Engineered receptors and methods of use thereof

Abstract

Provided herein are engineered receptors (e.g., CARs), dual CAR systems, immune cells expressing these engineered receptors or these dual CAR systems, and uses thereof.

Description

Engineered receptors and their application

[0001] Cross-references to related applications

[0002] This application claims priority to International Application No. PCT / CN2023 / 121096, filed on September 25, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence declaration

[0004] The following content, submitted as an XML file, is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (filename: IEC240638PCT_Sequence Listing, date of record: September 23, 2024, size: 133KB). Technical Field

[0005] This disclosure relates to engineered receptors (e.g., CARs), dual CAR systems, engineered immune effector cells containing them, and methods of using them. Background Technology

[0006] Chimeric antigen receptor (CAR)-based therapies are a rapidly emerging form of cancer treatment and have achieved significant responses in diseases such as refractory lymphomas. However, their clinical use is limited by toxicities associated with cytokine release syndrome and neurotoxicity, the modeling complexity of their manufacturing processes, the cost and time of treatment, and the risk of graft-versus-host disease (GvHD) associated with immunocellular therapy. Therefore, there is a need for CAR designs with improved efficacy and safety for clinical use. Summary of the Invention

[0007] This disclosure relates to engineered receptors comprising: an extracellular antigen-binding domain; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain derived from FcRγ, and one or more additional signaling domains selected from the CD28 co-stimulatory signaling domain, the Dap10 intracellular signaling domain, the OX40 intracellular signaling domain, the CD27 intracellular signaling domain, the CD137 (4-1BB) co-stimulatory signaling domain, and the ICOS intracellular signaling domain.

[0008] In some embodiments, the additional signal transduction domain is the CD28 co-stimulatory signal transduction domain.

[0009] In some embodiments, the CD28 co-stimulatory signal transduction domain is located at the N-terminus of the intracellular signal transduction domain derived from FcRγ.

[0010] In some embodiments, the additional signal transduction domain is the CD137 (4-1BB) co-stimulatory signal transduction domain.

[0011] In some embodiments, the CD137(4-1BB) co-stimulatory signal transduction domain is located at the N-terminus of the intracellular signal transduction domain derived from FcRγ.

[0012] In some embodiments, the transmembrane domain is derived from the following transmembrane domains: the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154.

[0013] In some embodiments, the engineered receptor further includes a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0014] In some embodiments, the hinge region is derived from the following hinge regions: CD8α, CD28, IgG1, IgG2, IgG3, or IgG4.

[0015] In some embodiments, the engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

[0016] In some embodiments, the engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

[0017] In some embodiments, the engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

[0018] In some embodiments, the engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

[0019] In some embodiments, the extracellular antigen-binding domain includes sdAb, scFv, (scFv)2, and V. HH-domain or VNAR-domain.

[0020] In some embodiments, the extracellular antigen-binding domain binds to tumor antigens.

[0021] In some embodiments, the tumor antigen is selected from the group consisting of: B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 gangliosides, glycoprotein 75, GPNMB, HER2 / neu, HGF, human dispersive factor receptor kinase, IGF-1 receptor, IGF-1, IgG1, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-hydroxyacetylneuraminic acid, NPC-1C, PDGF-RA, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGFβ2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin.

[0022] In some embodiments, the extracellular antigen-binding domain binds to CD19.

[0023] In some embodiments, the extracellular antigen-binding domain comprises: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains at least 80% identical amino acid sequences to the selected VH CDR1 amino acid sequence, the VH CDR2 region contains at least 80% identical amino acid sequences to the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains at least 80% identical amino acid sequences to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region contains at least 80% identical amino acid sequences to the selected VL CDR1 amino acid sequence, the VL CDR2 region contains at least 80% identical amino acid sequences to the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains at least 80% identical amino acid sequences to the selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 51, 52, and 53, respectively, and the selected VL CDRs 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 51, 52, and 53, respectively. The amino acid sequences of 1, 2, and 3 are shown in SEQ ID NO: 54, 55, and 56, respectively.

[0024] In some embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising at least 80% of the same amino acid sequence as the selected VH sequence, and the light chain variable region comprising at least 80% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO: 58.

[0025] In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7.

[0026] In some embodiments, the engineered receptor comprises an amino acid sequence shown in any one of SEQ ID NO: 33, 36-43 and 68, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 33, 36-43 and 68.

[0027] In some embodiments, the engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 36.

[0028] In some embodiments, the engineered receptor further comprises a signal peptide. In some embodiments, the engineered receptor comprises an amino acid sequence shown in any one of SEQ ID NO: 78-90, 94, 95, 97 and 98, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 78-90, 94, 95, 97 and 98.

[0029] In some embodiments, the extracellular antigen-binding domain binds to CD20.

[0030] In some embodiments, the extracellular antigen-binding domain includes V H The H antibody portion, this V H The H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains at least 80% of the same amino acid sequence as the selected CDR1 amino acid sequence, CDR2 contains at least 80% of the same amino acid sequence as the selected CDR2 amino acid sequence, and CDR3 contains at least 80% of the same amino acid sequence as the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 50.

[0031] In some embodiments, the extracellular antigen-binding domain includes V H The H antibody portion, this V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0032] In some embodiments, the engineered receptor comprises an amino acid sequence shown in any one of SEQ ID NO: 69-71 and 74, or an amino acid sequence having at least 90%, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 69-71 and 74.

[0033] In some embodiments, the engineered receptor further comprises a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the engineered receptor. In some embodiments, the signal peptide is derived from CD8α, and optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 91-93 and 96, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 91-93 and 96.

[0034] This disclosure also relates to a dual chimeric antigen receptor (CAR) system comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain; a first transmembrane domain; and a first intracellular domain comprising an intracellular signal transduction domain derived from FcRγ; and a CD28 co-stimulatory signal transduction domain or a CD137 (4-1BB) co-stimulatory signal transduction domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

[0035] In some embodiments, the second intracellular domain includes the CD3ζ primary intracellular signal transduction domain.

[0036] In some embodiments, the second engineered receptor further includes a co-stimulatory signal transduction domain located between the C-terminus of the transmembrane domain and the N-terminus of the CD3ζ primary intracellular signal transduction domain.

[0037] In some embodiments, the costimulatory signal transduction domain is a CD137 (4-1BB) costimulatory signal transduction domain or a CD28 costimulatory signal transduction domain.

[0038] In some embodiments, the first transmembrane domain and / or the second transmembrane domain are derived from the following transmembrane domains: the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154.

[0039] In some embodiments, the first transmembrane domain is a transmembrane domain derived from CD28, and the second transmembrane domain is a transmembrane domain derived from CD8α.

[0040] In some embodiments, the first transmembrane domain is a transmembrane domain derived from CD8α, and the second transmembrane domain is a transmembrane domain derived from CD28.

[0041] In some embodiments, the dual CAR system further includes a first hinge region located between the C-terminus of the first extracellular antigen-binding domain and the N-terminus of the first transmembrane domain; and / or a second hinge region located between the C-terminus of the second extracellular antigen-binding domain and the N-terminus of the second transmembrane domain.

[0042] In some embodiments, the first hinge region and / or the second hinge region are derived from the following hinge regions: CD8α, CD28, IgG1, IgG2, IgG3, or IgG4.

[0043] In some embodiments, the dual CAR system is specified as one of the following:

[0044] (1) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; or

[0045] (2) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0046] In some embodiments, the dual CAR system is specified as one of the following:

[0047] (1) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain;

[0048] (2) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; or

[0049] (3) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0050] In some embodiments, the dual CAR system is specified as one of the following:

[0051] (1) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain;

[0052] (2) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain;

[0053] (3) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain;

[0054] (4) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain;

[0055] (5) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; or

[0056] (6) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0057] In some embodiments, the first engineered receptor and the second engineered receptor are connected via a connector.

[0058] In some embodiments, the first extracellular antigen-binding domain and / or the second extracellular antigen-binding domain each bind to a tumor antigen.

[0059] In some embodiments, the tumor antigen is selected from the group consisting of: B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 gangliosides, glycoprotein 75, GPNMB, HER2 / neu, HGF, human dispersive factor receptor kinase, IGF-1 receptor, IGF-1, IgG1, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-hydroxyacetylneuraminic acid, NPC-1C, PDGF-RA, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGFβ2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin.

[0060] In some embodiments, the first extracellular antigen-binding domain binds to CD19.

[0061] In some embodiments, the first extracellular antigen-binding domain comprises: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 51, 52, and 53, respectively, and the selected VL CDRs The amino acid sequences of 1, 2, and 3 are shown in SEQ ID NO: 54, 55, and 56, respectively.

[0062] In some embodiments, the first extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising an amino acid sequence that is at least 80% identical to the selected VH sequence, and the light chain variable region comprising an amino acid sequence that is at least 80% identical to the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO: 58.

[0063] In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7.

[0064] In some embodiments, the second extracellular antigen-binding domain binds to CD20.

[0065] In some embodiments, the second extracellular antigen-binding domain includes V H The H antibody portion, this V HThe H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains at least 80% of the same amino acid sequence as the selected CDR1 amino acid sequence, CDR2 contains at least 80% of the same amino acid sequence as the selected CDR2 amino acid sequence, and CDR3 contains at least 80% of the same amino acid sequence as the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 50.

[0066] In some embodiments, the V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0067] In some embodiments, the dual CAR system is specified as one of the following:

[0068] (1) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 36; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 24.

[0069] (2) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 68; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 24.

[0070] In some embodiments, the first engineered receptor and / or the second engineered receptor further comprises a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the first engineered receptor. In some embodiments, the signal peptide is located at the N-terminus of the second engineered receptor. In some embodiments, the signal peptide is derived from CD8α, and optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO: 8.

[0071] In some embodiments, the dual CAR system comprises the amino acid sequence shown in SEQ ID NO: 19 or 60, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 19 or 60.

[0072] In some embodiments, the first extracellular antigen-binding domain binds to CD20.

[0073] In some embodiments, the first extracellular antigen-binding domain includes V H The H antibody portion, this V H The H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains at least 80% of the same amino acid sequence as the selected CDR1 amino acid sequence, CDR2 contains at least 80% of the same amino acid sequence as the selected CDR2 amino acid sequence, and CDR3 contains at least 80% of the same amino acid sequence as the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 50.

[0074] In some embodiments, the V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0075] In some embodiments, the second extracellular antigen-binding domain binds to CD19.

[0076] In some embodiments, the second extracellular antigen-binding domain includes:

[0077] A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains at least 80% identical amino acid sequence to the selected VH CDR1 amino acid sequence, the VH CDR2 region contains at least 80% identical amino acid sequence to the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains at least 80% identical amino acid sequence to the selected VH CDR3 amino acid sequence; and

[0078] The light chain variable region (VL) comprises CDRs 1, 2, and 3, wherein the VL CDR1 region contains at least 80% of the same amino acid sequence as the selected VL CDR1 amino acid sequence, the VL CDR2 region contains at least 80% of the same amino acid sequence as the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains at least 80% of the same amino acid sequence as the selected VL CDR3 amino acid sequence, wherein the selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 51, 52, and 53, respectively, and the selected VLCDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 54, 55, and 56, respectively.

[0079] In some embodiments, the second extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising an amino acid sequence that is at least 80% identical to the selected VH sequence, and the light chain variable region comprising an amino acid sequence that is at least 80% identical to the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO: 58.

[0080] In some embodiments, the second extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7.

[0081] In some embodiments, the dual CAR system is specified as one of the following:

[0082] (1) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 69; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 47.

[0083] (2) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 70, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 70; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 47.

[0084] (3) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 71; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 72.

[0085] (4) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 71; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 73.

[0086] (5) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 74; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 75.

[0087] (6) A first engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 74; and a second engineered receptor comprising the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 76.

[0088] In some embodiments, the first engineered receptor and / or the second engineered receptor further comprises a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the first engineered receptor. In some embodiments, the signal peptide is located at the N-terminus of the second engineered receptor. In some embodiments, the signal peptide is derived from CD8α, and optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO: 8.

[0089] In some embodiments, the dual CAR system comprises an amino acid sequence shown in any one of SEQ ID NO: 61-66, or an amino acid sequence having at least 90%, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 61-66.

[0090] This disclosure also relates to nucleic acids containing nucleotide sequences encoding any of the engineered receptors described herein or any of the dual CAR systems described herein.

[0091] This disclosure also relates to cells that comprise any of the engineered receptors described herein, any of the dual CAR systems described herein, or any of the nucleic acids described herein.

[0092] In some embodiments, the cell is an immune cell.

[0093] In some embodiments, the immune cell is selected from the group consisting of: T cells, NK cells, NKT cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, macrophages, monocytes, neutrophils, eosinophils, and combinations thereof.

[0094] In some embodiments, the T cell is an αβ T cell, a γδ T cell, or a panT cell.

[0095] This disclosure also relates to modified γδT cells comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain that binds to CD19; a first transmembrane domain; and a first intracellular domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain that binds to CD20; a second transmembrane domain; and a second intracellular domain.

[0096] In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7, and the second extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0097] This disclosure also relates to modified αβT cells comprising a first engineered receptor comprising: a first extracellular antigen-binding domain that binds to CD20; a first transmembrane domain; and a first intracellular domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain that binds to CD19; a second transmembrane domain; and a second intracellular domain.

[0098] In some embodiments, the first extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1, and the second extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7.

[0099] This disclosure also relates to pharmaceutical compositions comprising any of the cellular and pharmaceutically acceptable carriers described herein.

[0100] This disclosure also relates to methods for treating and / or preventing cancer, including administering an effective amount of any of the cells described herein or any of the pharmaceutical compositions described herein to a subject in need.

[0101] This disclosure also relates to methods for treating and / or preventing autoimmune diseases, including administering to a subject in need an effective amount of any of the cells described herein or any of the pharmaceutical compositions described herein.

[0102] As used herein, the terms “intracellular domain” and “cytoplasmic region” are used interchangeably to refer to the portion of the receptor within the cell. An intracellular domain may be the entire intracellular portion of the receptor or only a portion thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). An intracellular domain may be derived from an intracellular domain of a wild-type membrane protein (e.g., a receptor) or a functional variant thereof. An intracellular domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. An intracellular domain may be part of a chimeric antigen receptor (CAR).

[0103] As used herein, the term "DAP10 intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from DAP10 or a functional variant thereof. The DAP10 intracellular signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. The DAP10 intracellular signal transduction domain may contain the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20.

[0104] As used herein, the term "DAP12 intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from DAP12 or a functional variant thereof. The DAP12 intracellular signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. The DAP12 intracellular signal transduction domain may contain the sequence of SEQ ID NO: 17, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 17.

[0105] As used herein, the term "OX40 intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from OX40 or a functional variant thereof. The OX40 intracellular signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. The OX40 intracellular signal transduction domain may contain the sequence of SEQ ID NO: 21, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21.

[0106] As used herein, the term "CD27 intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from CD27 or a functional variant thereof. The CD27 intracellular signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. The CD27 intracellular signal transduction domain may contain the sequence of SEQ ID NO: 22, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 22.

[0107] As used herein, the term "ICOS intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from ICOS or a functional variant thereof. An ICOS intracellular signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. An ICOS intracellular signal transduction domain may contain the sequence of SEQ ID NO: 23, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 23.

[0108] As used herein, the term "FcRγ intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from FcRγ or a functional variant thereof. Intracellular signal transduction domains derived from FcRγ may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. Intracellular signal transduction domains derived from FcRγ may contain the sequence of SEQ ID NO: 16, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16.

[0109] As used herein, the term "costimulatory signaling domain" refers to a portion of a receptor that includes one or more domains (e.g., an endodomain) derived from a costimulatory protein so that the receptor persists after activation. Costimulatory signaling domains can be derived from the endodomain / cytoplasmic domain of a wild-type costimulatory protein or a functional variant thereof. Costimulatory signaling domains can have one or more mutations, including, for example, insertions, deletions, and / or substitutions.

