WO2025067205A1

WO2025067205A1 - Engineered receptors and methods of use thereof

Info

Publication number: WO2025067205A1
Application number: PCT/CN2024/120958
Authority: WO
Inventors: Qing Dai; Qingling JIANG; Yafeng Zhang
Original assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Current assignee: Nanjing Legend Biotechnology Co Ltd; Legend Biotech Ireland Ltd
Priority date: 2023-09-25
Filing date: 2024-09-25
Publication date: 2025-04-03
Anticipated expiration: 2026-03-25

Abstract

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

Description

ENGINEERED RECEPTORS AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

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

SEQUENCE STATEMENT

The content of the following submission on XML file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: IEC240638PCT_Sequence Listing, date recorded: September 23, 2024, size: 133KB) .

TECHNICAL FIELD

This disclosure relates to engineered receptors (e.g., CARs) , dual-CAR systems, engineered immune effector cells comprising the same, and methods of use thereof.

BACKGROUND

The chimeric antigen receptor (CAR) -based therapies are a rapidly emerging form of cancer treatment, and have resulted in remarkable responses in diseases such as refractory lymphoid malignancies. However, their clinical use is limited by toxicity related to cytokine release syndrome and neurotoxicity, the logistic complexity of their manufacturing, cost and time-to-treatment, and the risk of graft-versus-host disease (GvHD) associated with the immune cell therapy. Therefore, there exists a need for CAR designs with improved efficacy and safety for clinical uses.

SUMMARY

The present 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 domain selected from a CD28 co-stimulatory signaling domain, a Dap10 intracellular signaling domain, an OX40 intracellular signaling domain, a CD27 intracellular signaling domain, a CD137 (4-1BB) co-stimulatory signaling domain and an ICOS intracellular signaling domain.

In some embodiments, the additional signaling domain is CD28 co-stimulatory signaling domain.

In some embodiments, the CD28 co-stimulatory signaling domain is located at the N-terminus of the intracellular signaling domain derived from FcRγ.

In some embodiments, the additional signaling domain is CD137 (4-1BB) co-stimulatory signaling domain.

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

In some embodiments, the transmembrane domain is a transmembrane domain derived from: α chain of a T cell receptor, β chain of the T cell receptor, 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.

In some embodiments, 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.

In some embodiments, the hinge region is a hinge region derived from: CD8α, CD28, IgG1, IgG2, IgG3, or IgG4.

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

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

In some embodiments, the engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8αtransmembrane domain; a CD28 co-stimulatory signaling domain, and an intracellular signaling domain derived from FcRγ.

In some embodiments, the engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8αtransmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and an intracellular signaling domain derived from FcRγ.

In some embodiments, the extracellular antigen-binding domain comprises a sdAb, scFv, a (scFv) ₂, a V_HH domain, or a VNAR domain.

In some embodiments, the extracellular antigen-binding domain binds to a tumor antigen.

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-l, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP) , BAFF, B-lymphoma cell, 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 extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-l receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-l, VEGFR2, and vimentin.

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

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 comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.

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

In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 7.

In some embodiments, the engineered receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33, 36-43 and 68 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 33, 36-43 and 68.

In some embodiments, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 36.

In some embodiments, the engineered receptor further comprises a signal peptide. In some embodiments, the engineered receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 78-90, 94, 95, 97 and 98 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 78-90, 94, 95, 97 and 98.

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

In some embodiments, the extracellular antigen-binding domain comprises a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

In some embodiments, the extracellular antigen-binding domain comprises a V_HH antibody moiety comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the engineered receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 69-71, and 74 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 69-71, and 74.

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, optionally the signal peptide comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 91-93 and 96 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 91-93 and 96.

The present disclosure also relates to 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 signaling domain derived from FcRγ; and a CD28 co-stimulatory signaling domain or a CD137 (4-1BB) co-stimulatory signaling domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

In some embodiments, the second intracellular domain comprises a CD3ζ primary intracellular signaling domain.

In some embodiments, the second engineered receptor further comprises a co-stimulatory signaling domain located between the C-terminus of the transmembrane domain and the N-terminus of the CD3ζ primary intracellular signaling domain.

In some embodiments, the co-stimulatory signaling domain is CD137 (4-1BB) co-stimulatory signaling domain or CD28 co-stimulatory signaling domain.

