of said non-immunoglobulin antigen binding scaffolds is attached to one of said two chains of said T-cell constant chains and the other of said non-immunoglobulin antigen binding scaffolds domains is attached to the other of said two chains of said T-cell constant chains;
(f) two receptors or a fragment thereof specific for one or more target antigens, such that, when expressed, one of said receptors or a fragment thereof is attached to one of said two chains of said T-cell constant chains and the other of said receptors or a fragment thereof is attached to the other of said two chains of said T-cell constant chains;
(g) two ligands or a fragment thereof specific for one or more target antigens, such that, when expressed, one of said ligands or a fragment thereof is attached to one of said two chains of said T-cell constant chains and the other of said ligands or a fragment thereof is attached to the other of said two chains of said T-cell constant chains;
(h) two structurally distinct antigen binding fragments specific for one or more target antigens, such that, when expressed, one of said antigen binding fragments is attached to one of said two chains of said T-cell constant chains and the other of said antigen binding fragments is attached to the other of said two chains of said T-cell constant chains;
(i) two binding fragments one or both of which are bispecific or multispecific such that, when expressed, one of said antigen binding fragments is attached to one of said two chains of said T-cell constant chains and the other of said antigen binding fragments is attached to the other of said two chains of said T-cell constant chains; and
(j) two autoantigens or fragments thereof, such that, when expressed, one of said autoantigens or fragments thereof is attached to one of said two chains of said T-cell constant chains and the other of said autoantigens or fragments thereof is attached to the other of said two chains of said T-cell constant chains.
10 . The recombinant polynucleotide of claim 1 , wherein one or more non-natural TCR antigen binding domain(s) that bind to one or more of disease-associated antigens are selected from the group consisting of: CD19; CD5; CD123; CD22; CD30; CD171; CS-1 (also referred to 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; 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; a glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors; a glycosylated CD43 epitope expressed on non-hematopoietic cancers; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); tyrosinase; Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); mammary gland differentiation antigen (NY-BR-1); Wilms tumor protein (WT1); cancer/testis antigen 1 (NY-ES0-1); melanoma-associated antigen 1 (MAGE-A1); melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); C-type lectin domain family 12 member A (CLEC12A); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1); MPL; biotin; c-MYC epitope Tag; CD34; LAMP1 TROP2; GFRalpha4; CDH17; CDH6; CDH19; CD200R; Slea (CA19.9; sialyl Lewis antigen) Fucosyl-GM1; PTK7; CDH1-CD324; DLL3; CD276/B7H3; IL11Ra; IL13Ra2; CD179b-IGLl1; TCR gamma-delta; NKG2D; CD32 (FCGR2A); CSPG4-HMW-MAA; Tim1-/HVCR1; CSF2RA (GM-CSFR-alpha); TGFbetaR2; VEGFR2/KDR; Lewis Ag; TCR-beta1 chain; TCR-beta2 chain; TCR-gamma chain; TCR-delta chain; FITC; luteinizing hormone receptor (LHR); follicle stimulating hormone receptor (FSHR); chorionic gonadotropin hormone receptor (CGHR); CCR4; GD3; SLAMF6; SLAMF4; HIV1 envelope glycoprotein; HTLV1-Tax; CMV pp65; EBV-EBNA3c; influenza A hemagglutinin (HA); GAD; PDL1; guanylyl cyclase C (GCC); KSHV-K8.1 protein; KSHV-gH protein; auto antibody to desmoglein 3 (Dsg3); autoantibody to desmoglein 1 (Dsg1); HLA-A2; HLA-B; HLA-C; HLA-DP; HLA-DM; HLA-DOA; HLA-DOB; HLA-DQ; HLA-DR; HLA-G; IGE; CD99; Lym1; Lym2; RAS G12V; Tissue Factor 1 (TF1); AFP; GPRC5D; claudin18.2 (CLD18A2 or CLDN18A.2); STEAP1; LIV1; NECTIN-4; CRIPTO; GPA33; BST1/CD157; low conductance chloride channel; and an antigen recognized by TNT antibody.