[0110] As used herein, the term "CD137(4-1BB) costimulatory signal transduction domain" refers to an intracellular domain derived from CD137(4-1BB) or a functional variant thereof. The CD137(4-1BB) costimulatory signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. The CD137(4-1BB) costimulatory signal transduction domain may contain the sequence of SEQ ID NO: 13, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13.

[0111] As used herein, the term "CD28 costimulatory signaling domain" refers to an intracellular domain derived from CD28 or a functional variant thereof. A CD28 costimulatory signaling domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A CD28 costimulatory signaling domain may contain the sequence of SEQ ID NO: 14, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14.

[0112] As used herein, the term "primary intracellular signaling domain" refers to an intracellular signaling sequence that acts in a stimulatory manner to induce the function of immune effectors. Primary intracellular signaling domains may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. As used herein, "ITAM" is a conserved protein motif commonly found in the tail portion of signaling molecules expressed in many immune cells. This motif may consist of two repeats of the amino acid sequence YxxL / I, separated by 6–8 amino acids, where each x is any amino acid independently, resulting in the conserved motif YxxL / Ix(6–8)YxxL / I. The ITAM within the signaling molecule is important for intracellular signal transduction, which is at least partially mediated by phosphorylation of tyrosine residues in the ITAM upon activation of the signaling molecule. The ITAM may also serve as a docking site for other proteins involved in signaling pathways. Exemplary primary intracellular signaling sequences containing ITAM include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Primary intracellular signaling domains may be derived from CD3ζ. Intracellular signaling domains may consist of CD3ζ intracellular signaling domains. Primary intracellular signaling domains may be derived from wild-type CD3ζ intracellular signaling domains. Primary intracellular signaling domains may be functional mutants of CD3ζ intracellular signaling domains containing one or more mutations (such as Q65K).

[0113] As used herein, the term "CD3ζ primary intracellular signal transduction domain" refers to an intracellular signal transduction domain derived from CD3ζ or a functional variant thereof. A CD3ζ primary intracellular signal transduction domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A CD3ζ primary intracellular signal transduction domain may contain the sequence of SEQ ID NO: 15, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 15.

[0114] As used herein, the terms “transmembrane domain” or “transmembrane region” are used interchangeably to refer to a portion of a membrane protein (e.g., a receptor) that is embedded in the cell membrane. A transmembrane domain may be the entire portion of a protein embedded in the cell membrane or only a portion thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). A transmembrane domain may be derived from a wild-type receptor or a transmembrane domain of a functional variant thereof. A transmembrane domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions.

[0115] As used herein, the term "CD8α transmembrane domain" refers to a transmembrane domain derived from CD8α or a functional variant thereof. A CD8α transmembrane signaling domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A CD8α transmembrane signaling domain may contain the sequence of SEQ ID NO: 11, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11.

[0116] As used herein, the term "CD28 transmembrane domain" refers to a transmembrane domain derived from CD28 or a functional variant thereof. A CD28 transmembrane signaling domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A CD28 transmembrane signaling domain may contain the sequence of SEQ ID NO: 12, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12.

[0117] As used herein, the terms “hinge domain” or “hinge region” are used interchangeably to refer to the portion of a membrane protein (e.g., a receptor) that connects the transmembrane region and the extracellular domain. The hinge region may be part of the extracellular region. The hinge region may be derived from the hinge region of a wild-type receptor or a functional variant thereof. The hinge region may have one or more mutations, including, for example, insertions, deletions, and / or substitutions.

[0118] As used herein, the term "CD8α hinge region" refers to a hinge region derived from CD8α or a functional variant thereof. A CD8α hinge region may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A CD8α hinge region may contain the sequence of SEQ ID NO: 9, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9.

[0119] As used herein, the term "CD28 hinge region" refers to a hinge region derived from CD28 or a functional variant thereof. A CD28 hinge region may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A CD28 hinge region may contain the sequence of SEQ ID NO: 10, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10.

[0120] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An “expression vector” is capable of delivering one or more polynucleotides of interest into a host cell that has been introduced into the expression vector and expressing those polynucleotides as encoded polypeptides. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by operatively linking to regulatory elements such as promoters, enhancers, and / or poly-A tails, which are located within the vector or in the genome of the host cell at or near the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell in which the expression vector has been introduced.

[0121] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells, such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs may contain extracellular ligand-binding domains or extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains that are specific to one or more antigens, such as tumor antigens. "CAR-T cell" refers to a T cell that expresses a CAR. "CAR-NK cell" refers to an NK cell that expresses a CAR.

[0122] As used herein, the term "cancer" or "cancer cell" refers to cells that divide in an uncontrolled manner, such as forming a solid tumor or forming an excessive number of tumor cells in the blood. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growth, such as tumors; carcinogenic processes, metastatic tissue, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. Cancer cells can form solid tumors or form an excessive number of tumor cells in the blood (e.g., blood cancers). Alternatively or additionally, it can include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. Examples of solid tumors include malignant tumors of different organ systems, such as sarcomas, adenocarcinomas, and carcinomas, such as those affecting the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidneys, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small bowel cancers, and esophageal cancers. Examples of cancers that can be treated using the methods described herein include, for example, bone cancer; pancreatic cancer; skin cancer (e.g., melanoma); head and neck cancer; malignant melanoma of the skin or eye; uterine cancer; ovarian cancer; rectal cancer; anal cancer; stomach cancer; testicular cancer; fallopian tube cancer; endometrial cancer; cervical cancer; vaginal cancer; vulvar cancer; Hodgkin's disease; non-Hodgkin's lymphoma; esophageal cancer; small bowel cancer; endocrine system cancers; thyroid cancer; parathyroid cancer; adrenal cancer; and soft tissue sarcoma. ; urethral cancer; penile cancer; chronic or acute leukemia including acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; lymphocytic lymphoma; bladder cancer; kidney or ureter cancer; renal pelvis cancer; central nervous system (CNS) vegetations; primary CNS lymphoma; tumor angiogenesis; spinal cord axon tumors; brainstem gliomas; pituitary adenomas; Kaposi's sarcoma; epidermoid cancer; squamous cell carcinoma; and / or T-cell lymphoma.

[0123] As used herein, the terms “extracellular domain” or “extracellular region” are used interchangeably to refer to the portion of the receptor outside the cell membrane. An extracellular domain may be the entire portion of the receptor outside the cell membrane or only a portion thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). An extracellular domain may be derived from the extracellular domain of a wild-type receptor or a functional variant thereof. An extracellular domain may have one or more mutations, including, for example, insertions, deletions, or substitutions.

[0124] As used herein, the terms "antigen-binding domain" or "extracellular antigen-binding domain" refer to a portion of a full-length antibody that is capable of specifically binding to an antigen. The antigen-binding fragment may contain at least one variable domain (e.g., a variable domain of a heavy chain, a single-domain antibody, or V...). H H). Non-limiting examples of antibody fragments include, for example, VH-VL pairs, scFv, Fab, Fab', F(ab')2, and Fv fragments.

[0125] As used herein, the term "derived from" indicates the relationship between the first and second molecules. It generally refers to the structural similarity between the first and second molecules and does not imply or include any restriction on the process or origin of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ sequence / structure to enable it to perform the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include any restriction on the specific process for generating the intracellular signaling domain; for example, it does not imply that, in order to provide the intracellular signaling domain, it is necessary to start with a CD3ζ sequence and delete unwanted sequences, or to impose mutations. Domains derived from a particular protein may have at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical sequences to the relevant functional portion of the particular protein.

[0126] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the following: the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the AbM numbering system (Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art can readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). The CDR of the antibodies disclosed herein can be defined according to the Kabat, AbM, IMGT, or Chothia numbering systems, or any combination thereof. Unless otherwise specified or clear from the context, the CDR of the antibodies disclosed herein is preferably defined according to the AbM numbering system.

[0127] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human, or non-human, to which treatment is administered according to the methods disclosed herein. This disclosure contemplates both veterinary and non-veterinary applications. Human patients may be adults or adolescents (e.g., humans under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, this includes non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0128] 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 disclosure pertains. This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0129] Other features and advantages of this disclosure will become apparent from the following detailed description, the accompanying drawings, and the claims. Attached Figure Description

[0130] Figure 1 is a schematic diagram of the CD20 CAR construct.

[0131] Figure 2 is a schematic diagram of the CD19 CAR construct.

[0132] Figure 3 is a schematic diagram of the CD20 / CD19 dual CAR construct.

[0133] Figure 4 shows the expression of CD20 and CD19 in tumor cell lines.

[0134] Figures 5A-5C illustrate the cytotoxicity of cytokine release from CD20 CAR-T cells co-cultured with CD20-positive Raji cells.

[0135] Figures 6A-6C illustrate the antitumor efficacy and proliferation of CD20 sdAb1 CAR-T cells in the Raji xenograft model, which were superior to those of other CD20 CAR-T cells. There was no significant change in body weight compared to the cohort treated with UnT.

[0136] Figures 7A-7B illustrate the cytotoxicity of cytokine release from CD19 CAR-T cells co-cultured with CD19-positive Raji cells.

[0137] Figure 8 is a diagram showing the cytotoxicity of CD19 CAR-T cells carrying FcRγ with different CAR components.

[0138] Figures 9A-9C show that CD19 CAR-T cells carrying FcRγ have stronger proliferation and lower cytokine release compared to CD19 CAR-T cells carrying CD3ζ and Dap12.

[0139] Figure 10 shows that CD19 CAR-T cells carrying the CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain, and FcRγ intracellular domain exhibit stronger proliferation than CD19 CAR-T cells with other modalities.

[0140] Figures 11A-11F illustrate the antitumor activity of CD20 / CD19 dual CAR-γδT cells against CD20 and / or CD19 positive tumor cells.

[0141] Figures 12A-12F show that CD20 / CD19 double CAR-γδT cells did not induce cytotoxicity against CD20 / CD19 double negative cells.

[0142] Figures 13A-13B show that CD20 / CD19 dual CAR-T cells outperform single-target CAR-T controls in terms of persistence and long-term proliferation.

[0143] Figures 14A-14D illustrate the superior efficacy and proliferation of CD20 / CD19 dual CAR-T cells from two separate γδT cells compared to the CD20 single CAR control.

[0144] Figures 15A-15B illustrate the superior anti-tumor efficacy of CD20 / CD19 dual CAR in vivo compared to CD20 single CAR and CD19 single CAR.

[0145] Figures 16A-16D illustrate that CD20 / CD19 dual CARs exhibit better persistence and proliferation in γδT cells than tandem CARs.

[0146] Figures 17A-17B illustrate that CD20 / CD19 dual CARs exhibit better persistence and proliferation in αβT cells than tandem CARs.

[0147] Figure 18 is a schematic diagram of CD20 / CD19 dual CAR constructs with different modalities.

[0148] Figures 19A-19F illustrate the antitumor activity of CD20 / CD19 dual CAR constructs with different modalities against CD20 and / or CD19 positive tumor cells.

[0149] Figures 20A-20B illustrate the persistent killing and robust proliferation of CD20 / CD19 dual CAR constructs with different modalities.

[0150] Figure 21 shows the percentage of B cells in PBMCs before co-culturing with CAR-T cells.

[0151] Figure 22 shows the percentage of B cells in PBMCs after co-culturing with CAR-T cells at different ratios for 72 hours.

[0152] Figure 23 shows the release of INF-γ from CAR-T cells co-cultured with PBMCs.

[0153] Figure 24 shows the percentage of B cells in PBMCs after 72 hours of co-culturing with CAR-T cells at a ratio of 1:50.

[0154] Figure 25 shows the selected sequences listed in this disclosure. Detailed Implementation

[0155] This disclosure relates to engineered receptors (e.g., CARs) and immune cells (e.g., CAR-T cells) that express these engineered receptors. In particular, immune cells that express engineered receptors can be used in immunocellular therapies for the treatment of cancer.

[0156] Chimeric antigen receptor (CAR)

[0157] Chimeric antigen receptors (CARs) typically comprise an extracellular domain capable of binding to an antigen and an intracellular domain comprising one or more intracellular signaling domains derived from signal transduction proteins. These intracellular signaling domains are typically distinct from the polypeptides that derive the extracellular domains. The extracellular domain can be any protein molecule or portion thereof capable of specifically binding to a predetermined antigen. The extracellular domain may contain an antibody or its antigen-binding fragment. The intracellular signaling domain can be any known oligopeptide or polypeptide domain that functions to transmit signals that induce activation or inhibition of biological processes within the cell, such as the activation of immune cells (e.g., T cells or NK cells).

[0158] Chimeric antigen receptors (CARs) integrate many facets that activate normal T or NK cells into a single protein. They link an extracellular antigen-recognition domain with an intracellular signaling domain, thereby activating T or NK cells upon antigen binding. CARs typically contain the following regions: an antigen-binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular domain. The intracellular region may contain an intracellular signaling domain or an intracellular signaling region.

[0159] Detailed reviews of CAR and CAR-expressing cells can be found in, for example, J Exp Clin Cancer Res, March 31, 2022; 41(1):119; Mol Cancer, January 30, 2023; 22(1):20; and Br J Haematol, April 2021; 193(2):216-230. doi: 10.1111 / bjh.17186. Epub November 20, 2020, each of which is incorporated herein by reference in its full text. Exemplary structures of antigen receptors (including hinges, transmembrane domains, and intracellular signaling domains) and methods for engineering such receptors and introducing them into cells are described, for example, in: Chandran et al., “T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance.” Immunological reviews 290.1 ​​(2019): 127-147; Cartellieri, Marc et al., “Chimeric antigen receptor-engineered T cells for immunotherapy of cancer.” BioMed Research International 2010 (2010); and PCT publications WO2017173256A1; US ​​2002 / 131960, US 2013 / 287748, US 2013 / 0149337, US 6,451,995, US 7,446,190, US 8,252,592; each of which is incorporated herein by reference in its entirety.

[0160] In one aspect, this article provides engineered receptors (e.g., CARs) comprising: an extracellular antigen-binding domain; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain derived from FcRγ, and one or more additional signaling domains selected from the CD28 co-stimulatory signaling domain, the Dap10 intracellular signaling domain, the OX40 intracellular signaling domain, the CD27 intracellular signaling domain, the CD137 (4-1BB) co-stimulatory signaling domain, and the ICOS intracellular signaling domain.

[0161] In one aspect, this paper provides dual chimeric antigen receptor (CAR) systems comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain; a first transmembrane domain; and a first intracellular domain comprising an intracellular signal transduction domain derived from FcRγ; and a CD28 co-stimulatory signal transduction domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

[0162] In one aspect, this paper provides dual chimeric antigen receptor (CAR) systems comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain; a first transmembrane domain; and a first intracellular domain comprising an intracellular signal transduction domain derived from FcRγ; and a CD137(4-1BB) co-stimulatory signal transduction domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

[0163] Intracellular domains may contain intracellular signaling domains derived from FcRγ or functional variants thereof. Intracellular signaling domains derived from FcRγ may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. The Fc receptor common γ chain (FcRgamma or FcRγ) is a widely expressed adaptor carrying an immune receptor tyrosine-based activation motif (ITAM), which transduces activation signals from various immune receptors. Through its transmembrane portion, FcRγ constitutively associates with the common β chain of the IL-3 receptor and signals by recruiting the kinase Syk. Retrovirus-mediated complementation demonstrates the essential function of the FcRγ ITAM in IL-3 signal transduction. The function of FcRγ is to direct selectively cytokine-triggered signals to the ITAM-mediated IL-4 production pathway. The intracellular domains of FcRγ are shown in SEQ ID NO: 16. The intracellular signal transduction domain of FcRγ may contain the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16.