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

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.

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.

In some embodiments, 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.

In some embodiments, the first and/or second hinge region is a hinge region derived from: CD8α, CD28, IgG1, IgG2, IgG3, or IgG4.

In some embodiments, the dual-CAR system is specified as one of the following:

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

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

In some embodiments, the dual-CAR system is specified as one of the following:

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

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

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

In some embodiments, the dual-CAR system is specified as one of the following:

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

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

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

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

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

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

In some embodiments, the first engineered receptor and the second engineered receptor is connected via a linker.

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

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

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 a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.

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

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

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

In some embodiments, the second extracellular antigen-binding domain comprises a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

In some embodiments, the V_HH antibody moiety comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the dual-CAR system is specified as one of the following:

(1) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 36; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 24;

(2) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 68 , or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 68; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 24.

In some embodiments, the first engineered receptor and/or the second engineered receptor further comprise 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, optionally the signal peptide comprises the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the dual-CAR system comprises an amino acid sequence set forth in SEQ ID NO: 19 or 60, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 19 or 60.

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

In some embodiments, the first extracellular antigen-binding domain comprises a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

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

In some embodiments, 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 a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.

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

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

In some embodiments, the dual-CAR system is specified as one of the following:

(1) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 69; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 47 ;

(2) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 70, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 70; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 47;

(3) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 71; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 72;

(4) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 71; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 73;

(5) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 74; the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 75;

(6) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 74; or the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 76.

In some embodiments, the dual-CAR system comprises an amino acid sequence set forth in any one of SEQ ID NOs: 61-66, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 61-66.

The present disclosure is also related to nucleic acids comprising a nucleotide sequence encoding any one of the engineered receptor described herein, or any one of the dual-CAR systems described herein.

The present disclosure is also related to cells comprising any one of the engineered receptor described herein, any one of the dual-CAR systems described herein, or any one of the nucleic acids described herein.

In some embodiments, the cell is an immune cell.

In some embodiments, the immune cell is selected from the group consisting of T cell, NK cell, NKT, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, a macrophage, a monocyte, a neutrophil, an eosinophil and a combination thereof.

In some embodiments, the T cell is a αβT cell, γδT cell or panT cell.

The present 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.

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

The present 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.

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

The present disclosure also relates to pharmaceutical compositions comprising any one of the cells described herein, and a pharmaceutical acceptable carrier.

The present disclosure also relates to methods for treating and/or preventing cancer comprising administering an effective amount of any one of the cells described herein or any one of the pharmaceutical compositions described herein to a subject in need thereof.

The present disclosure also relates to methods for treating and/or preventing autoimmune disease comprising administering an effective amount of any one of the cells described herein or any one of the pharmaceutical compositions described herein to a subject in need thereof.

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

As used herein, the term “DAP10 intracellular signaling domain” refers to the intracellular signaling domain derived from DAP10 or a functional variant thereof. The DAP10 intracellular signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The DAP10 intracellular signaling domain may comprise a sequence of SEQ ID NO: 20 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 20.

As used herein, the term “DAP12 intracellular signaling domain” refers to the intracellular signaling domain derived from DAP12 or a functional variant thereof. The DAP12 intracellular signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The DAP12 intracellular signaling domain may comprise a sequence of SEQ ID NO: 17 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 17.

As used herein, the term “OX40 intracellular signaling domain” refers to the intracellular signaling domain derived from OX40 or a functional variant thereof. The OX40 intracellular signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The OX40 intracellular signaling domain may comprise a sequence of SEQ ID NO: 21 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 21.

As used herein, the term “CD27 intracellular signaling domain” refers to the intracellular signaling domain derived from CD27 or a functional variant thereof. The CD27 intracellular signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD27 intracellular signaling domain may comprise a sequence of SEQ ID NO: 22 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 22.

As used herein, the term “ICOS intracellular signaling domain” refers to the intracellular signaling domain derived from ICOS or a functional variant thereof. The ICOS intracellular signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The ICOS intracellular signaling domain may comprise a sequence of SEQ ID NO: 23 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 23.

As used herein, the term “FcRγ intracellular signaling domain” refers to the intracellular signaling domain derived from FcRγ or a functional variant thereof. The intracellular signaling domain derived from FcRγ can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The intracellular signaling domain derived from FcRγ may comprise a sequence of SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16.