11 . The recombinant polynucleotide of claim 1 , wherein the one or more non-natural TCR antigen binding domain(s) is selected from the group consisting of:
(i) a heavy chain variable region (vH) comprising a sequence as set forth in any of SEQ ID NOs:2506 to 2680 or 12160 to 12278, or sequences with at least 95% identity thereto and containing the CDRs of any of the foregoing polypeptides and which binds to its antigen and a complementary light chain variable region (vL) comprising a sequence as set forth in any one of SEQ ID NOs 2307 to 2482 or 12042 to 12159 or sequences with at least 95% identity thereto and containing the CDRs of any of the foregoing polypeptides and which binds to its antigen;
(ii) a single chain variable fragment (scFv) comprising a sequence as set forth in any one SEQ ID NOs: 2770 to 2939, 12303 to 12357, or 18162 to 18224, or sequences with at least 95% identity thereto and containing the CDRs of any of the forgoing polypeptides and which binds to its antigen;
(iii) a camelid VHH domain comprising a sequence as set forth in any one of SEQ ID NOs: 2701 to 2725 or 12279 to 12294, or sequences with at least 95% identity thereto and containing the CDRs of any of the forgoing polypeptides and which binds to its antigen;
(iv) a non-immunoglobulin scaffold encoded by a polynucleotide of any one of SEQ ID NOs 439 to 443, or sequences with at least 95% identity thereto and containing the CDRs of any of the forgoing polypeptides and which binds to its antigen.
(v) a receptor comprising a sequence as set forth in any one of SEQ ID NOs 2736 to 2748, or sequences with at least 95% identity thereto and which binds to its cognate;
(vi a ligand comprising a sequence as set forth in any one of SEQ ID NOs 2758 to 2768 or 12359 to 12361, or sequences with at least 95% identity thereto containing the CDRs of any of the forgoing polypeptides and which binds to its cognate; and
(vii) a light chain variable region (vL) comprising one or more of light chain complementary determining region for a selected target antigen as set forth in any of SEQ ID NOs:13999 to 14879 or 14880 and a complementary heavy chain variable region (vH) comprising one or more of heavy chain complementary determining region for a selected target antigen as set forth in any of SEQ ID NOs:14881 to 15761 or 15762.
12 . A vector comprising the at least one recombinant polynucleotide of claim 1 , wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, a retrovirus vector, a baculovirus vector, a sleeping beauty transposon vector, and a piggybac transposon vector.
13 . A synthetic immune receptor (SIR) polypeptide heterodimer encoded by at least one recombinant polynucleotide of claim 1 .
14 . A recombinant T cell or a stem cell that can differentiate into a T cell comprising at least one recombinant polynucleotide encoding a synthetic immune receptor (SIR), wherein the SIR comprises a heterodimer of T-cell receptor (TCR) constant chains or functional variants thereof selected from (a)(i) and (a)(ii), (a)(i) and (a)(iii), (a)(iv) and (a)(v), (a)(vi) and (a)(vi), (a)(i) and (a)(vi), a(ii) and a(vi), a(iii) and a(vi), a(iv) and a(vi), and a(v) and a(vi) below,
wherein the T cell has impaired or abolished expression of one or more endogenous TCR chains or the stem cell has been engineered to result in impaired or abolished expression of one or more endogenous TCR chains in the T cell, and/or
wherein the polynucleotide encoding the TCR constant chains or functional variants of the TCR constant chains comprise one or more mutations that enhance the expression and/or pairing of encoded TCR constant chains or functional variants thereof, and/or reduce the pairing of the encoded TCR constant chains or functional variants thereof with the endogenous TCR chains;
wherein the encoded SIR comprises
(a) a T-cell receptor (TCR) constant chain or a functional variant thereof having an amino acid sequence selected from the group consisting of
(i) a T-cell receptor α (Cα) constant chain or region which may comprise an optional accessory module;
(ii) a T-cell receptor beta (Cβ) constant chain or region which may comprise an optional accessory module;
(iii) a pre-TCRα constant chain or region which may comprise an optional accessory module;
(iv) a T cell receptor γ (Cγ) constant chain or region which may comprise an optional accessory module;
(v) a T cell receptor δ (Cδ) constant chain or region which may comprise an optional accessory module; and
(vi) a T cell receptor constant chain region fused to a CD3z chain region and which may comprise an optional accessory module;
(b) an optional linker; and
(c) one or more non-natural TCR antigen binding domain(s) linked to (a) selected from the group consisting of:
(1) an antibody;
(2) an antibody fragment selected from the group consisting of a Fv, a Fab, and a (Fab′)2;
(3) a heavy chain variable region of an antibody (vH domain) and a light chain variable region of an antibody (vL domain) specific for a target antigen, such that one of said vH and vL domains is attached to a first of said two TCR constant chains of (a) and the other of said vH and vL domains is attached to a second of said TCR constant chains of (a);
(4) a single chain variable fragment (scFv);
(5) a single domain antibody (SDAB);
(6) a camelid VHH domain;
(7) a monomeric variable region of an antibody;
(8) a non-immunoglobulin antigen binding scaffold optionally selected from the group consisting of a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obody, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a Kunitz domain, an armadillo repeat protein and a functional fragment thereof;
(9) a receptor or a fragment thereof, and
(10) a ligand or a fragment thereof.