[0164] Intracellular domains may contain one or more additional intracellular signaling domains. Intracellular signaling domains can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells). "Immune effector function or immune effector response" refers to a function or response of immune effector cells that enhances or promotes the immune attack of target cells. For example, an immune effector function or response can refer to the property of T cells or NK cells to promote the killing of target cells or inhibit the growth or proliferation of target cells. Examples of immune effector functions (e.g., in CAR-T cells) include cytolytic activity and helper activities (such as the secretion of cytokines). Intracellular signaling domains can generate signals that promote the proliferation and / or survival of CAR-containing cells. Signaling domains of naturally occurring molecules can contain the entire intracellular (or cytoplasmic) portion of the molecule, or the entire naturally occurring intracellular signaling domain, or a fragment or derivative thereof. Intracellular signal transduction domains may be, but are not limited to, the Dap10 intracellular signal transduction domain, the Dap12 intracellular signal transduction domain, the OX40 intracellular signal transduction domain, the CD27 intracellular signal transduction domain, and / or the ICOS (CD278) intracellular signal transduction domain.

[0165] The intracellular domain may have a Dap10 intracellular signal transduction domain. The Dap10 intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 20.

[0166] The intracellular domain may have a Dap12 intracellular signal transduction domain. The Dap12 intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 17.

[0167] The intracellular domain may have an OX40 intracellular signal transduction domain. The OX40 intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 21.

[0168] The intracellular domain may have a CD27 intracellular signal transduction domain. The CD27 intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 22.

[0169] The intracellular domain may have an ICOS (CD278) intracellular signal transduction domain. The ICOS intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 23.

[0170] The intracellular signaling domains of CARs may contain primary intracellular signaling domains. These primary intracellular signaling domains contain intracellular signaling sequences that act in a stimulatory manner to induce immune effector function. These primary intracellular signaling domains may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs.

[0171] The intracellular domain may have a CD3ζ primary intracellular signal transduction domain. The CD3ζ primary intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 15.

[0172] Intracellular domains may contain one or more costimulatory signaling domains. Costimulatory signaling domains may be intracellular portions of costimulatory molecules. The term "costimulatory molecule" refers to a homologous binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the immune cell, such as, but not limited to, proliferation and survival. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an efficient immune response. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), and activating NK cell receptors. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, L... FA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0173] After the antigen-binding domain binds to the tumor antigen, the co-stimulatory signal transduction domain of the CAR can transduce signals to enhance the proliferation, survival, and differentiation of CAR-modified immune cells (such as T cells) and inhibit activation-induced cell death.

[0174] The intracellular domain may include a co-stimulatory signal transduction domain derived from CD28. The CD28 co-stimulatory signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 14.

[0175] The intracellular domain may include a co-stimulatory signal transduction domain derived from CD28 and an intracellular signal transduction domain derived from FcRγ. The CD28 co-stimulatory signal transduction domain may be located at the N-terminus of the intracellular signal transduction domain derived from FcRγ. The CD28 co-stimulatory signal transduction domain may be located at the C-terminus of the intracellular signal transduction domain derived from FcRγ.

[0176] The intracellular domain may include a co-stimulatory signal transduction domain derived from 4-1BB (i.e., CD137). The 4-1BB co-stimulatory signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 13.

[0177] The CAR may comprise an intracellular signal transduction domain derived from FcRγ and a 4-1BB co-stimulatory signal transduction domain. The 4-1BB co-stimulatory signal transduction domain may be located at the N-terminus of the intracellular signal transduction domain derived from FcRγ. The 4-1BB co-stimulatory signal transduction domain may be located at the C-terminus of the intracellular signal transduction domain derived from FcRγ.

[0178] The antigen-binding domain (e.g., extracellular antigen-binding domain) of an engineered receptor (e.g., CAR) can be an antibody or antibody fragment, such as scFv, Fv, Fab, (Fab')2, single-domain antibody (sdAb), or V. H The H domain. The antigen-binding domain may contain a ligand or extracellular portion that allows the receptor to specifically bind to a tumor antigen. Engineered receptors (e.g., CARs) can be monospecific, bispecific, or multispecific receptors. The antigen-binding domain can specifically bind to a single tumor antigen. The antigen-binding domain can bind to two or more tumor antigens.

[0179] Tumor antigens can be selected from the following groups: B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 gangliosides, glycoprotein 75, GPNMB, HER2 / neu, HGF, human dispersive factor receptor kinase, IGF-1 receptor, IGF-1, IgG1, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-hydroxyacetylneuraminic acid, NPC-1C, PDGF-RA, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH Tumor antigens include 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGFβ2, TGF-b, TRAIL-R1, TRAIL-R2, CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin, as well as other clinically significant tumor antigens and combinations thereof. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells.

[0180] The extracellular antigen-binding domain can bind to CD19. The dual CAR system described herein may have a first extracellular antigen-binding domain that binds to CD19. The dual CAR system described herein may have a second extracellular antigen-binding domain that binds to CD20.

[0181] The extracellular antigen-binding domain can bind to CD20. The dual CAR system described herein may have a first extracellular antigen-binding domain that binds to CD20. The dual CAR system described herein may also have a second extracellular antigen-binding domain that binds to CD19.

[0182] In one aspect, this article provides chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD19; a transmembrane domain; and one or more of the following: (1) an intracellular domain comprising an intracellular signal transduction domain derived from FcRγ, a Dap12 intracellular signal transduction domain, a Dap10 intracellular signal transduction domain, an OX40 intracellular signal transduction domain, a CD27 intracellular signal transduction domain, or an ICOS intracellular signal transduction domain; (2) a CD28 co-stimulatory signal transduction domain or a CD137 (4-1BB) co-stimulatory signal transduction domain; and (3) an activation domain comprising a CD3ζ primary intracellular signal transduction domain.

[0183] In one aspect, this article provides chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD20; a transmembrane domain; and one or more of the following: (1) an intracellular domain comprising an intracellular signal transduction domain derived from FcRγ, a Dap12 intracellular signal transduction domain, a Dap10 intracellular signal transduction domain, an OX40 intracellular signal transduction domain, a CD27 intracellular signal transduction domain, or an ICOS intracellular signal transduction domain; (2) a CD28 co-stimulatory signal transduction domain or a CD137 (4-1BB) co-stimulatory signal transduction domain; and (3) an activation domain comprising a CD3ζ primary intracellular signal transduction domain.

[0184] In one respect, this article provides chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD19; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain derived from FcRγ.

[0185] In one respect, this article provides chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD20; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain derived from FcRγ.

[0186] The extracellular antigen-binding domain that binds to CD19 may be or contains a CD19-binding fragment (e.g., FMC63, SJ25C1, or those disclosed in various patents such as WO 2022 / 012683). The antigen-binding domain that binds to CD19 may comprise: a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains at least 80% identical amino acid sequences to the selected VH CDR1 amino acid sequence, the VH CDR2 region contains at least 80% identical amino acid sequences to the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains at least 80% identical amino acid sequences to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) containing CDRs 1, 2, and 3, wherein the VL CDR1 region contains at least 80% identical amino acid sequences to the selected VL CDR1 amino acid sequence, the VL CDR2 region contains at least 80% identical amino acid sequences to the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains at least 80% identical amino acid sequences to the selected VL CDR3 amino acid sequence, wherein the selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 51, 52, and 53, respectively, and the selected VL… The amino acid sequences of CDR 1, 2, and 3 are shown in SEQ ID NO: 54, 55, and 56, respectively.

[0187] The antigen-binding domain that binds to CD19 may include a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region contains at least 80% of the same amino acid sequence as the selected VH sequence and the light chain variable region contains at least 80% of the same amino acid sequence as the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO: 58.

[0188] The antigen-binding domain that binds to CD19 may contain the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7.

[0189] The antigen-binding domain that binds to CD19 may contain the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 6.

[0190] The antigen-binding domain that binds to CD20 may contain V H The H antibody portion, this V HThe H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains an amino acid sequence that is at least 80% identical to the selected CDR1 amino acid sequence, CDR2 contains an amino acid sequence that is at least 80% identical to the selected CDR2 amino acid sequence, and CDR3 contains an amino acid sequence that is at least 80% identical to the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 50.

[0191] The antigen-binding domain that binds to CD20 may contain V H The H antibody portion, this V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0192] The transmembrane domain of a CAR may contain transmembrane domains selected from the following: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIRDS2, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD4 ... 19. IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane domains of CARs can be derived from the α chain, β chain, ζ chain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154 transmembrane domains.

[0193] The transmembrane domain of the CAR may include the transmembrane domain of CD8α. The CD8α transmembrane domain may have the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11.

[0194] The transmembrane domain of the CAR may include the transmembrane domain of CD28. The CD28 transmembrane domain may have the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12.

[0195] The extracellular antigen-binding domain can be connected to the transmembrane domain via a hinge region. The hinge region can be derived from CD8α, CD28, IgG1, IgG2, IgG3, or IgG4. The hinge region can include the hinge region of CD8α. The CD8α hinge region can have the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 9. The hinge region can include the hinge region of CD28. The CD28 hinge region can have the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10. The hinge region can include the hinge region of IgG4. The IgG4 hinge region may have the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 67.

[0196] CARs may contain an intracellular signal transduction domain derived from FcRγ and a CD28 co-stimulatory signal transduction domain. The intracellular signal transduction domain derived from FcRγ may be located at the C-terminus of one or more additional signal transduction domains.

[0197] The CAR may contain a signal peptide (SP), such as the CD8α signal peptide. The CD8α signal peptide may have the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0198] Engineered receptors (e.g., CARs) may comprise the amino acid sequence shown in any one of SEQ ID NO: 33, 36-43, and 68, or a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO: 33, 36-43, and 68. CARs can specifically bind to CD19-positive tumor cells.

[0199] Engineered receptors (e.g., CARs) may comprise the amino acid sequence shown in any one of SEQ ID NOs: 69-71 and 74, or a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NOs: 69-71 and 74. CARs can specifically bind to CD20-positive tumor cells.

[0200] The dual CAR system may contain the amino acid sequence shown in any one of SEQ ID NO: 19 and 60-66, or a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO: 19 and 60-66. The dual CAR system can specifically bind to CD19-positive tumor cells, CD20-positive tumor cells, and / or both CD19-positive and CD20-positive tumor cells.

[0201] In the dual CAR system described herein, the first engineered receptor may comprise, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor may comprise, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0202] In the dual CAR system described herein, the first engineered receptor may comprise, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor may comprise, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0203] In the dual CAR system described herein, the first engineered receptor may comprise, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor may comprise, from the N-terminus to the C-terminus, the following: a second extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0204] In the dual CAR system described herein, the first engineered receptor may comprise, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor may comprise, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0205] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0206] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0207] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0208] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0209] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0210] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0211] In the dual CAR system described herein, the first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

[0212] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13), and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 24. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 77.

[0213] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain 2F2 that specifically binds to CD20 (e.g., SEQ ID NO: 3, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 3), a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 13), and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 13). 15. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical sequences. Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 26.

[0214] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain 3H7 (e.g., SEQ ID NO: 4, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). 15. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical amino acid sequences. Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 27.

[0215] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 6), an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and an intracellular domain comprising a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 15). Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 28.

[0216] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 6), an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and an intracellular domain comprising an intracellular signal transduction domain of Dap12 (e.g., SEQ ID NO: 17, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 17). Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 29.

[0217] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 6), an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and an intracellular domain comprising an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 16). Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 30.

[0218] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 6) that specifically binds to CD19, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 13), and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 31. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 78, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 78.

[0219] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6) that specifically binds to CD19, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a Dap12 intracellular signal transduction domain (e.g., SEQ ID NO: 17, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). 17. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 32. An engineered receptor (e.g., a CAR) may further contain a signal peptide. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 79, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 79.

[0220] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6) that specifically binds to CD19, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 33. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 80, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 80.

[0221] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 12), and an intracellular domain comprising an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 16). Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 34.

[0222] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 7), a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and an intracellular domain comprising an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 16). Engineered receptors (e.g., CARs) may contain the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 35.

[0223] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 36. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 81.

[0224] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 12), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 13) from the N-terminus to the C-terminus, and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to the sequence in SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 37. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 82.

[0225] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 12), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 16) and a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 16) from the N-terminus to the C-terminus. An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 38. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 83, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 83.

[0226] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and an intracellular signal transduction domain derived from FcRγ (e.g., SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16) and a Dap10 intracellular signal transduction domain (SEQ ID NO: 20, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 39. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 84, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 84.

[0227] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and an intracellular signaling domain derived from FcRγ (e.g., SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16) and an OX40 intracellular signaling domain (SEQ ID NO: 21, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16). 21. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 40. An engineered receptor (e.g., a CAR) may further contain a signal peptide. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 85, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 85.

[0228] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and an intracellular signaling domain from the N-terminus to the C-terminus comprising a CD27 intracellular signaling domain (e.g., SEQ ID NO: 22, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 22) and an intracellular signaling domain derived from FcRγ (e.g., SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 41. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 86, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 86.

[0229] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and an intracellular signal transduction domain derived from FcRγ (e.g., SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16) and a CD27 intracellular signal transduction domain (SEQ ID NO: 22, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16) from the N-terminus to the C-terminus. 22. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 42. An engineered receptor (e.g., a CAR) may further contain a signal peptide. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 87, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 87.

[0230] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and an intracellular signal transduction domain derived from FcRγ (e.g., SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16) and an ICOS intracellular signal transduction domain (SEQ ID NO: 23, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16). 23. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 43. An engineered receptor (e.g., a CAR) may further contain a signal peptide. An engineered receptor (e.g., a CAR) may contain the amino acid sequence shown in SEQ ID NO: 88, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 88.

[0231] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 47. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 89, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 89.

[0232] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and an intracellular signal transduction domain derived from FcRγ (e.g., SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 68. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 90, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 90.

[0233] The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 69. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 91, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 91.

[0234] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 70, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 70. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 92, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 92.

[0235] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 71. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 93, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 93.

[0236] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 72. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 94, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 94.

[0237] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 73, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 73. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 95, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 95.

[0238] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequences. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 74. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 96.

[0239] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 75. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 97.

[0240] The engineered receptors (e.g., CARs) provided herein may comprise an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). An intracellular domain comprising at least 80%, 85%, 90%, or 95% identical amino acid sequence. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 76. An engineered receptor (e.g., a CAR) may further comprise a signal peptide. An engineered receptor (e.g., a CAR) may comprise the amino acid sequence shown in SEQ ID NO: 98, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 98.

[0241] A dual-CAR system may comprise a CD19-specific CAR as described herein, linked to a CD20-specific CAR as described herein. The CD19-specific CAR and the CD20-specific CAR as described herein may be linked via a P2A element (e.g., SEQ ID NO: 18, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 18).

[0242] A dual-CAR system may comprise a first engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO:7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:7), a CD28 hinge region (e.g., SEQ ID NO:10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:10), a CD28 transmembrane domain (e.g., SEQ ID NO:12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:12), and an intracellular signal transduction domain comprising a CD28 co-stimulatory signal transduction domain (SEQ ID NO:14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:14) and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO:16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:14). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 36. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 81.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 24. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 77. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 19.

[0243] A dual CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO:7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:7), an IgG4 hinge region (e.g., SEQ ID NO:67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:67), a CD28 transmembrane domain (e.g., SEQ ID NO:12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:12), a CD28 co-stimulatory signal transduction domain (SEQ ID NO:14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO:16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:14). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 68. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 90, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 90.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 24. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 77. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 60, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 60.

[0244] A dual-CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 69. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 91, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 91.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 13). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 47. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 89, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 89. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 61, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 61.

[0245] A dual-CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 70, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 70. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 92, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 92.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), and a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 13). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 47. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 89, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 89. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 62, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 62.

[0246] A dual-CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 71. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 93, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 93.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 72. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 94, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 94. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 63, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 63.

[0247] A dual-CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 71. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 93, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 93.A dual-CAR system may comprise a second engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain CD19scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 73, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 73. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 95, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 95. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 64, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 64.