As used herein, the term “co-stimulatory signaling domain” refers to the portion of the receptor that includes one or more domains (e.g., endodomains) from co-stimulatory proteins for the receptor to persist after activation. The co-stimulatory signaling domain can be derived from the endodomains (cytoplasmic domains) of a wild-type co-stimulatory protein or a functional variant thereof. The co-stimulatory signaling domain may have one or more mutations, including e.g., insertions, deletions, and/or substitutions.

As used herein, the term “CD137 (4-1BB) co-stimulatory signaling domain” refers to the cytoplasmic domains derived from CD137 (4-1BB) or a functional variant thereof. The CD137 (4-1BB) co-stimulatory signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD137 (4-1BB) co-stimulatory signaling domain may comprise a sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13.

As used herein, the term “CD28 co-stimulatory signaling domain” refers to the cytoplasmic domains derived from CD28 or a functional variant thereof. The CD28 co-stimulatory signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD28 co-stimulatory signaling domain may comprise a sequence of SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14.

As used herein, the term “primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary intracellular signaling domain may contain a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM. An “ITAM, ” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif can comprise two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix (6-8) YxxL/I. ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs can also function as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcR gamma (FCER1G) , FcR beta (Fc Epsilon Rib) , CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. The primary intracellular signaling domain may derive from CD3ζ. The intracellular signaling domain may consist of a CD3ζ cytoplasmic signaling domain. The primary intracellular signaling domain may derive from a cytoplasmic signaling domain of wildtype CD3ζ. The primary intracellular signaling domain may be a functional mutant of the cytoplasmic signaling domain of CD3ζ containing one or more mutations, such as Q65K.

As used herein, the term “CD3ζ primary intracellular signaling domain” refers to the cytoplasmic signaling domains derived from CD3ζ or a functional variant thereof. The CD3ζprimary intracellular signaling domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD3ζ primary intracellular signaling domain may comprise a sequence of SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15.

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

As used herein, the term “CD8α transmembrane domain” refers to the transmembrane domain derived from CD8α or a functional variant thereof. The CD8α transmembrane domain can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD8αtransmembrane domain may comprise a sequence of SEQ ID NO: 11 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11.

As used herein, the term “CD28 transmembrane domain” refers to the transmembrane domain derived from CD28 or a functional variant thereof. The CD28 transmembrane domain may have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD28 transmembrane domain can comprise a sequence of SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12.

As used herein, the terms “hinge domain” or “hinge region” are used interchangeably herein to refer to the portion of a membrane protein (e.g., a receptor) that connects the transmarine region and the extracellular domain. The hinge region can be part of an extracellular region. The hinge region can 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 e.g., insertions, deletions, and/or substitutions.

As used herein, the term “CD8α hinge region” refers to the hinge region derived from CD8α or a functional variant thereof. The CD8α hinge region can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD8α hinge region may comprise a sequence of SEQ ID NO: 9 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 9.

As used herein, the term “CD28 hinge region” refers to the hinge region derived from CD28 or a functional variant thereof. The CD28 hinge region can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The CD28 hinge region may comprise a sequence of SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10.

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 to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

As used herein, the term “chimeric antigen receptor” or “CAR” as used herein refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells. Some CARs are also known as “artificial T-cell receptors, ” “chimeric T cell receptors, ” or “chimeric immune receptors. ” The CAR may comprise an extracellular ligand binding domain or an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens) , a transmembrane domain, and an intracellular signaling domain. “CAR-T cell” refers to a T cell that expresses a CAR. “CAR-NK cell” refers to an NK cell that expresses a CAR.

As used herein, the term “cancer” or “cancer cell” refers to the cells dividing in an uncontrolled manner, e.g., forming the solid tumors or the excessive tumor cells in blood. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The cancer cells can form the solid tumors or the excessive tumor cells in blood (e.g., hematologic cancer) . Alternatively or additionally, it can include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon) , genitourinary tract (e.g., renal, urothelial cells) , prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. Examples of cancers that can be treated by the methods described herein include e.g., bone cancer, pancreatic cancer, skin cancer (e.g., melanoma) , cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia (AML) , chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, and/or T cell lymphoma.