15 . The recombinant cell of claim 14 , wherein the TCR constant chain(s) or functional variants thereof of the encoded SIR comprise an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor α (Cα) constant chain or region that comprises a) one or more mutations at positions 10, 15, 45, 48, 61, 91, 92, 93, and/or 94 corresponding to SEQ ID NO: 3010, and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; or b) an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3010, 3012, 3015 to 3020, 3022, 3023, 3053, 12384 to 12386, or 18234 to 18235 and comprises one or more of the following position-amino acids 10C, 15C, 45C, 48C, 61R, 91S, 92D, 93V, and/or 94P corresponding to SEQ ID NO: 3010, and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(ii) a T-cell receptor beta (Cβ) constant chain or region that comprises a) one or more mutations at positions 15, 17, 18, 22, 57, 79, 133, 136 and/or 139 corresponding to SEQ ID NO: 3024 or 3025 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; or b) an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3024, 3025, 3027, 3028, 3030, 3033 to 3044, 3054 to 3055, 12387 to 12390, or 18236 to 18237, and comprises one or more of the following position-amino acids 15C, 17C, 18K or R, 22A, 57C, 59C, 77C, 79G, 133I, 136A and/or 139H, and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iii) a pre-TCRα chain constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3046, 3047 or 3048 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iv) a T-cell receptor γ (Cγ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NO: 3049 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(v) a T-cell receptor δ (Cδ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3051 or 3052 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(vi) a T cell receptor α (Cα) constant chain region fused to a CD3z chain region with an optional costimulatory domain with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3021, or 12421 to 12428 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; and
(vii) a T cell receptor β (Cβ) constant chain region fused to a CD3z chain region with an optional costimulatory domain with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3045, or 12401 to 12409 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized.
16 . The recombinant T cell or stem cell that can differentiate into a T cell of claim 14 , wherein (a) the T cell is selected from the group consisting of an alpha/beta T cell, a gamma/delta T cell, a regulatory T cell (TREG), a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, and a synthetic T cell; (b) the stem cell is selected from the group consisting of a hematopoietic stem cell, a peripheral blood stem cell, a bone marrow-derived stem cell, an immune stem cell, a lymphoid stem cell, an embryonic stem cell, and an induced pluripotent stem cell (iPSC); and (c) the T cell of (a) or the stem cell of (b) is autologous, allogeneic, syngeneic, or xenogeneic.
17 . The recombinant T cell or stem cell that can differentiate into a T cell of claim 14 , wherein the cell further comprises:
(a) at least one chimeric antigen receptor (CAR) polypeptide, wherein the CAR comprises:
(i) an antigen binding domain, a transmembrane domain and an intracellular domain of an inhibitory molecule,
(ii) an antigen binding domain, a transmembrane domain, and a primary signaling domain,
(iii) an antigen binding domain, a transmembrane domain, a costimulatory signaling domain but no primary signaling domain, or
(iv) an antigen binding domain, a transmembrane domain, a primary signaling domain and a costimulatory domain;
(b) reduced or eliminated expression of an endogenous TCR;
(c) reduced or eliminated expression of one or more subunits that comprise a functional endogenous TCR;
(d) reduced or eliminated expression of one or more TCR chains selected from the group consisting of TCRα, TCRβ1, TCRβ2, TCRγ, TCRδ, and pre-TCRα