[0248] A dual-CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 74. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 96.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 75. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 97. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 65, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 65.

[0249] A dual-CAR system may comprise a first engineered receptor (e.g., CAR) comprising an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11), a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13), and an intracellular signal transduction domain derived from FcRγ (SEQ ID NO: 16, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 74. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 96.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), an IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 67), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 76. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 98, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 98. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 66.

[0250] This article also provides a dual CAR system comprising a first engineered receptor (e.g., CAR), wherein the first engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 13) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 13). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 24. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 77.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and a Dap12 intracellular signal transduction domain (SEQ ID NO: 17, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). 17. An intracellular domain containing at least 80%, 85%, 90%, or 95% identical sequence. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The dual CAR system may comprise the amino acid sequence shown in SEQ ID NO:44, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO:44.

[0251] This article also provides a dual CAR system comprising a first engineered receptor (e.g., CAR), wherein the first engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 1) that specifically binds to CD20, a CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), a CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and a 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 13) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 13). The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 24. The first engineered receptor (e.g., CAR) may further contain a signal peptide. The first engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 77.A dual-CAR system may comprise a second engineered receptor (e.g., a CAR) comprising an extracellular antigen-binding domain CD19 scFv (FMC63) that specifically binds to CD19 (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7), a CD28 hinge region (e.g., SEQ ID NO: 10, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 14, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signal transduction domain (e.g., SEQ ID NO: 15, or at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14). The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 75. The second engineered receptor (e.g., CAR) may further contain a signal peptide. The second engineered receptor (e.g., CAR) may contain the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 97. The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 59, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 59.

[0252] This article also provides a dual CAR system comprising, from the N-terminus to the C-terminus, tandem CD20 binding scFv (Leu16) (e.g., SEQ ID NO: 5, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 5), CD19 scFv (FMC63) (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 7), CD8 / CD8α hinge region (e.g., SEQ ID NO: 9, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 9), CD8 / CD8α transmembrane domain (e.g., SEQ ID NO: 11, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11), and 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11). The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 45, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 45.

[0253] This article also provides a dual CAR system comprising, from the N-terminus to the C-terminus, tandem CD20 binding scFv (2F2) (e.g., SEQ ID NO: 3, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 3), CD19 scFv (FMC63) (e.g., SEQ ID NO: 7, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 7), IgG4 hinge region (e.g., SEQ ID NO: 67, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 67), CD28 transmembrane domain (e.g., SEQ ID NO: 12, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 12), and 4-1BB co-stimulatory signal transduction domain (e.g., SEQ ID NO: 13, or at least 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 12). The dual CAR system may contain the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 15.

[0254] Genetically engineered cells

[0255] This disclosure provides cells expressing the engineered receptor (CAR) or dual CAR system described herein (e.g., genetically engineered immune cells, T cells, NK cells, tumor-infiltrating lymphocytes). These engineered cells can be used to treat various disorders or diseases as described herein (e.g., cancer).

[0256] In one aspect, this article provides modified γδT cells comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain that binds to CD19; a first transmembrane domain; and a first intracellular domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain that binds to CD20; a second transmembrane domain; and a second intracellular domain.

[0257] In one aspect, this article provides modified αβT cells comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain that binds to CD20; a first transmembrane domain; and a first intracellular domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain that binds to CD19; a second transmembrane domain; and a second intracellular domain.

[0258] The first engineered receptor and / or the second engineered receptor may be any of the engineered receptors described herein (e.g., CAR).

[0259] Cells can be immune cells. Immune cells can be selected from the following groups: T cells, NK cells, NKT cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, macrophages, monocytes, neutrophils, eosinophils, and combinations thereof.

[0260] T cells can be αβT cells or γδT cells. Modified γδT cells can be Vδ1 γδT cells. Modified γδT cells can be Vδ2 γδT cells. Modified γδT cells can be Vδ1-Vδ2- γδT cells. Modified γδT cells can include a polyclonal population of γδT cells, including Vδ1 γδT cells, Vδ2 γδT cells, and Vδ1-Vδ2- γδT cells. Modified γδT cells can be panT cells.

[0261] Modified cells (e.g., modified γδT cells) can express either the engineered receptor described herein (e.g., CAR) or a dual CAR system.

[0262] Engineered cells can be obtained from, for example, humans and non-human animals. Engineered cells can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. Cells can be derived from humans, rats, or mice. These cells can be mouse lymphocytes and engineered (e.g., transduced) to express the engineered receptor (CAR) or dual CAR system described herein. Cells can be obtained from humans. Engineered cells can be blood cells. Cells can be CD8+ T cells, CD4+ T cells, memory T cells, Treg cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, or macrophages / monocytes.

[0263] The preparation of engineered cells may include one or more culture and / or preparation steps. Cells used to introduce binding molecules (e.g., CARs) can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. The subject from whom cells are isolated may be a subject suffering from a disease or condition, requiring cell therapy, or to whom cell therapy will be administered. The subject may be someone requiring a specific therapeutic intervention, such as adoptive cell therapy in which engineered cells are being isolated, processed, and / or engineered.

[0264] The cells can be NK cells. Human natural killer cells (NK cells) play an important role in the innate immune defense against malignant lymphoma cells and are therefore suitable for adoptive immunotherapy (i.e., adoptive cell immunotherapy). However, the use of NK cells is difficult due to the difficulty in expanding cells in vitro and the variability in NK cell activity among individual patients.

[0265] NK cells are part of the innate immune system, providing the first line of defense against pathogens and cancer cells. They produce cytokines and mediate cytotoxicity without prior sensitization, and they have the ability to interact with and activate other immune cells. NK cells used in immunotherapy can be generated from multiple sources, such as expanded autologous or allogeneic peripheral blood, umbilical cord blood, hematopoietic stem cells, induced pluripotent stem cells, and cell lines.

[0266] NK cell activation and effector function are complex processes because they depend on the integration of signals from two different types of receptors—activating and inhibitory receptors. Normal, healthy cells express MHC class I molecules on their surface, which act as ligands for inhibitory receptors and contribute to NK cell self-tolerance. Cellular stresses associated with viral infection or tumor development (such as DNA damage, senescence, or tumor suppressor genes) upregulate ligands for activating receptors. This causes a shift in the balance toward NK cell activation. Transmembrane and intracellular stimulatory / activating molecules in NK cells can influence NK cell differentiation pathways, the metabolic cycle, apoptosis, and activation-induced cell death.

[0267] The cell may be a T cell. T cells may express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. The cell surface receptor may be a wild-type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing an antigenic moiety associated with a target cell. T cells may be obtained by various methods known in the art, such as in vitro culture of T cells isolated from a patient (e.g., tumor-infiltrating lymphocytes). Genetically modified T cells may be obtained by transducing T cells with a viral vector (e.g., isolated from peripheral blood of a patient). T cells may be CD4+ T cells, CD8+ T cells, or regulatory T cells. T cells may be T helper type 1 T cells and T helper type 2 T cells. T cells expressing this receptor may be αβ T cells. T cells expressing this receptor may be γδ T cells. T cells may be central memory T cells. T cells may be effector memory T cells. T cells may be naive T cells.

[0268] The cells can be stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cells can be primary cells, such as those isolated directly from the subject and / or isolated from the subject and frozen. Stem cells can be cultured with additional differentiation factors to obtain the desired cell type (e.g., NK cells).

[0269] Different cell types can be obtained from appropriate isolation methods. Isolation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cells, such as surface markers, like surface proteins, intracellular markers, or nucleic acids. Any known isolation method based on such markers can be used. Isolation can be based on affinity or immunoaffinity. For example, in some aspects, isolation involves separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers), such as by incubating with antibodies or binding couplers that specifically bind to such markers, followed typically by a washing step and separating cells that bind to the antibody or binding coupler from those that do not.

[0270] Such separation steps can be based on positive selection (where cells that bind to the reagent are retained for further use) and / or negative selection (where cells that do not bind to the antibody or binding coupler are retained). In some instances, both portions are retained for further use. Negative selection can be particularly useful when no antibody is available to specifically identify cell types in a heterogeneous population, allowing for optimal separation based on markers expressed by cells other than the desired cell population.

[0271] Also provided are cell populations (e.g., genetically engineered cell populations), compositions containing such cells and / or enriched with such cells, such as wherein cells expressing a CAR or dual CAR system comprise at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher percentages of the total cells in the composition or of a certain type of cells (such as NK cells, T cells, CD8+ or CD4+ cells).

[0272] Engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) are co-cultured with target cells (e.g., tumor cells) for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days or longer, such that the engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) can be activated.

[0273] Engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) can have cytotoxic effects on tumor cells. When exposed to tumor cells, engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) can kill approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or more of the tumor cells. Engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) can be contacted with tumor cells at ratios of approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0274] Engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) can be exposed to tumor cells for approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or longer.

[0275] Engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) can induce the production of one or more cytokines upon contact with target cells. Cytokines may include interferon-γ (IFNγ). Cytokines may include granulocyte-macrophage colony-stimulating factor (GM-CSF). Engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) may induce fewer cytokines production than cells expressing other engineered receptors or dual CAR systems. Engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) may induce more cytokines production than cells not expressing the engineered receptor or dual CAR system.

[0276] Engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) can reduce or slow tumor progression in subjects with cancer. Cancer progression can be evaluated every 1, 2, 3, 4, 5, 6, or 7 days. Cancer progression can be evaluated every 1, 2, 3, or 4 weeks. Cancer progression can be evaluated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more. Engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) can retain therapeutic activity, efficacy, and durability after approximately 1, 2, 3, 4, 5, 6, or 7 days. Engineered cells (e.g., cells expressing engineered receptors (e.g., CAR) or dual CAR systems as described herein) can retain therapeutic activity, efficacy, and durability after approximately 1, 2, 3, or 4 weeks. Engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) can retain therapeutic activity, efficacy, and durability after approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. Approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) can be retained after approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

[0277] Compared to cells that do not express the engineered receptor (e.g., CAR) or dual CAR system described herein, engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) may have increased proliferative capacity. The proliferation of engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) may be increased by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to cells that do not express the engineered receptor (e.g., CAR) or dual CAR system described herein. The proliferation of engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) may be increased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more compared to cells that do not express the engineered receptor (e.g., CAR) or dual CAR system described herein.

[0278] Compared to cells that do not express CARs containing intracellular signaling domains derived from FcRγ (e.g., CARs expressing other intracellular signaling domains), engineered cells expressing CARs containing intracellular signaling domains derived from FcRγ (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) can have increased proliferative capacity. The proliferation of engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) can be increased by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Compared to cells that do not express CARs containing intracellular signaling domains derived from FcRγ (e.g., CARs expressing other intracellular signaling domains), the proliferation of engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems described herein) can be increased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

[0279] Compared to cells that do not express the engineered receptor (e.g., CAR) or dual CAR system described herein, engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein) may have lower cytokine (e.g., IFNγ) release. The cytokine release of engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein may be reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. The cytokine release of engineered cells (e.g., cells expressing the engineered receptor (e.g., CAR) or dual CAR system described herein may be reduced by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more compared to cells that do not express the engineered receptor (e.g., CAR) or dual CAR system described herein.

[0280] Compared to cells that do not express CARs containing intracellular signaling domains derived from FcRγ (e.g., CARs expressing other intracellular signaling domains), engineered cells expressing CARs containing intracellular signaling domains derived from FcRγ (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) may have lower cytokine (e.g., IFNγ) release. Cytokine release in engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) may be reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Compared to cells that do not express CARs containing intracellular signaling domains derived from FcRγ (e.g., CARs expressing other intracellular signaling domains), engineered cells (e.g., cells expressing engineered receptors (e.g., CARs) or dual CAR systems as described herein) may have reduced cytokine release by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

[0281] Compared to cells that do not express engineered receptors (e.g., CAR) or dual CAR systems, or cells that express a single-target receptor (e.g., CAR), engineered cells expressing the dual CAR system described herein can exhibit increased antitumor efficacy (e.g., cytotoxicity against tumor cells). The antitumor efficacy (cytotoxicity against tumor cells) of engineered cells expressing the dual CAR system described herein can be increased by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. The antitumor efficacy (cytotoxicity against tumor cells) of engineered cells expressing the dual CAR system described herein can be increased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more compared to cells that do not express engineered receptors (e.g., CAR) or dual CAR systems, or cells that express a single-target receptor (e.g., CAR).

[0282] Compared to cells that do not express engineered receptors (e.g., CAR) or dual CAR systems, or cells that express a single-target receptor (e.g., CAR), engineered cells expressing the dual CAR system described herein can exhibit increased long-term proliferation. The long-term proliferation of engineered cells expressing the dual CAR system described herein can be increased by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. The long-term proliferation of engineered cells expressing the dual CAR system described herein can be increased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more compared to cells that do not express engineered receptors (e.g., CAR) or dual CAR systems, or cells that express a single-target receptor (e.g., CAR). Cell proliferation can be observed after approximately 1, 2, 3, 4, 5, 6 or 7 days, approximately 1, 2, 3, 4 weeks, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer.

[0283] Nucleic acids (polynucleotides)

[0284] This disclosure provides nucleic acids containing a multinucleotide sequence encoding an engineered receptor (e.g., CAR) or dual CAR system as described herein.

[0285] The polynucleotide disclosed herein may comprise a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker. The linker used in this disclosure allows multiple proteins encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), which are translated into polyproteins that dissociate into individual protein components. The polynucleotide may comprise a first polynucleotide sequence, a linker, and a second polynucleotide sequence from 5' to 3'. The polynucleotide may comprise a second polynucleotide sequence, a linker, and a first polynucleotide sequence from 5' to 3'. The first polynucleotide sequence may encode a first engineered receptor (e.g., a first CAR) as described herein, and the second polynucleotide sequence may encode a second engineered receptor (e.g., a second CAR) as described herein. The first polynucleotide sequence may encode a second engineered receptor (e.g., a second CAR) as described herein, and the second polynucleotide sequence may encode a first engineered receptor (e.g., a first CAR) as described herein.

[0286] The adapter may contain a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct entry of an internal ribosome into a protein-coding region of a start codon (such as ATG), thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES derived from viral or cellular mRNA sources such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES derived from, for example, heart virus, rhinovirus, foot-and-mouth disease virus, HCV, Friend mouse leukemia virus (FrMLV), and Moloney mouse leukemia virus (MoMLV). Those skilled in the art will be able to select an appropriate IRES.

[0287] The linker may contain a nucleic acid sequence encoding a self-cleaving peptide. As used herein, a “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. The use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), TaV, and swine tussock virus-1 (PTV-1); and cardiogenic viruses, such as Theylvirus and encephalomyocarditis virus. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. Those skilled in the art will be able to select appropriate self-cleaving peptides.

[0288] The adapter may contain spacer sequences. Various spacer sequences are known in the art, including but not limited to glycine-serine (GS) spacers (also known as GS adapters), such as (GS)n, (SG)n, (GSGGS)n (SEQ ID NO: 2), and (GGGS)n (SEQ ID NO: 25), where n represents an integer of at least 1. Those skilled in the art will be able to select appropriate spacer sequences.

[0289] The polynucleotides disclosed herein can be operatively linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those skilled in the art.

[0290] The promoter can be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter. For example, the CD4 gene promoter can be used; see, for example, Salmon et al., Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al., (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al., Blood (2011) 117:1565.