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

As used herein, the term “antigen binding domain” or “extracellular antigen binding domain” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. An antigen binding fragment may comprise at least one variable domain (e.g., a variable domain of a heavy chain, single domain antibody or V_HH) . Non-limiting examples of antibody fragments include, e.g., VH-VL pair, scFv, Fab, Fab’, F (ab’) ₂, and Fv fragments.

“Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connotate or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta sequence/structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connotate or include a limitation to a particular process of producing the intracellular signaling domain, for example, it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain. A domain derived from a particular protein may have a sequence that is 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 to the relevant functional portion of the particular protein.

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 definitions in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) , Chothia numbering system (Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) , AbM numbering system (Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag) or IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) . For a given antibody, those skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, 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 CDRs of the antibodies of the disclosure may be defined according to Kabat, AbM, IMGT, or Chothia numbering system, or any combination thereof. Unless otherwise indicated or clear from the context, the CDRs of the antibodies of the disclosure are preferably defined according to AbM numbering system.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided. Veterinary and non-veterinary applications are contemplated by the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

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 belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of CD20 CAR constructs.

FIG. 2 is a schematic representation of CD19 CAR constructs.

FIG. 3 is a schematic representation of CD20/CD19 dual CAR constructs.

FIG. 4 is a graph showing the expression of CD20 and CD19 in tumor cell lines.

FIG. 5A-5C are graphs demonstrating the cytotoxicity of cytokine release of CD20 CAR-T cells in co-culture with CD20 positive Raji cells.

FIG. 6A-6C are graphs demonstrating CD20 sdAb1 CAR-T cells have better anti-tumor efficacy and proliferation than other CD20 CAR-T cells in Raji xenograft model, with no significant change in body weight comparing to cohorts treated with UnT.

FIG. 7A-7B are graphs demonstrating the cytotoxicity of cytokine release of CD19 CAR-T cells in co-culture with CD19 positive Raji cells.

FIG. 8 is a graph demonstrating the cytotoxicity of FcRγ bearing CD19 CAR-T cells with different CAR components.

FIG. 9A-9C are graphs demonstrating FcRγ bearing CD19 CAR-T cells have greater proliferation with lower cytokine release comparing to CD3ζ and Dap12 bearing CD19 CAR-T cells.

FIG. 10 is a graph demonstrating CD19 CAR-T cells bearing CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain, and FcRγ cytoplasmic domain exhibited greater proliferation than CD19 CAR-T cells with other modalities.

FIG. 11A-11F are graphs demonstrating the anti-tumor activity of CD20/CD19 dual CAR-γδT cells against CD20 and/or CD19 positive tumor cells.

FIG. 12A-12F are graphs demonstrating CD20/CD19 dual CAR-γδT cells induced no cytotoxicity against CD20/CD19 double negative cells.

FIG. 13A-13B are graphs demonstrating CD20/CD19 dual CAR-T cells are superior to single targeting CAR-T counterparts in durability and long-term proliferation.

FIGs. 14A-14D are graphs demonstrating CD20/CD19 dual CAR-T cells have superior efficacy and proliferation to CD20 single CAR counterpart in γδT cells from two individual donors.

FIGs. 15A-15B are graphs demonstrating CD20/CD19 dual CAR has better anti-tumor efficacy than CD20 single CAR and CD19 single CAR in vivo.

FIGs. 16A-16D are graphs demonstrating CD20/CD19 dual CAR has better durability and proliferation than tandem CAR in γδT cells.

FIGs. 17A-17B are graphs demonstrating CD20/CD19 dual CAR has better durability and proliferation than tandem CAR in αβT cells.

FIGs. 18 is a schematic representation of CD20/CD19 dual CAR constructs with different modalities.

FIGs. 19A-19F are graphs demonstrating the anti-tumor activity of CD20/CD19 dual CAR constructs with different modalities against CD20 and/or CD19 positive tumor cells.

FIGs. 20A-20B are graphs demonstrating the durable killing and great proliferation of CD20/CD19 dual CAR constructs with different modalities.

FIG. 21 shows the percentage of B cells in PBMCs before co-culture with CAR-T cells.

FIG. 22 shows the percentage of B cells in PBMCs after co-culture with CAR-T cells at different ratios for 72 hours.

FIG. 23 shows the INF-γ release of CAR-T cells in co-culture with PBMCs.