(e) an impaired endogenous TCR
(f) loss of expression or decreased expression of a functional HLA;
(g) loss of expression or decreased expression of β2 microglobulin;
(h) a deletion or mutation in one or more target antigens to a form that is no longer recognized by the SIR
(i) a deletion of diaglycerol kinase (DGK) or ikaros;
(j) an agent that increases the expression and/or activity of the SIR, wherein the agent is selected from the group consisting of CD3ζ, CD3δ, CD3ε, CD3γ, or combination thereof;
(k) an agent that enhances the activity of SIR-expressing cell;
(l) an agent that inhibits an inhibitory molecule
(m) an agent that inhibits the expression of one or more target antigens
(n) an agent that provides co-stimulation to a SIR-expressing cell
(o) a cytokine and/or a chemokine;
(p) an agent that promotes the proliferation, persistence, expansion, and activation of an SIR-expressing cell
(q) a soluble receptor
(r) a second SIR;
(s) an accessory module selected from the group consisting of 41BBL, CD40L, K13, MC159, cFLIP-L/MRITa, cFLIP-p22, HTLV1 Tax, HTLV2 Tax, HTLV2, Tax-RS mutant, FKBP×2-K13, FKBP×2-HTLV2-Tax, FKBP×2-HTLV2-Tax-RS, IL6R-304-vHH-Alb8-vHH, IL12f, PD1-4H1 scFV, PD1-5C4 scFV, PD1-4H1-Alb8-vHH, PD1-5C4-Alb8-vHH, CTLA4-Ipilimumab-scFv, CTLA4-Ipilimumab-Alb8-vHH, IL6-19A-scFV, IL6-19A-scFV-Alb8-vHH, sHVEM, sHVEM-Alb8-vHH, hTERT, Fx06, CD3z, CD3z-GGGS-41 BB, CD3-BBz, CD3-CD28z, CD3-CD28-Lck fusion protein, shRNA targeting Brd4, hTERT, heparinase, cytokine, chemokine, IL2, IL-7, IL-15, IL12f, IL-21, a costimulatory agent, a soluble receptor, and any combination thereof; or
(t) a therapeutic control, wherein the therapeutic control is optionally selected from the group consisting of a 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 phosphoribosyl transferase, human caspase 8, human caspase 9, inducible caspase 9 (icaspase9), purine nucleoside phosphorylase, linamarase/linamarin/glucose oxidase, deoxyribonucleoside kinase, horseradish peroxidase (HRP)/indole-3-acetic (IAA), Gamma-glutamylcysteine synthetase, CD20/alphaCD20, CD34/thymidine kinase chimera, dox-dependent caspase-2, mutant thymidine kinase (HSV-TKSR39), AP1903/Fas system, a chimeric cytokine receptor (CCR), a selection marker, dihydroxyfolate receptor (DHFR), a mutant DHFR, methylated-DNA-protein-cysteine methyltransferase, inosine monophosphate dehydrogenase II (IMDHP2), puromycin acetyle transferase (PAC), blasticidin-resistance gene, a mutant calcinueurin a/b (Can/b), CNa12, CNb30, a suicide gene, and any combination thereof.
18 . The cell of claim 14 , wherein the SIR comprises:
(a) TCRα and TCRβ constant chains or variants thereof wherein the cell lacks the expression of or has impaired expression of the endogenous TCRα chain or TCRβ chain or both; or
(b) TCRγ and TCRδ constant chains or variants thereof wherein the cell lacks the expression of or has impaired expression of the endogenous TCRγ chain or TCRδ chain or both; or
(c) TCRα and TCRβ constant chains or variants thereof wherein the cell is an alpha/beta T cell, a gamma/delta T cell, a regulatory T cell (T REG ), a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, a synthetic T cell, or a T cell derived from a stem cell; or
(d) TCRγ and TCRδ constant chains or variants thereof wherein the cell is an alpha/beta T cell, a gamma/delta T cell, a regulatory T cell (T REG ), a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, a synthetic T cell, or a T cell derived from a stem cell; or
(e) a T cell receptor α (Cα) constant chain region fused to a CD3z chain and a T cell receptor β (Cβ) constant chain region fused to a CD3z chain wherein the cell is an alpha/beta T cell, a gamma/delta T cell, a regulatory T cell (TREG), a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, a synthetic T cell, or a T cell derived from a stem cell; and/or
f) a linker between one or both T constant chain regions and the non-TCR antigen binding domain(s).