[0291] Other examples of suitable promoters include immediate early cytomegalovirus (CMV) promoter sequences. These are strongly constitutive promoter sequences capable of driving high-level expression of any polynucleotide sequence operatively linked to them. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Raoult sarcoma virus promoter, the EF-1α promoter, and human gene promoters (such as, but not limited to, actin, myosin, hemoglobin, and creatine kinase promoters). Furthermore, this disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to it when such expression is desired, or turn off such expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0292] Expression vectors (e.g., lentiviral vectors) can be used to introduce engineered receptors (e.g., CARs) or dual-CAR systems into immune cells or their precursors (e.g., T cells). Therefore, the expression vectors (e.g., lentiviral vectors) disclosed herein may contain polynucleotides encoding engineered receptors (e.g., CARs) or dual-CAR systems. Expression vectors (e.g., lentiviral vectors) may contain additional elements that facilitate the functional expression of the engineered receptor (e.g., CAR) or dual-CAR system encoded therein. Expression vectors containing polynucleotides encoding engineered receptors (e.g., CARs) or dual-CAR systems may further contain mammalian promoters. The vector may contain an elongation factor-1-α promoter (EF-1α promoter). Using an EF-1α promoter can improve the expression efficiency of downstream transgenes (e.g., engineered receptors (e.g., CARs) or dual-CAR systems). Physiological promoters (e.g., EF-1α promoters) are less likely to induce integration-mediated genotoxicity and may eliminate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and can be incorporated into the vectors disclosed herein. Vectors (e.g., lentiviral vectors) may further comprise non-essential cis-acting sequences that can improve titers and gene expression.

[0293] The polynucleotide may encode an amino acid sequence shown in any one of SEQ ID NO: 19, 24, 26-47, 59-66 and 68-98, or an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to an amino acid sequence shown in any one of SEQ ID NO: 19, 24, 26-47, 59-66 and 68-98.

[0294] This disclosure also provides nucleic acid sequences that are at least 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleotide sequence as described herein, and amino acid sequences that are at least 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any amino acid sequence as described herein. In some cases, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence as described herein. Nucleic acid sequences may be fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, or 5000 nucleotides. The amino acid sequence can be less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 amino acid residues.

[0295] An amino acid sequence may (i) contain an amino acid sequence; or (ii) consist of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0296] A nucleic acid sequence may (i) contain a nucleic acid sequence; or (ii) consist of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0297] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., vacancies can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of identical positions shared by the two sequences, taking into account the number of vacancies that need to be introduced to achieve optimal alignment and the length of each vacancies. For example, a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5 can be used to perform sequence comparisons and determine the percentage of identity between two sequences.

[0298] Introducing polynucleotides into host cells

[0299] The polynucleotides described herein (e.g., vectors) can be introduced as one or more polynucleotides or constructs, which optionally contain markers that will allow selection of host cells containing the one or more constructs. Genes and regulatory regions can be isolated, ligated, cloned into appropriate cloning hosts, and analyzed by restriction enzyme digestion or sequencing as needed. In particular, using PCR, individual fragments comprising all or part of functional units can be isolated, where one or more mutations can be introduced as needed using primer repair, ligation, in vitro mutagenesis, etc. The obtained polynucleotides, confirmed to have appropriate sequences, can then be introduced into host cells by any convenient method. Polynucleotides can be integrated and packaged into non-replicating defective viral genomes (such as lentiviruses, adenoviruses, adeno-associated viruses (AAVs), or herpes simplex virus (HSVs) or others, including retroviral vectors), for infection or transduction into cells. If desired, the polynucleotides can contain viral sequences for transfection. Alternatively, polynucleotides can be introduced by fusion, electroporation, bioprojectiles, transfection, lipid transfection, etc. Before introducing one or more constructs, host cells can be grown and expanded in a culture, followed by appropriate treatment for the introduction and integration of one or more constructs. Cells are then amplified and screened using markers present in the constructs. Various markers that can be successfully used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0300] Engineered receptors (e.g., CARs) or dual-CAR systems can be introduced as RNA into modified cells for transient expression. The RNA can be delivered to the immune cells disclosed herein via various means, including, for example, microinjection, electroporation, and lipid-mediated transfection. The construct can be introduced into the cell's genome via transposons. An example of a synthetic transposon used is the Sleeping Beauty transposon, which contains an expression cassette containing the appropriate gene for its active fragment. The construct can be integrated at a specific locus in the host cell's genome. Homologous recombination can be used to replace an endogenous gene with the gene encoded by the construct.

[0301] A lentiviral delivery system can be used to introduce constructs encoding engineered receptors (e.g., CAR) or dual CAR systems into host cells. A retroviral delivery system can be used to introduce constructs encoding engineered receptors (e.g., CAR) or dual CAR systems into host cells.

[0302] The host cell can be a human cell. The host cell can be a human T cell. Human T cells can be purified from commercially available PBMCs. The host cell can be an αβ T cell. The host cell can be a γδ T cell. The host cell can be an NK cell. The host cell can be a panT cell.

[0303] Treatment

[0304] The engineered receptor (CAR) or dual CAR system described herein, the polynucleotides described herein, and the modified cells described herein can be used in a variety of experimental, therapeutic, and commercial applications.

[0305] In one aspect, this disclosure provides a pharmaceutical composition comprising an engineered receptor (e.g., CAR) or dual CAR system as described herein, a cell or nucleic acid as described herein, and a pharmaceutically acceptable carrier.

[0306] In one aspect, this disclosure provides a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein.

[0307] In one respect, this disclosure provides a method for modulating an immune response, the method comprising administering an effective amount of the modified cells described herein to a subject in need.

[0308] The disease or disorder is cancer, an inflammatory disease, or an autoimmune disease.

[0309] Cancer can be a solid tumor or a blood cancer. Cancer can be liver cancer, stomach cancer, colon cancer, lymphoma, acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0310] As used in this article, the term "effective amount" refers to the amount that is effective at the dose and duration necessary to achieve the desired result.

[0311] Examples of treatable cancers include, but are not limited to, leukemia (including chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and T-cell and B-cell leukemia), lymphoma (Hodgkin's and non-Hodgkin's), lymphoproliferative disorders, plasmacytoma, histiocytoma, melanoma, adenoma, sarcoma, solid tissue cancer, hypoxic tumors, squamous cell carcinoma, genitourinary cancers (such as cervical and bladder cancer), hematopoietic cancers, head and neck cancers, and nervous system cancers.

[0312] This disclosure further includes the use of the modified cells described herein in the manufacture of medicaments or pharmaceutical compositions for treating diseases or disorders or modulating immune responses, treating infections, or treating cancers as described above.

[0313] Modified cells can also be used in experimental models, for example, to further study and elucidate cell function.

[0314] One or more of the modified cells described herein can be administered to a subject in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). In some cases, the modified cells can be expanded in the subject's body (in vivo) after administration. The modified cells can be frozen to provide cells for multiple treatments with the same cell preparation. The modified cells and pharmaceutical compositions comprising them disclosed herein can be packaged as kits (reagents). Kits may include instructions (e.g., written instructions) regarding the use of the modified cells and compositions comprising them.

[0315] In one aspect, this disclosure provides a treatment method comprising administering a therapeutically effective amount of modified cells to a subject. The therapeutically effective amount of modified cells may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. The therapeutically effective amount of modified cells may be administered for at least one week. The therapeutically effective amount of modified cells may be administered for at least two weeks.

[0316] The modified cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the modified cells can vary. For example, the modified cells can be used as a preventative agent and can be administered continuously to subjects with a condition or predisposition to reduce the likelihood of the disease or condition occurring. The modified cells can be administered to subjects during or as soon as possible after the onset of symptoms. Administration of the modified cells can begin immediately upon the onset of symptoms, within 3 hours before the onset of symptoms, within 6 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 48 hours before the onset of symptoms, or at any time after the onset of symptoms. The initial administration can be via any practical route (e.g., intravenous infusion or injection), such as any of the routes described herein using any of the formulations described herein. In some instances, the administration of the modified cells disclosed herein is intravenous administration. Following the onset of cancer or an infectious disease, one or more doses of modified cells may be administered as soon as feasible, for the duration required to treat the disease, such as approximately 24 hours to approximately 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, or approximately 1 month to approximately 3 months. For cancer treatment, one or more doses of modified cells may be administered several years after the onset of cancer and before or after other treatments. In some instances, modified cells may be administered for at least approximately 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The treatment duration can vary for each subject.

[0317] Methods for administering modified cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T-cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) NatRev Clin Oncol. 8(10): 577-85. See, for example, Themeli et al., (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al., (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al., (2013) PLoS ONE 8(4): e61338. Cell therapy (e.g., adoptive T-cell therapy) can be performed via autologous transfer, wherein cells are isolated from and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Thus, in some respects, cells are derived from a subject requiring treatment (e.g., a patient), and after isolation and processing, the cells are administered to the same subject.

[0318] Cell therapy (e.g., adoptive T-cell therapy) can be performed via allogeneic transfer, where cells are isolated and / or otherwise prepared from a subject other than the one to be or ultimately receive the cell therapy (e.g., a first subject). In such cases, the cells are then administered to a different subject of the same species (e.g., a second subject). The first and second subjects can be genetically identical. The first and second subjects can be genetically similar. The second subject can express the same HLA class or supertype as the first subject.

[0319] Prior to administration of the cells or cell-containing compositions, the subject may have been treated with a therapeutic agent targeting a disease or condition (e.g., tumor). In some respects, the subject may be refractory or unresponsive to other therapeutic agents. The subject may have a persistent or relapsing disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even though the subject has become resistant to another therapy, administration may still be effective in treating the subject.

[0320] The subject may respond to another treatment agent, and treatment with that agent reduces the disease burden. In some cases, the subject initially responds to the treatment agent but exhibits a relapse of the disease or condition over time. The subject may not experience a relapse. It can be determined that the subject is at risk of relapse, such as being at high risk of relapse, and therefore cells are administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse. In some cases, the subject has not previously received treatment with another treatment agent.

[0321] Subjects may have persistent or recurrent diseases, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even though the subject has become resistant to another therapy, administration can still effectively treat the subject.

[0322] The modified cells described herein can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. Furthermore, the modified cells can be used to treat any condition related to cancer, particularly a cell-mediated immune response against one or more tumor cells, where treatment or ablation of the disease is desired. Types of cancer to be treated with the modified cells or pharmaceutical compositions include carcinoma, blastoma, and sarcoma, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, etc. Cancer can be a non-solid tumor (such as a hematologic malignancy) or a solid tumor. Adult oncology / cancer and pediatric oncology / cancer are also included. Cancer can be a solid tumor or a hematologic malignancy. Cancer can be carcinoma. Cancer can be sarcoma. Cancer can be leukemia. Cancer can be a solid tumor.

[0323] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the types of cells that form them (such as sarcoma, epithelial carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, seminomas, bladder cancer, melanomas, and CNS tumors (such as gliomas (such as brainstem gliomas and mixed gliomas), glioblastomas (also known as multimorphic glioblastomas), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0324] Cancers suitable for treatment using the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (various tissues), bladder cancer, including transitional cell carcinoma (a malignant tumor of the bladder), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell carcinoma and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, osteoblastoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0325] Sarcomas suitable for treatment using the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0326] The modified cells described herein (e.g., immune cells, T cells, or NK cells) can be included in compositions for immunotherapy. The composition may comprise a pharmaceutical composition and further comprise a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified cells can be administered.

[0327] The modified cells can be used immediately for the above-described treatments, experimental or commercial applications, or they can be cryopreserved for later use. The pharmaceutical composition may be included in a container, package or dispenser along with the instructions for use.

[0328] The modified cells disclosed herein can be formulated into unit dosage forms suitable for precise single-dose administration. In some cases, the unit dosage form includes additional lymphocytes. In a unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit doses can be in the form of packages containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose, non-resealable containers. Multi-dose, resealable containers can be used, for example, with or without preservatives. In some instances, the pharmaceutical composition does not contain preservatives. Formulations for parenteral injection can be presented in unit dosage forms, such as in ampoules or in multi-dose containers with preservatives.

[0329] Example

[0330] The disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0331] Example 1: Generation of CD20 CAR constructs

[0332] CD20 sdAb1 (SEQ ID NO: 1) is an anti-CD20 V inhibitor disclosed in WO 2022 / 012680 A1. H H. 2F2scFv (SEQ ID NO: 3) is an anti-CD20 binding protein derived from the anti-CD20 antibody oframumab. 3H7 scFv (SEQ ID NO: 4) is the anti-CD20 binding protein used in ADI-001, a CD20 CAR-γδT cell product developed by Adicet. Leu16 scFv (SEQ ID NO: 5) is an anti-CD20 binding protein widely used clinically in conjunction with CD20 CAR-T cell therapy. Exemplary CD20 CAR constructs were designed as shown in Table 1, and a schematic diagram of the CD20 CAR construct is shown in Figure 1. The CAR fragment was then cloned into a retroviral or lentiviral vector to construct a CD20 CAR construct in a single coding frame, expressed using the human EF1α promoter.

[0333] Table 1. Exemplary CD20 CAR Construction

[0334]

[0335] Example 2: Generation of CD19 CAR constructs

[0336] CD19 sdAb (SEQ ID NO: 6) is an anti-CD19 V antibody disclosed in WO 2022 / 012683 A1. H H. FMC63 scFv (SEQ ID NO: 7) is an anti-CD19 binding protein widely used clinically in conjunction with CD19 CAR-T cell therapy. FcRγ (SEQ ID NO: 16), Dap12 (SEQ ID NO: 17), and CD3ζ (SEQ ID NO: 15) are adaptor molecules coupled to native cytotoxic receptors (NCRs) such as NKp46, NKp44, and NKp30. These adaptor molecules allow intracellular activation signaling via immune receptor tyrosine-based activation motifs (ITAMs) after the NCR is linked to its putative ligand (Kelly Hudspeth et al., Frontiers in Immunology. 2013). Exemplary CD19 CAR constructs were designed as shown in Table 2, and a schematic diagram of the CD19 CAR construct is shown in Figure 2. The CAR fragment was then cloned into a retroviral or lentiviral vector to construct a CD19 CAR construct in a single coding frame, expressed using the human EF1α promoter.

[0337] Table 2. Exemplary CD19 CAR Construction

[0338]

[0339] Example 3: Generation of CD20 / CD19 dual CAR constructs

[0340] The CD20 / CD19 dual CAR construct was generated via a bicistronic vector, with the CD19 CAR linked to the CD20 CAR at the N- or C-terminus via a P2A self-cleaving element (SEQ ID NO: 18). A schematic diagram of the CD20 / CD19 dual CAR construct is shown in Figure 3. TanCAR7 (SEQ ID NO: 45) and C-CAR039 (SEQ ID NO: 46), which target CD20 and CD19 by tandemly linking anti-CD20 scFv Leu16 or 2F2 with anti-CD19 scFv FMC63, were also generated as reference controls. Exemplary CD20 / CD19 dual CAR constructs were designed as shown in Table 3. The CD20 / CD19 dual CAR fragment was then cloned into a retroviral or lentiviral vector to construct the CD20 / CD19 dual CAR construct within a single coding frame, and expressed using the human EF1α promoter.

[0341] Table 3. Exemplary CD20 / CD19 Dual CAR Constructs

[0342]

[0343] Example 4: Preparation of Virus and CAR-T Cells

[0344] Preparation of retroviruses

[0345] After thawing and amplification of the packaged cells, they were transiently transfected with transfer plasmids at a pre-optimized ratio using polyethyleneimine, followed by culture medium exchange. After harvesting, the cells were transferred to a concentration step and then ultracentrifuged. The viral pellet was washed with formulation buffer. The virus was appropriately aliquoted and immediately stored at -80°C. Viral titers were determined by flow cytometry by measuring the efficiency of transduction into Jurkat cell lines.

[0346] Preparation of lentiviruses

[0347] A mixture of lentiviral packaging plasmids (Addgene, catalog number 12259) containing pCMV-ΔR-8.47 and pMD2.G was premixed with a vector carrying the CAR gene at a pre-optimized ratio using polyethyleneimine and then added to HEK-293 cells. The supernatant was collected after overnight incubation. The virus-containing supernatant was filtered through a 0.45 µm PES filter and then ultracentrifuged to concentrate the lentivirus. The viral pellet was washed with pre-cooled DPBS. The virus was appropriately aliquoted and immediately stored at -80°C. Viral titers were determined by measuring the efficiency of transduction into supT1 cell lines via flow cytometry.