FIG. 24 shows the percentage of B cells in PBMCs after co-culture with CAR-T cells at the ratio of 1: 50 for 72 hours.

FIG. 25 shows selected sequences listed in the present disclosure.

DETAILED DESCRIPTION

The present disclosure relates to engineered receptors (e.g., CARs) and immune cells (e.g., CAR T cells) that express the engineered receptors. Specifically, the immune cells expressing the engineered receptors can be used in immune cell therapies for treating cancer.

Chimeric Antigen Receptors (CARs)

A chimeric antigen receptor (CAR) typically comprises an extracellular domain capable of binding to an antigen, and an intracellular domain comprising one or more intracellular signaling domains derived from signal transducing proteins. These intracellular signaling domains are typically different from the polypeptide from which the extracellular domain is derived. The extracellular domain can be any proteinaceous molecule or part thereof that can specifically bind to a predetermined antigen. The extracellular domain may comprise an antibody or antigen binding fragment thereof. The intracellular signaling domain may be any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell.

Chimeric antigen receptors (CARs) combine many facets of normal T cell or NK cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell or NK cell when an antigen is bound. CARs typically have the following regions: an antigen binding domain, an extracellular hinge region, a transmembrane domain, and an intracellular domain. The intracellular region comprises an intracellular signaling domain or an intracellular signaling region.

A detailed review of CARs and CAR-expressing cells can be found in, e.g., J Exp Clin Cancer Res, 2022 Mar 31; 41 (1) : 119; Mol Cancer, 2023 Jan 30; 22 (1) : 20; and Br J Haematol, 2021 Apr; 193 (2) : 216-230. doi: 10.1111/bjh. 17186. Epub 2020 Nov 20, each of which is incorporated by reference in its entirety. Exemplary structure of antigen receptors, including the hinge, the transmembrane domain, and the intracellular cell signaling domain, and methods for engineering and introducing such receptors 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 publication No. WO2017173256A1; US2002/131960, US2013/287748, US2013/0149337, U.S. 6,451,995, U.S. 7,446,190, U.S. 8,252,592; each of which is incorporated herein by reference in its entirety.

In one aspect, provided herein are 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 domain selected from a CD28 co-stimulatory signaling domain, a Dap10 intracellular signaling domain, an OX40 intracellular signaling domain, a CD27 intracellular signaling domain, a CD137 (4-1BB) co-stimulatory signaling domain and an ICOS intracellular signaling domain.

In one aspect, provided herein are 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 signaling domain derived from FcRγ; and a CD28 co-stimulatory signaling domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

In one aspect, provided herein are 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 signaling domain derived from FcRγ; and a CD137 (4-1BB) co-stimulatory signaling domain; and a second engineered receptor comprising: a second extracellular antigen-binding domain; a second transmembrane domain; and a second intracellular domain.

The intracellular domain may comprise an intracellular signaling domain derived from FcRγ or a functional variant thereof. The intracellular signaling domain derived from FcRγ can have one or more mutations, including e.g., insertions, deletions, and/or substitutions. The Fc receptor common gamma-chain (FcRgamma, or FcRγ) is a widely expressed adaptor bearing an immunoreceptor tyrosine-based activation motif (ITAM) that transduces activation signals from various immunoreceptors. Through its transmembrane portion, FcRγ associated constitutively with the common beta-chain of the IL-3 receptor and signaled by recruiting the kinase Syk. Retrovirus-mediated complementation demonstrated the essential function of the ITAM of FcRγin IL-3 signal transduction. FcRγ functions to route selective cytokine-triggered signals into the ITAM-mediated IL-4 production pathway. The cytoplasmic domain of FcRγ is shown in SEQ ID NO: 16. The intracellular signaling domain of FcRγ may comprise an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 16.

The intracellular domain may comprise one or more additional intracellular signaling domain (s) . The intracellular signaling domain may generate a signal that promotes an immune effector function of the CAR-containing cell, e.g., a CAR-T cell. “Immune effector function or immune effector response” refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response can refer to a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity and helper activity (such as the secretion of cytokines) . The intracellular signaling domain may generate a signal that promotes proliferation and/or survival of the CAR containing cell. The signaling domain of a naturally occurring molecule can comprise the entire intracellular or cytoplasmic portion, or the entire native intracellular signaling domain, of the molecule, or a fragment or derivative thereof. The intracellular signaling domain may be, but are not limited to, a Dap10 intracellular signaling domain, a Dap12 intracellular signaling domain, an OX40 intracellular signaling domain, a CD27 intracellular signaling domain, and/or an ICOS (CD278) intracellular signaling domain.