19 . A method of making a SIR-expressing T cell or stem cell that can differentiate into a T cell, comprising steps:
A)—introducing into a T cell or stem cell that can differentiate into a T cell at least one recombinant polynucleotide encoding a synthetic immune receptor (SIR), wherein the SIR comprises a heterodimer of T-cell receptor (TCR) constant chains or functional variants thereof selected from (a)(i) and (a)(ii), (a)(i) and (a)(iii), (a)(iv) and (a)(v), (a)(vi) and (a)(vi), (a)(i) and (a)(vi), a(ii) and a(vi), a(iii) and a(vi), a(iv) and a(vi), and a(v) and a(vi) below,
wherein the polynucleotide encoding the TCR constant chains or functional variants of the TCR constant chains comprise one or more mutations that:
enhance the expression and/or pairing of encoded TCR constant chains or functional variants thereof, and/or reduce the pairing of the encoded TCR constant chains or functional variants thereof with the endogenous TCR chains;
wherein the encoded SIR comprises:
(a) a T-cell receptor (TCR) constant chain or a functional variant thereof having an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor α (Cα) constant chain or region which may comprise an optional accessory module;
(ii) a T-cell receptor beta (Cβ) constant chain or region which may comprise an optional accessory module;
(iii) a pre-TCRα constant chain or region which may comprise an optional accessory module;
(iv) a T cell receptor γ (Cγ) constant chain or region which may comprise an optional accessory module;
(v) a T cell receptor δ (Cδ) constant chain or region which may comprise an optional accessory module; and
(vi) a T cell receptor constant chain region fused to a CD3z chain region and which may comprise an optional accessory module; and
(b) an optional linker; and
(c) one or more non-natural TCR antigen binding domain(s) linked to (a) selected from the group consisting of:
(1) an antibody;
(2) an antibody fragment selected from the group consisting of a Fv, a Fab, and a (Fab′)2;
(3) a heavy chain variable region of an antibody (vH domain) and a light chain variable region of an antibody (vL domain) specific for a target antigen, such that one of said vH and vL domains is attached to a first of said two TCR constant chains of (a) and the other of said vH and vL domains is attached to a second of said TCR constant chains of (a);
(4) a single chain variable fragment (scFv);
(5) a single domain antibody (SDAB);
(6) a camelid VHH domain;
(7) a monomeric variable region of an antibody;
(8) a non-immunoglobulin antigen binding scaffold optionally selected from the group consisting of a DARPIN, an affibody, an affilin, an adnectin, an affitin, an obody, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a Kunitz domain, an armadillo repeat protein and a functional fragment thereof;
(9) a receptor or a fragment thereof; and
(10) a ligand or a fragment thereof; and
optionally
B)—reducing or abolishing the expression of one or more endogenous TCR chains in the T cell or stem cell that can differentiate into a T cell, wherein optionally the endogenous TCR chain is selected from the group consisting of TCRα, TCRβ1, TCRβ2, TCRγ and TCRδ; and/or
C)—reducing or abolishing the expression of an HLA and/or β2 microglobulin; and/or
D)—reducing or abolishing the expression of one or more target antigens to a form that is no longer recognized by the SIR;
wherein, where indicated, steps A, B, C and/or D can be carried out concurrently or sequentially in any order.
20 . The method of claim 18 , wherein the encoded TCR constant chain(s) or functional variants thereof comprise an amino acid sequence selected from the group consisting of:
(i) a T-cell receptor a (Cα) constant chain or region that comprises a) one or more mutations at positions 10, 15, 45, 48, 61, 91, 92, 93, and/or 94 corresponding to SEQ ID NO: 3010, and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; or b) an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3010, 3012, 3015 to 3020, 3022, 3023, 3053, 12384 to 12386, or 18234 to 18235 and comprises one or more of the following position-amino acids 10C, 15C, 45C, 48C, 61R, 91S, 92D, 93V, and/or 94P corresponding to SEQ ID NO: 3010, and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(ii) a T-cell receptor beta (Cβ) constant chain or region that comprises a) one or more mutations at positions 15, 17, 18, 22, 57, 79, 133, 136 and/or 139 corresponding to SEQ ID NO: 3024 or 3025 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; or b) an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3024, 3025, 3027, 3028, 3030, 3033 to 3044, 3054 to 3055, 12387 to 12390, or 18236 to 18237, and comprises one or more of the following position-amino acids 15C, 17C, 18K or R, 22A, 57C, 59C, 77C, 79G, 133I, 136A and/or 139H, and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iii) a pre-TCRα chain constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3046, 3047 or 3048 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(iv) a T-cell receptor γ (Cγ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NO: 3049 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(v) a T-cell receptor δ (Cδ) constant chain or region with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3051 or 3052 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized;
(vi) a T cell receptor α (Cα) constant chain region fused to a CD3z chain region with an optional costimulatory domain with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3021, or 12421 to 12428 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized; and
(vii) a T cell receptor β (Cβ) constant chain region fused to a CD3z chain region with an optional costimulatory domain with an amino acid sequence that is at least 85% identical to SEQ ID NOs: 3045, or 12401 to 12409 and is optionally encoded by a polynucleotide sequence that is not wild-type and/or is codon-optimized.
21 . A pharmaceutical composition comprising the recombinant cell of claim 14 and a pharmaceutically acceptable carrier.
22 . A method of treating or preventing a disease in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 21 , wherein the disease is selected from the group consisting of a cancer, an autoimmune disease, an infectious disease, an allergic disease, or a degenerative disease.