[0348] Collection and transduction of αβT lymphocytes

[0349] Leukocytes were collected from healthy donors via apheresis. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque™ PLUS medium, according to the manufacturer's protocol. Human T cells were purified from PBMCs using the Pan T Cell Isolation Kit (Miltenyi, catalog number 130-096-535), according to the manufacturer's protocol. The purified T cells were then pre-activated for 48 hours using the Human T Cell Activation / Expansion Kit (Miltenyi, catalog number 130-091-441), according to the manufacturer's protocol, with anti-CD3 / CD28 MACSiBead particles added at a bead-to-cell ratio of 1:2. The pre-activated T cells were transduced with lentiviral stock in the presence of 7 µg / mL polyglobulin. The transduced cells were then transferred to a cell culture incubator for transgene expression under appropriate conditions.

[0350] Collection and transduction of γδ T lymphocytes

[0351] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using a Ficoll-Paque-based density gradient centrifugation protocol. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and antibiotics. To activate cells, ZOL (50 μM working concentration, Sigma) was added to the medium on day 0. The medium also contained recombinant human IL-2 (100 IU / mL) (Beijing Four Rings Bio-Pharm Co.), recombinant human IL-15 (100 IU / mL), and vitamin C (70 μM) (Sigma). Cells were transduced with lentivirus at an MOI of 5 24–96 hours post-activation. Cells were harvested 10 days post-transduction to determine total cell count, purity, and transduction efficiency. Cells were further enriched using a negative TCRγ / δ+ T cell isolation kit (Miltenyi Biotec) before future use or cryopreservation.

[0352] Example 5: CD20 and CD19 expression in tumor cell lines

[0353] The expression of CD20 and CD19 in tumor cell lines was determined by flow cytometry. As shown in Figure 4, CD20 and CD19 were simultaneously expressed in Raji cells, Z-138 cells, and NALM6-CD20 cells. CD20 was expressed in Raji-CD19 KO cells, while CD19 was not expressed. CD19 was expressed in NALM6 cells, while CD20 was not expressed.

[0354] Example 6: In vitro cytotoxicity and cytokine release of CD20 CAR constructs

[0355] Different anti-CD20 binding proteins were integrated with the CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3ζ signaling pathway, respectively, to generate CD20 CAR constructs carrying different anti-CD20 binding proteins within the same CAR architecture (as shown in Table 1). These CAR constructs were transduced into γδT cells to prepare CAR-γδT cells. On day 7 post-transduction, CAR-T cells were co-cultured with the CD20-positive NHL cell line Raji at E:T (effective to target cell) ratios of 3:1 and 1:1 for 20–24 hours. Untransduced γδT cells (UnT) were used as a negative control. CAR-T cells were analyzed using ONE-Glo from Promega. TM Cytotoxicity was assessed using a luciferase assay system. As shown in Figure 5A, all CAR constructs exhibited potent cytotoxicity against Raji cells.

[0356] In addition, co-culture supernatants were collected to measure cytokines using a assay kit from Cisbio according to the manufacturer's instructions. As shown in Figures 5B-5C, increased secretion of IFNγ and TNFα was detected in the supernatant collected from the co-culture of CAR-T cells and Raji cells, while no increased cytokine release was observed in the supernatant collected from the co-culture of UnT cells and Raji cells. In summary, CD20 CAR-T cells exhibited cytotoxicity and cytokine release against CD20-positive Raji cells in vitro.

[0357] Example 7: In vivo evaluation of CD20 CAR construct

[0358] In the Raji xenograft model, the antitumor activity of CD20 CAR-γδT cells was evaluated in vivo. On day 0, 2 × 10⁶ cells were transplanted. 6 Raji cells were subcutaneously implanted into NCG mice. Once the tumor reached 100-150 mm... 3 (12 days after tumor implantation), mice were randomly assigned to the treatment group. 5 × 10⁶ mice were placed in the treatment group. 6 CAR-positive T cells (CD20 sdAb1 BBZ, 2F2 BBZ, 3H7 BBZ) were suspended in 400 µL of HBSS medium and then administered to mice via tail vein. Untransduced γδ T cells (UnT) were used as a negative control. Tumor volume and body weight were measured twice weekly. Human CD3+ cells in peripheral blood viable cells were identified weekly by flow cytometry. +The percentage of T cells. As shown in Figures 6A-6C, CD20 sdAb1 BBZ CAR-T cells exhibited better anti-tumor efficacy than 2F2 BBZ CAR-T cells and 3H7 BBZ CAR-T cells. Mice treated with CD20 sdAb1 BBZ CAR-T cells achieved tumor-free status and showed no significant change in body weight compared to the cohort treated with UnT. Furthermore, compared to the cohorts treated with 2F2 BBZ CAR-T cells and 3H7 BBZ CAR-T cells, the cohort treated with CD20 sdAb1 BBZ CAR-T cells showed a higher percentage of CD3+ cells. + T cell proliferation was enhanced. Three weeks after CAR-T cell infusion, human CD3... + T cell contraction. No tumor suppression was observed by UnT infusion. The results indicate that in a xenograft mouse model, CD20 CAR-T cells carrying CD20 sdAb1 have better anti-tumor efficacy than CD20 CAR-T cells carrying other anti-CD20 binding proteins.

[0359] Example 8: In vitro cytotoxicity and cytokine release of CD19 CAR construct in αβT cells

[0360] With or without juxtamembranous insertion of the 4-1BB intracellular domain (second-generation or first-generation CAR), three different domains carrying ITAM (immunoreceptor tyrosine-based activation motifs) (CD3ζ intracellular domain, Dap12 intracellular domain, and FcRγ intracellular domain) were integrated with the CD19 sdAb, CD8 hinge, and CD8 transmembrane domain, respectively. These CD19CAR constructs (as shown in Table 2) were transduced into αβT cells to prepare CAR-αβT cells. On day 7 post-transduction, CAR-T cells were co-cultured with the CD19-positive NHL cell line Raji at a 5:1 E:T ratio for 20–24 hours. Untransduced αβT cells (UnT) were used as a negative control. CAR-T cells were analyzed using ONE-Glo from Promega. TM Cytotoxicity was assessed using a luciferase assay system. As shown in Figure 7A, CAR-T cells carrying CD3ζ, Dap12, and FcRγ exhibited cytotoxicity against Raji cells. Proximal insertion of the 4-1BB domain inhibited the killing efficacy of CARs carrying Dap12 and FcRγ, while CARs carrying CD3ζ showed comparable cytotoxicity with or without 4-1BB.

[0361] In addition, co-culture supernatants were collected to measure cytokines using a assay kit from Cisbio according to the manufacturer's instructions. As shown in Figure 7B, increased IFNγ secretion was detected in the supernatant collected from the co-culture of CAR-T cells and Raji cells, while no increased IFNγ release was observed in the supernatant collected from the co-culture of UnT cells and Raji cells. CAR-T cells carrying FcRγ secreted less IFNγ than CAR-T cells carrying CD3ζ and CAR-T cells carrying Dap12, which is consistent with the cytotoxicity data.

[0362] Example 9: In vitro cytotoxicity of CD19 CAR construct in γδT cells

[0363] FMC63 scFv was integrated with a CD8 or CD28 hinge, a CD8 or CD28 transmembrane domain, a 4-1BB, CD28, Dap10 (SEQ ID NO: 20) or OX40 (SEQ ID NO: 21) intracellular domain, and an FcRγ intracellular domain to generate a set of CAR constructs targeting CD19, as shown in Table 4. To demonstrate the effect of the distance between the FcRγ intracellular domain and the cell membrane, 4-1BB was generated in parallel, inserted upstream or downstream of FcRγ. FMC63 BBZ constructs with conventional CD8 hinge, CD8 transmembrane domain, and CD3ζ intracellular domain, with or without the 4-1BB intracellular domain, were used as positive controls.

[0364] Table 4. CAR constructs targeting CD19

[0365]

[0366] These CD19 CAR constructs were transduced into γδT cells to prepare CAR-γδT cells. On day 7 post-transduction, CAR-T cells were co-cultured with the CD19-positive NHL cell line Raji at E:T ratios of 5:1 and 1:1 for 20–24 hours. Untransduced γδT cells (UnT) were used as a negative control. The results were obtained using ONE-Glo from Promega. TMCytotoxicity was assessed using a luciferase assay system. As shown in Figure 8, each type of CD19 CAR-T cell exhibited dose-dependent cytotoxicity against Raji cells. CAR-T cells carrying the CD28 hinge and transmembrane domain showed superior cytotoxicity compared to those carrying the CD8 hinge and transmembrane domain, indicating that the CD28 hinge and transmembrane domain provide a greater benefit for FcRγ cytotoxicity than the CD8 hinge and transmembrane domain. Furthermore, FMC63 FcRγBB (4-1BB downstream of FcRγ) CAR-T cells were superior to FMC63 BBFcRγ (4-1BB upstream of FcRγ) CAR-T cells, indicating that juxtamembrane localization promotes FcRγ cytotoxicity. This is consistent with the fact that FMC63 FcRγBB, FMC63 FcRγDap10, and FMC63 FcRγOX40 showed comparable cytotoxicity to FMC63 FcRγ. Interestingly, cytotoxicity was well maintained when the CD28 intracellular domain was inserted upstream of FcRγ. In other words, FcRγ can withstand membrane insertion of the CD28 intracellular domain with minimal reduction in cytotoxicity.

[0367] Example 10: In vitro proliferation of CD19 CAR constructs in αβ T cells

[0368] CD19 sdAbs were integrated with intracellular domains of 4-1BB and CD3ζ, Dap12, or FcRγ to generate second-generation CAR constructs targeting CD19. The constructs include CD19 sdAb BBZ (SEQ ID NO. 31), CD19sdAb BBDap12 (SEQ ID NO. 32), and CD19 sdAb BBFcRγ (SEQ ID NO. 33).

[0369] These CD19 CAR constructs were transduced into αβT cells to prepare CAR-αβT cells. On day 7 post-transduction, CAR-T cells were co-cultured with the CD19-positive NHL cell line Raji at an E:T ratio of 1:1 or 1:3 for 3 days as the first co-culture cycle. Untransduced αβT cells (UnT) were used as a negative control. After co-culture, cells were collected, and the number of CAR-T cells was determined by cell counting and flow cytometry. The collected CAR-T cells from the previous co-culture cycle were then added to freshly prepared Raji cells that had been plated in new 24-well plates for the next co-culture cycle. Each cycle lasted 2–3 days, for a total of 4 cycles (9 days of co-culture in total). The cumulative fold increase of CAR-T cells was determined by multiplying the fold increase in each co-culture cycle. As shown in Figures 9A-9B, at E:T ratios of 1:1 and 1:3, CD19 CAR-T cells carrying the FcRγ intracellular domain exhibited superior proliferation compared to CAR-T cells carrying both CD3ζ and Dap12 intracellular domains. At an E:T ratio of 1:3, CD19 CAR-T cells carrying only FcRγ showed proliferative capacity even at such a low E:T ratio.

[0370] Additionally, supernatants from the first co-culture cycle were collected to measure cytokines using a assay kit from Cisbio according to the manufacturer's instructions. As shown in Figure 9C, at a 1:1 E:T ratio, CD19 CAR-T cells carrying the FcRγ intracellular domain secreted significantly less IFNγ than IFNγ CAR-T cells carrying the CD3ζ and Dap12 intracellular domains. At a 1:3 E:T ratio, comparable levels of IFNγ release were detected for all three types of CD19 CAR-T cells. No increased IFNγ release was observed in the supernatant collected from the co-culture of UnT cells and Raji cells. In conclusion, CD19 CAR-T cells carrying the FcRγ intracellular domain exhibited stronger proliferation and lower cytokine release compared to CD19 CAR-T cells carrying the CD3ζ and Dap12 intracellular domains, demonstrating the potential advantages of using the FcRγ intracellular domain in CAR design for clinical applications.

[0371] Example 11: In vitro proliferation of CD19 CAR constructs in γδT cells

[0372] FMC63 scFv was integrated with a CD8 or CD28 hinge, a CD8 or CD28 transmembrane domain, an intracellular domain of 4-1BB, CD28, Dap10, OX40, CD27 (SEQ ID NO: 22) or ICOS (SEQ ID NO: 23), and an intracellular domain of FcRγ to generate a set of CAR constructs targeting CD19, as shown in Table 5. An FMC63 BBZ construct with a conventional CD8 hinge, CD8 transmembrane domain, 4-1BB intracellular domain, and CD3ζ intracellular domain was used as a positive control.

[0373] Table 5. CAR constructs targeting CD19

[0374]

[0375] These CD19 CAR constructs were transduced into γδT cells to prepare CAR-γδT cells. On day 7 post-transduction, CAR-T cells were co-cultured with the CD19-positive NHL cell line Raji at a 1:1 E:T ratio for 4 days as the first co-culture cycle. After co-culture, cells were collected, and the number of CAR-T cells was determined by cell counting and flow cytometry. The collected CAR-T cells from the previous co-culture cycle were then added to freshly prepared Raji cells that had been plated in new 24-well plates, and co-cultured for another 4 days as the second co-culture cycle. Two cycles were performed (a total of 8 days of co-culture). The cumulative fold increase of CAR-T cells was determined by multiplying the fold increase in each co-culture cycle. As shown in Figure 10, CD19 CAR-T cells carrying the CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain, and FcRγ intracellular domain exhibited stronger proliferation than CD19 CAR-T cells with other modalities.

[0376] Example 12: In vitro cytotoxicity and cytokine release of CD20 / CD19 dual CAR constructs in γδT cells

[0377] Table 6. Builds for generating dual-CAR systems

[0378]

[0379] The CAR constructs shown in Table 6 were transduced into γδT cells to prepare CAR-γδT cells. The FMC63 BBZ construct (SEQ ID NO: 47) was transduced into αβT cells to prepare CAR-αβT cells as a reference control. Untransduced γδT cells (Un-γδT) and untransduced αβT cells (Un-αβT) were used as negative controls. On day 7 post-transduction, CAR-T cells were co-cultured with CD20 and CD19 double-positive Raji cells, CD20-positive CD19-negative Raji-CD19 KO cells, and CD20-negative CD19-positive NALM6 cells at E:T ratios of 2:1 and 1:2 for 20–24 hours. Cells were analyzed using ONE-Glo from Promega. TM Cytotoxicity was assessed using a luciferase assay system. As shown in Figures 11A-11C, CD20 / CD19 dual CAR-T cells exhibited potent cytotoxicity against all three cell lines, while CD20 sdAb1 BBZ CAR-T cells killed only CD20-positive cell lines, and FMC63 28FcRγ and FMC63 BBZ CAR-T cells killed only CD19-positive cell lines.

[0380] In addition, co-culture supernatants were collected to measure cytokines using a assay kit from Cisbio according to the manufacturer's instructions. As shown in Figures 11D-11F, increased INFγ secretion was detected in the supernatants collected from co-cultures of CD20 / CD19 dual CAR-T cells with CD20-positive Raji cells and Raji-CD19 KO cells. However, no increased INFγ secretion was detected in CD20 / CD19 dual CARs with CD20-negative CD19-positive NALM6 cells. This is consistent with the low cytokine release of CD19 CARs carrying the FcRγ intracellular domain. No increased INFγ release was observed in the supernatants collected from co-cultures of UnT cells with each cell line type. In conclusion, CD20 / CD19 dual CAR-T cells exhibited antitumor activity against CD20 and / or CD19-positive tumor cells.

[0381] Example 13: CD20 / CD19 double CAR-γδ T cells did not induce cytotoxicity against CD20 / CD19 double negative cells.

[0382] To confirm the killing specificity of the CD20 / CD19 dual CAR construct, CD20 / CD19 dual CAR-γδT cells from two separate donors were co-cultured with three types of tumor cell lines (A-371, A549, and H929) that did not express CD20 or CD19. Untransduced γδT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured with CD20 and CD19 double-negative cell lines A-371, A549, and H929 at E:T ratios of 10:1, 3:1, and 1:1, respectively, for 20–24 hours. The cells were analyzed using ONE-Glo from Promega. TM Cytotoxicity was assessed using a luciferase assay system. As shown in Figures 12A-12C, CD20 / CD19 dual CAR-T cells did not exhibit cytotoxicity against any of the three cell lines.

[0383] In addition, co-culture supernatants were collected to measure cytokines using a detection kit from Cisbio according to the manufacturer's instructions. As shown in Figures 12D-12F, no increase in INFγ secretion was observed in the supernatants collected from co-cultures of CD20 / CD19 dual CAR-T cells with each cell line type. In summary, the CD20 / CD19 dual CAR construct did not exhibit cytotoxicity or cytokine release in co-cultures with CD20 and CD19 double-negative cells.

[0384] Example 14: In vitro proliferation of CD20 / CD19 dual CAR constructs in γδT cells

[0385] CAR-γδT cells were prepared by transducing CD20 / CD19 dual CAR constructs, CD20 sdAb1 BBZ constructs, FMC63 28FcRγ constructs, and FMC63 BBZ constructs into γδT cells. Untransduced γδT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured with the CD20 and CD19 double-positive cell line NALM6-CD20 at a 1:1 E:T ratio for 3 days as the first co-culture cycle. After co-culture, cells were collected, and CD3+ levels in total viable cells were determined by flow cytometry. +The percentage of T cells was determined by cell counting and flow cytometry to identify the number of CAR-T cells. CAR-T cells collected from previous co-culture cycles were then added to freshly prepared NALM6-CD20 cells already plated in new 24-well plates for the next co-culture cycle. Seven cycles were performed (a total of 21 days of co-culture). The cumulative fold increase of CAR-T cells was determined by multiplying the fold increase in each co-culture cycle. As shown in Figures 13A-13B, CD20 / CD19 dual CAR-T cells exhibited the most durable killing capacity and the largest proliferation. This demonstrates that CD20 / CD19 dual CAR-T cells outperformed the single-target CAR-T control in terms of durability and long-term proliferation.

[0386] Example 15: In vivo evaluation of CD20 / CD19 dual CARs in γδT cells

[0387] CAR-γδT cells were prepared by transducing CD20 / CD19 dual CAR constructs and CD20 sdAb1 BBZ constructs into γδT cells from two separate donors. CAR-αβT cells were prepared as a reference control by transducing FMC63 BBZ constructs into αβT cells. Untransduced γδT cells (Un-γδT) were used as a negative control. The antitumor activity of these CAR-T cells was evaluated in vivo in a Z-138 xenograft model. On day 0, 3 × 10⁻⁶ cells were transduced into αβT cells. 6 Z-138 cells were subcutaneously implanted into NCG mice. Once the tumor volume reached 100-150 mm... 3 (14 days after tumor implantation), mice were randomly assigned to the treatment group. 2 × 10⁶ mice were then placed in the treatment group. 6CAR-positive T cells were suspended in 400 µL of HBSS medium and then administered to mice via the tail vein. HBSS medium without T cells was used as a control. Tumor volume was measured twice weekly. The percentage of CAR-T cells in peripheral blood viable cells was determined weekly by flow cytometry. As shown in Figures 14A-14B, for CAR-γδT cells from donor 1, treatment with CD20 sdAb1 BBZ CAR-T cells resulted in tumor-free status in 3 out of 4 mice, while tumor growth was not suppressed in 1 mouse. However, in all 3 mice that achieved tumor-free status, tumor recurrence occurred 30 days after CAR-T infusion. In contrast, CD20 / CD19 dual CAR-T cells achieved tumor-free status in all 4 mice, with tumor recurrence in only 2 out of 4 mice, and the other 2 mice remaining tumor-free for 60 days after CAR-T infusion. Furthermore, compared to the cohort treated with CD20 sdAb1 BBZ CAR-T cells, the cohort treated with CD20 / CD19 dual CAR-T cells exhibited stronger CAR-T cell proliferation. As shown in Figures 14C-14D, all four mice achieved tumor-free status after treatment with CD20 sdAb1 BBZ CAR-T cells for CAR-γδT cells from donor 2. However, tumor recurrence occurred in all four mice 40 days after CAR-T infusion. In contrast, CD20 / CD19 dual CAR-T cells achieved and maintained tumor-free status in all four mice within 60 days after CAR-T infusion. Additionally, compared to the cohort treated with CD20 sdAb1 BBZ CAR-T cells, the cohort treated with CD20 / CD19 dual CAR-T cells showed stronger CAR-T cell proliferation. As shown in Figure 14C, after treatment with FMC63 BBZ CAR-αβT cells, one of the three mice failed to effectively inhibit tumor growth, while the other two mice achieved tumor-free status. No tumor suppression was observed with Un-γδT cells from two donors, as a control group administered via infusion of the mediator. In summary, the efficacy and proliferation of CD20 / CD19 dual CAR-T cells in γδT cells were demonstrated to be superior to the CD20 single CAR control. CD20 / CD19 dual CAR-T cells provided long-term protection against tumor recurrence in xenograft mouse models, consistent with the long PFS achieved through CD20 / CD19 dual CAR-T cell therapy in clinical trials.

[0388] Example 16: CD20 / CD19 dual CAR has better anti-tumor efficacy in vivo than CD20 single CAR and CD19 single CAR.

[0389] CAR-γδT cells were prepared by transducing CD20 / CD19 dual CAR constructs, CD20 sdAb1 BBZ constructs, and FMC63 28FcRγ into γδT cells. Untransduced γδT cells (Un-γδT) were used as a negative control. The antitumor activity of these CAR-T cells was evaluated in vivo in a Z-138 xenograft model. On day 0, 3 × 10⁻⁶ cells were transduced into γδT cells. 6 Z-138 cells were subcutaneously implanted into NCG mice. Once the tumor volume reached 100-150 mm... 3 (14 days after tumor implantation), mice were randomly assigned to the treatment group. 2 × 10⁶ mice were placed in the treatment group. 6 CAR-positive T cells were suspended in 400 µL of HBSS medium and then administered to mice via the tail vein. Tumor volume was measured twice weekly. The percentage of CAR-T cells in peripheral blood viable cells was determined weekly by flow cytometry. As shown in Figures 15A-15B, CD20 / CD19 dual CAR-T cells achieved better tumor suppression than CD20 sdAb1BBZ CAR-T cells and FMC63 28FcRγ CAR-T cells. Furthermore, the cohort treated with CD20 / CD19 dual CAR-T cells exhibited stronger CAR-T cell proliferation compared to the cohorts treated with CD20 sdAb1BBZ CAR-T cells and FMC63 28FcRγ CAR-T cells.

[0390] Example 17: CD20 / CD19 dual CARs exhibit better persistence and proliferation in γδT cells than tandem CARs.

[0391] CAR-γδT cells were prepared by transducing the CD20 / CD19 dual CAR construct and two tandem CAR constructs (TanCAR7 and C-CAR039) shown in Table 3 into γδT cells from two separate donors. Untransduced γδT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured for 3 days with the CD20 single-positive cell line Raji-CD19 KO, the CD19 single-positive cell line NALM6, or K-562-CD19 at a 2:1:1 E:T:T (effecton to target cell to target cell) ratio as the first co-culture cycle. After co-culture, cells were collected, and CD3+ in total viable cells was determined by flow cytometry. +The percentage of T cells was determined by cell counting and flow cytometry to identify the number of CAR-T cells. CAR-T cells collected from previous co-culture cycles were then added to freshly prepared NALM6-CD20 cells already plated in new 24-well plates for the next co-culture cycle. Three cycles were performed (a total of 9 days of co-culture). The cumulative fold increase of CAR-T cells was determined by multiplying the fold increase in each co-culture cycle. As shown in Figures 16A-16D, CD20 / CD19 dual CAR-T cells showed more durable killing ability and better proliferation than TanCAR7 CAR-T cells and C-CAR039 CAR-T cells in both donors. The superiority of CD20 / CD19 dual CAR-T cells over tandem CAR-T cells in terms of durability and long-term proliferation is demonstrated in γδT cells.

[0392] Example 18: CD20 / CD19 dual CARs exhibit better persistence and proliferation in αβT cells than tandem CARs.

[0393] CAR-γδT cells were prepared by transducing a CD20 / CD19 dual CAR construct and two tandem CAR constructs (TanCAR7 and C-CAR039) into αβT cells from two separate donors. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured with the CD20 and CD19 double-positive cell line Raji cells at a 1:1 E:T ratio for 3 days as the first co-culture cycle. After co-culture, cells were harvested, and CD3+ levels in total viable cells were determined by flow cytometry. + The percentage of T cells was determined by cell counting and flow cytometry to identify the number of CAR-T cells. CAR-T cells collected from previous co-culture cycles were then added to freshly prepared NALM6-CD20 cells already plated in new 24-well plates for the next co-culture cycle. Seven cycles were performed (a total of 16 days of co-culture, with 2–3 days per cycle). The cumulative fold increase of CAR-T cells was determined by multiplying the fold increase in each co-culture cycle. As shown in Figures 17A–17B, CD20 / CD19 dual CAR-T cells exhibited more durable killing ability and better proliferation than TanCAR7 and C-CAR039 CAR-T cells. The superiority of CD20 / CD19 dual CAR-T cells over tandem CAR-T cells in terms of durability and long-term proliferation is demonstrated in αβT cells.

[0394] Example 19: Generation of CD20 / CD19 dual CAR constructs with different modalities

[0395] CD20 / CD19 dual-CAR constructs with different modalities were generated using bicistronic vectors, with the CD19 CAR linked to the CD20 CAR at the N- or C-terminus via a P2A self-cleaving element (SEQ ID NO: 18). A schematic diagram of CD20 / CD19 dual-CAR constructs with different modalities is shown in Figure 18. Exemplary CD20 / CD19 dual-CAR constructs with different modalities were designed as shown in Table 7. The CD20 / CD19 dual-CAR fragments were then cloned into retroviral or lentiviral vectors to construct CD20 / CD19 dual-CAR constructs within a single coding frame, expressed using the human EF1α promoter.

[0396] Table 7. Examples of Dual CAR Constructs

[0397]

[0398] Example 20: In vitro cytotoxicity and cytokine release of CD20 / CD19 dual CAR constructs with different modalities

[0399] CAR-αβT cells were prepared by transducing CD20 / CD19 dual CAR constructs with different modalities into αβT cells. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured for 20–24 hours with CD20 and CD19 double-positive Raji cells, CD20-positive CD19-negative Raji-CD19 KO cells, and CD20-negative CD19-positive K-562-CD19 cells at E:T ratios of 8:1 and 2:1, respectively. The cells were analyzed using ONE-Glo from Promega. TM Cytotoxicity was assessed using a luciferase assay system. As shown in Figures 19A-19C, CD20 / CD19 dual CAR-T cells with different modalities exhibited potent cytotoxicity against all three cell lines, while the CD20 / CD19 dual CAR with the CD20 sdAb1CD8HTM BB FcRγ / FMC63 IgG4 hinge CD28TM CD28Z modality showed superior cytotoxicity.

[0400] In addition, co-culture supernatants were collected to measure cytokines using a assay kit from Cisbio according to the manufacturer's instructions. As shown in Figures 19D-19F, increased INFγ secretion was detected in the supernatants collected from co-cultures of CD20 / CD19 dual CAR-T cells with all three cell lines. No increased INFγ release was observed in the supernatants collected from co-cultures of UnT cells with each cell line. Notably, compared to other CD20 / CD19 dual CAR modalities, the CD20 / CD19 dual CAR modality using the FcRγ domain of a CD20-targeting CAR secreted significantly lower levels of INFγ in co-cultures with Raji cells and Raji-CD19 KO cells. In conclusion, CD20 / CD19 dual CAR-T cells with different modalities exhibited antitumor activity against CD20 and / or CD19-positive tumor cells.

[0401] Example 21: In vitro proliferation of CD20 / CD19 dual CAR constructs with different modalities

[0402] CAR-αβT cells were prepared by transducing CD20 / CD19 dual CAR constructs with different modalities into αβT cells. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured with the CD20 and CD19 double-positive cell line Raji cells at a 1:1 E:T ratio for 3 days as the first co-culture cycle. After co-culture, cells were collected, and CD3+ levels in total viable cells were determined by flow cytometry. + The percentage of T cells was determined by cell counting and flow cytometry to identify the number of CAR-T cells. CAR-T cells collected from previous co-culture cycles were then added to freshly prepared NALM6-CD20 cells already plated in new 24-well plates for the next co-culture cycle. Ten cycles were performed (a total of 25 days of co-culture, 2–3 days per cycle). The cumulative fold increase of CAR-T cells was determined by multiplying the fold increase in each co-culture cycle. As shown in Figures 20A–20B, CD20 / CD19 dual CAR-T cells with different modalities exhibited durable killing ability and robust proliferation. Notably, the CD20 / CD19 dual CAR modality with CD20 sdAb1 BBZ / FMC63 CD28HTMCD28Z (a widely used form of parallel CAR with two CD3ζ domains) showed less proliferation than other CD20 / CD19 dual CAR modalities with a combination of one CD3ζ domain and one FcRγ domain or one Dap 12 domain.

[0403] Example 22: Co-culture of CD20 / CD19 dual CARs with PBMCs

[0404] B cells play a crucial role in the pathogenesis and development of a range of autoimmune diseases. Emerging data have demonstrated that B cell depletion targeting CD19 or CD20 may be an effective strategy for treating autoimmune diseases such as systemic lupus erythematosus. To explore the potential of CD20 / CD19 dual CARs in autoimmune diseases, in vitro studies were conducted to investigate the capabilities of CD20 / CD19 dual CARs in depleted primary B cells. Two different CD20 / CD19 dual CAR constructs, as shown in Table 8, were transduced into αβT cells to prepare CAR-αβT cells. The TanCAR7 construct (SEQ ID NO: 45), the C-CAR039 construct (SEQ ID NO: 46), and the FMC63 BBZ construct (SEQ ID NO: 47), targeting CD20 and CD19, were generated as reference controls. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post-transduction, CAR-T cells were co-cultured with human PBMCs at E:T ratios of 1:5, 1:20, and 1:50 for 72 hours. The percentage of B cells was determined by FACS staining with anti-CD19 and anti-CD20 antibodies. As shown in Figure 21, B cells comprised 16.89% of PBMCs before co-culture. After 72 hours of co-culture with CAR-T cells, as shown in Figure 22, B cells were exhausted to varying degrees by CAR-T cells. CD20 sdAb1 BBZ / FMC63 CD28HTM CD28 FcRγ and CD20 sdAb1 CD8HTM BBFcRγ / FMC63 IgG4 hinge CD28TM CD28Z showed superior B cell exhaustion capacity compared to the C-CAR039 and FMC63BBZ constructs at a 1:50 ratio.

[0405] In addition, co-culture supernatants were collected to measure cytokines using a assay kit from Cisbio according to the manufacturer's instructions. As shown in Figure 23, increased INFγ secretion was detected in the supernatant collected from the co-culture of CAR-T cells with PBMCs. No increased INFγ release was observed in the supernatant collected from the co-culture of UnT cells with PBMCs or from PBMCs alone. Notably, CD20 sdAb1 BBZ / FMC63 CD28HTMCD28 FcRγ and CD20 sdAb1 CD8HTM BB FcRγ / FMC63 IgG4 hinge CD28TM CD28Z secreted lower levels of INFγ in co-culture with PBMCs compared to the FMC63 BBZ construct, suggesting that the risk of CRS with CD20 / CD19 dual CARs may be lower in clinical settings.

[0406] To confirm the conclusions of this study, parallel CD20 sdAb1 BBZ / FMC63 CD28HTM CD28FcRγ, CD20 sdAb1 CD8HTM BB FcRγ / FMC63 IgG4 hinge CD28TM CD28Z, TanCAR7, C-CAR039, and UnT were generated and co-cultured with human PBMCs from another donor at an E:T ratio of 1:50 for 72 hours. As shown in Figure 24, CD20 sdAb1 BBZ / FMC63 CD28HTM CD28FcRγ and CD20 sdAb1 CD8HTM BB FcRγ / FMC63 IgG4 hinge CD28TM CD28Z exhibited superior B cell depletion capacity compared to TanCAR7 and C-CAR039. In conclusion, CD20 / CD19 dual CAR-T cells can effectively eliminate B cells from PBMCs under conditions of low cytokine release.

[0407] Table 8. Examples of Dual CAR Constructs

[0408]

[0409] Other embodiments

[0410] It should be understood that although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An engineered receptor comprising: an extracellular antigen-binding domain; a transmembrane domain; and an intracellular domain, the intracellular domain comprising an intracellular signal transduction domain derived from FcRγ, and one or more additional signal transduction domains selected from the CD28 co-stimulatory signal transduction domain, the Dap10 intracellular signal transduction domain, the OX40 intracellular signal transduction domain, the CD27 intracellular signal transduction domain, the CD137 (4-1BB) co-stimulatory signal transduction domain, and the ICOS intracellular signal transduction domain.

2. The engineered receptor of claim 1, wherein the additional signal transduction domain is the CD28 co-stimulatory signal transduction domain.

3. The engineered receptor of claim 2, wherein the CD28 co-stimulatory signal transduction domain is located at the N-terminus of the intracellular signal transduction domain derived from FcRγ.

4. The engineered receptor of claim 1, wherein the additional signal transduction domain is a CD137 (4-1BB) co-stimulatory signal transduction domain.

5. The engineered receptor of claim 4, wherein the CD137 (4-1BB) co-stimulatory signal transduction domain is located at the N-terminus of the intracellular signal transduction domain derived from FcRγ.

6. The engineered receptor according to any one of claims 1-5, wherein the transmembrane domain is derived from the following transmembrane domains: the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154.

7. The engineered receptor according to any one of claims 1-6, wherein the engineered receptor further comprises a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

8. The engineered receptor according to any one of claims 1-7, wherein the hinge region is derived from the hinge region of CD8α, CD28, IgG1, IgG2, IgG3 or IgG4.

9. The engineered receptor according to any one of claims 1-8, wherein the engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

10. The engineered receptor according to any one of claims 1-8, wherein the engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

11. The engineered receptor according to any one of claims 1-8, wherein the engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

12. The engineered receptor according to any one of claims 1-8, wherein the engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ.

13. The engineered receptor according to any one of claims 1-12, wherein the extracellular antigen-binding domain comprises sdAb, scFv, (scFv)2, and V. H H-domain or VNAR-domain.

14. The engineered receptor according to any one of claims 1-13, wherein the extracellular antigen-binding domain binds to a tumor antigen.

15. The engineered receptor of claim 14, wherein the tumor antigen is selected from the group consisting of: B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 gangliosides, glycoprotein 75, GPNMB, HER2 / neu, HGF, human dispersive factor receptor kinase, IGF-1 receptor, IGF-1, IgG1, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-hydroxyacetylneuraminic acid, NPC-1C, PDGF-RA, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGFβ2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin.

16. The engineered receptor of claim 15, wherein the extracellular antigen-binding domain binds to CD19.

17. The engineered receptor of claim 16, wherein the extracellular antigen-binding domain comprises: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences are respectively in SEQ ID NO: As shown in SEQ ID NO: 51, 52, 53, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 54, 55, 56, respectively.

18. The engineered receptor of claim 17, wherein the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the selected VH sequence, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO:

58.

19. The engineered receptor of claim 18, wherein the extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

7.

20. The engineered receptor according to any one of claims 1-19, wherein the engineered receptor comprises an amino acid sequence shown in any one of SEQ ID NO: 33, 36-43 and 68, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 33, 36-43 and 68.

21. The engineered receptor according to any one of claims 1-20, wherein the engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

36.

22. The engineered receptor of claim 15, wherein the extracellular antigen-binding domain binds to CD20.

23. The engineered receptor of claim 22, wherein the extracellular antigen-binding domain comprises V H H antibody portion, the V H The H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains at least 80% of the same amino acid sequence as the selected CDR1 amino acid sequence, CDR2 contains at least 80% of the same amino acid sequence as the selected CDR2 amino acid sequence, and CDR3 contains at least 80% of the same amino acid sequence as the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO:

50.

24. The engineered receptor of claim 23, wherein the extracellular antigen-binding domain comprises V H H antibody portion, the V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

25. The engineered receptor according to any one of claims 1-15 and 22-24, wherein the engineered receptor comprises an amino acid sequence shown in any one of SEQ ID NO: 69-71 and 74, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 69-71 and 74.

26. The engineered receptor according to any one of claims 1-25, wherein the engineered receptor further comprises a signal peptide.

27. The engineered receptor of claim 26, wherein the signal peptide is located at the N-terminus of the engineered receptor.

28. The engineered receptor of claim 26 or 27, wherein the signal peptide is derived from CD8α, and wherein optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO:

8.

29. A chimeric antigen receptor (CAR) system comprising: a first engineered receptor comprising: a first extracellular antigen-binding domain; a first transmembrane domain; and a first intracellular domain comprising an intracellular signal transduction domain derived from FcRγ; and a CD28 co-stimulatory signal transduction domain or a CD137 (4-1BB) co-stimulatory signal transduction domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

30. The dual CAR system of claim 29, wherein the second intracellular domain comprises the CD3ζ primary intracellular signal transduction domain.

31. The dual CAR system of claim 29 or 30, wherein the second engineered receptor further comprises a co-stimulatory signal transduction domain located between the C-terminus of the transmembrane domain and the N-terminus of the CD3ζ primary intracellular signal transduction domain.

32. The dual CAR system of claim 31, wherein the co-stimulatory signal transduction domain is a CD137 (4-1BB) co-stimulatory signal transduction domain or a CD28 co-stimulatory signal transduction domain.

33. The dual CAR system of any one of claims 29-32, wherein the first transmembrane domain and / or the second transmembrane domain is derived from the transmembrane domain of: the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154.

34. The dual CAR system of any one of claims 29-33, wherein the first transmembrane domain is a transmembrane domain derived from CD28, and the second transmembrane domain is a transmembrane domain derived from CD8α.

35. The dual CAR system of any one of claims 29-34, wherein the first transmembrane domain is a transmembrane domain derived from CD8α, and the second transmembrane domain is a transmembrane domain derived from CD28.

36. The dual CAR system of any one of claims 29-35, wherein the dual CAR system further comprises a first hinge region located between the C-terminus of the first extracellular antigen-binding domain and the N-terminus of the first transmembrane domain; and / or a second hinge region located between the C-terminus of the second extracellular antigen-binding domain and the N-terminus of the second transmembrane domain.

37. The dual CAR system of any one of claims 29-36, wherein the first hinge region and / or the second hinge region are derived from the following hinge regions: CD8α, CD28, IgG1, IgG2, IgG3 or IgG4.

38. The dual CAR system of any one of claims 29-37, wherein (1) the first engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3... The first engineered receptor comprises, from the N-terminus to the C-terminus, a first extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

39. The dual CAR system of any one of claims 29-38, wherein (1) the first engineered receptor comprises, from the N-terminus to the C-terminus: a first extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus: an extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; (2) the first engineered receptor comprises, from the N-terminus to the C-terminus: a first extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ. The second engineered receptor comprises, from the N-terminus to the C-terminus, an intracellular signal transduction domain of γ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; or (3) the first engineered receptor comprises, from the N-terminus to the C-terminus, a first extracellular antigen-binding domain that binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain that binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain.

40. The dual CAR system of any one of claims 29-39, wherein (1) the first engineered receptor comprises, from the N-terminus to the C-terminus, a first extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; (2) the first engineered receptor comprises, from the N-terminus to the C-terminus, a first extracellular antigen-binding domain; I The first engineered receptor comprises, from its N-terminus to its C-terminus: a gG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from its N-terminus to its C-terminus: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; (3) the first engineered receptor comprises, from its N-terminus to its C-terminus: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second ... The first engineered receptor, from its N-terminus to its C-terminus, comprises: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; (4) the first engineered receptor, from its N-terminus to its C-terminus, comprises: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor, from its N-terminus to its C-terminus, comprises: an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; (5) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signal transduction domain; and an intracellular signal transduction domain derived from FcRγ; and the second engineered receptor comprises, from the N-terminus to the C-terminus, the following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signal transduction domain; and a CD3ζ primary intracellular signal transduction domain; or (6) The first engineered receptor comprises, from the N-terminus to the C-terminus, the following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain;The second engineered receptor comprises, from its N-terminus to its C-terminus, an extracellular antigen-binding domain; an IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain; and a CD3ζ primary intracellular signaling domain.

41. The dual CAR system of any one of claims 29-40, wherein the first engineered receptor and the second engineered receptor are connected via a connector.

42. The dual CAR system according to any one of claims 29-41, wherein the first extracellular antigen-binding domain and / or the second extracellular antigen-binding domain each bind to a tumor antigen.

43. The dual CAR system of claim 42, wherein the tumor antigen is selected from the group consisting of: B cell maturation antigen (BCMA), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain-B, folate receptor 1, GD2, GD3 gangliosides, glycoprotein 75, GPNMB, HER2 / neu, HGF, human dispersive factor receptor kinase, IGF-1 receptor, IGF-1, IgG1, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-hydroxyacetylneuraminic acid, NPC-1C, PDGF-RA, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tendinin C, TGFβ2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin.

44. The dual CAR system of claim 43, wherein the first extracellular antigen-binding domain binds to CD19.

45. The dual CAR system of claim 44, wherein the first extracellular antigen-binding domain comprises: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences are respectively in SEQ ID NO: As shown in SEQ ID NO: 51, 52, 53, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 54, 55, 56, respectively.

46. ​​The dual CAR system of claim 45, wherein the first extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising an amino acid sequence that is at least 80% identical to the selected VH sequence, the light chain variable region comprising an amino acid sequence that is at least 80% identical to the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO:

58.

47. The dual CAR system of claim 46, wherein the first extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

7.

48. The dual CAR system according to any one of claims 43-47, wherein the second extracellular antigen-binding domain binds to CD20.

49. The dual CAR system of claim 48, wherein the second extracellular antigen-binding domain comprises V H H antibody portion, the V H The H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains an amino acid sequence that is at least 80% identical to the selected CDR1 amino acid sequence, CDR2 contains an amino acid sequence that is at least 80% identical to the selected CDR2 amino acid sequence, and CDR3 contains an amino acid sequence that is at least 80% identical to the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO:

50.

50. The dual CAR system of claim 49, wherein the V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

51. The dual CAR system according to any one of claims 44-50, wherein (1) the first engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 36; and the second engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 24; or (2) the first engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 68; and the second engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

24.

52. The dual CAR system according to any one of claims 44-51, wherein the dual CAR system comprises the amino acid sequence shown in SEQ ID NO: 19 or 60, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 19 or 60.

53. The dual CAR system of claim 43, wherein the first extracellular antigen-binding domain binds to CD20.

54. The dual CAR system of claim 53, wherein the first extracellular antigen-binding domain comprises V H H antibody portion, the V H The H antibody portion includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains an amino acid sequence that is at least 80% identical to the selected CDR1 amino acid sequence, CDR2 contains an amino acid sequence that is at least 80% identical to the selected CDR2 amino acid sequence, and CDR3 contains an amino acid sequence that is at least 80% identical to the selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 48, the selected CDR2 amino acid sequence contains the amino acid sequence shown in SEQ ID NO: 49, and the selected CDR3 amino acid sequence contains the amino acid sequence shown in SEQ ID NO:

50.

55. The dual CAR system of claim 54, wherein the V H The H antibody portion contains the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

56. The dual CAR system of any one of claims 53-55, wherein the second extracellular antigen-binding domain binds to CD19.

57. The dual CAR system of claim 56, wherein the second extracellular antigen-binding domain comprises: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences are respectively in SEQ ID NO: As shown in SEQ ID NO: 51, 52, 53, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO: 54, 55, 56, respectively.

58. The dual CAR system of claim 57, wherein the second extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the selected VH sequence, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57 and the selected VL sequence is SEQ ID NO:

58.

59. The dual CAR system of claim 58, wherein the second extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

7.

60. The dual CAR system according to any one of claims 53-59, wherein (1) the first engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 69; and the second engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 47; (2) the first engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 70, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 70; and the second engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 47; (3) the first engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 71; The amino acid sequence shown in SEQ ID NO: 71 has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 72; (4) The first engineered receptor contains the amino acid sequence shown in SEQ ID NO: 71, or has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 71; and the second engineered receptor contains the amino acid sequence shown in SEQ ID NO: 73, or has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 73; (5) The first engineered receptor contains the amino acid sequence shown in SEQ ID NO: 74, or has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 74; and the second engineered receptor contains the amino acid sequence shown in SEQ ID NO: 75, or has at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 74; The amino acid sequence shown in SEQ ID NO: 74 has at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 74; or (6) the first engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence shown in SEQ ID NO: 74.Furthermore, the second engineered receptor comprises the amino acid sequence shown in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

76.

61. The dual CAR system according to any one of claims 53-60, wherein the dual CAR system comprises an amino acid sequence shown in any one of SEQ ID NO: 61-66, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 61-66.

62. The dual CAR system of any one of claims 29-61, wherein the first engineered receptor and / or the second engineered receptor further comprise a signal peptide.

63. The dual CAR system of claim 62, wherein the signal peptide is located at the N-terminus of the first engineered receptor and / or the N-terminus of the second engineered receptor.

64. The dual CAR system of claim 62 or 63, wherein the signal peptide is derived from CD8α, and wherein optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO:

8.

65. A nucleic acid comprising a nucleotide sequence encoding an engineered receptor as described in any one of claims 1-28 or a dual CAR system as described in any one of claims 29-64.

66. A cell comprising an engineered receptor as described in any one of claims 1-28, a dual CAR system as described in any one of claims 29-64, or a nucleic acid as described in claim 65.

67. The cell of claim 66, wherein the cell is an immune cell.

68. The cell of claim 67, wherein the immune cell is selected from the group consisting of: T cells, NK cells, NKT cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, macrophages, monocytes, neutrophils, eosinophils, and combinations thereof.

69. The cell of claim 68, wherein the T cell is an αβ T cell, a γδ T cell, or a panT cell.

70. A modified γδT cell, the modified γδT cell comprising: a first engineered receptor, the first engineered receptor comprising: a first extracellular antigen-binding domain binding to CD19; a first transmembrane domain; and a first intracellular domain; and a second engineered receptor, the second engineered receptor comprising: a second extracellular antigen-binding domain binding to CD20; a second transmembrane domain; and a second intracellular domain.

71. The modified γδT cells of claim 70, wherein the first extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7, and the second extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

72. A modified αβT cell, the modified αβT cell comprising: a first engineered receptor, the first engineered receptor comprising: a first extracellular antigen-binding domain binding to CD20; a first transmembrane domain; and a first intracellular domain; and a second engineered receptor, the second engineered receptor comprising: a second extracellular antigen-binding domain binding to CD19; a second transmembrane domain; and a second intracellular domain.

73. The modified αβT cells of claim 72, wherein the first extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1, and the second extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

7.

74. A pharmaceutical composition comprising cells as described in any one of claims 66-73, and a pharmaceutically acceptable carrier.

75. A method for treating and / or preventing cancer, the method comprising administering to a subject in need an effective amount of cells as described in any one of claims 66-73 or a pharmaceutical composition as described in claim 74.

76. A method for treating and / or preventing autoimmune diseases, the method comprising administering to a subject in need an effective amount of cells as described in any one of claims 66-73 or a pharmaceutical composition as described in claim 74.

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