The intracellular domain may have a Dap10 intracellular signaling domain. The Dap10 intracellular signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 20.

The intracellular domain may have a Dap12 intracellular signaling domain. The Dap12 intracellular signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 17.

The intracellular domain may have a OX40 intracellular signaling domain. The OX40 intracellular signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 21.

The intracellular domain may have a CD27 intracellular signaling domain. The CD27 intracellular signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 22.

The intracellular domain may have a ICOS (CD278) intracellular signaling domain. The ICOS intracellular signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 23.

The intracellular signaling domain of a CAR may comprise a primary intracellular signaling domain. Primary intracellular signaling domain contains cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary intracellular signaling domain may contain a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM.

The intracellular domain may have a CD3ζ primary intracellular signaling domain. The CD3ζ primary intracellular signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 15.

The intracellular domain may comprise one or more co-stimulatory signaling domains. A co-stimulatory signaling domain can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. A co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) , and 4-1BB (CD137) . Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, 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, LFA-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 a ligand that specifically binds with CD83.

Upon binding of the antigen binding domain to tumor antigen, the co-stimulatory signaling domains of the CAR can transduce signals for enhanced proliferation, survival and differentiation of the modified immune cells having the CAR (such as T cells) , and inhibit activation induced cell death.

The intracellular domain may comprise a co-stimulatory signaling domain derived from CD28. The CD28 co-stimulatory signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 14.

The intracellular domain may comprise a co-stimulatory signaling domain derived from CD28 and an intracellular signaling domain derived from FcRγ. The CD28 co-stimulatory signaling domain may be located at the N-terminus of the intracellular signaling domain derived from FcRγ. The CD28 co-stimulatory signaling domain may be located at the C-terminus of the intracellular signaling domain derived from FcRγ.

The intracellular domain may comprise a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137) . The 4-1BB co-stimulatory signaling domain may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 13.

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

The antigen-binding domain (e.g., extracellular antigen-binding domain) of the engineered receptor (e.g., CAR) may be an antibody or an antibody fragment, such as an scFv, a Fv, a Fab, a (Fab′) ₂, a single domain antibody (sdAb) , or a V_HH domain. The antigen-binding domain may comprise a ligand or an extracellular portion of a receptor that specifically binds to a tumor antigen. The engineered receptor (e.g., CAR) may be a monospecific, bispecific or multispecific receptor. The antigen binding domain may specifically bind a single tumor antigen. The antigen binding domain may bind two or more tumor antigens.

The tumor antigen may be selected from the group consisting of tumor antigen 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-l, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP) , BAFF, B-lymphoma cell, 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 extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-l receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-l, VEGFR2, and vimentin, and other tumor antigens with clinical significance, and combinations thereof. The tumor antigen may be derived from an intracellular protein of tumor cells. The tumor antigen may be expressed on the surface of tumor cells.

The extracellular antigen-binding domain may 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.

The extracellular antigen-binding domain may 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 have a second extracellular antigen-binding domain that binds to CD19.

In one aspect, provided herein are chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD19; a transmembrane region; and one or more of the following: (1) an intracellular domain comprising an intracellular signaling domain derived from FcRγ, a Dap12 intracellular signaling domain, a Dap10 intracellular signaling domain, an OX40 intracellular signaling domain, a CD27 intracellular signaling domain or an ICOS intracellular signaling domain; (2) a CD28 co-stimulatory signaling domain or a CD137 (4-1BB) co-stimulatory signaling domain; and (3) an activation domain comprising a CD3ζprimary intracellular signaling domain.

In one aspect, provided herein are chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD20; a transmembrane region; and one or more of the following: (1) an intracellular domain comprising an intracellular signaling domain derived from FcRγ, a Dap12 intracellular signaling domain, a Dap10 intracellular signaling domain, an OX40 intracellular signaling domain, a CD27 intracellular signaling domain or an ICOS intracellular signaling domain; (2) a CD28 co-stimulatory signaling domain or a CD137 (4-1BB) co-stimulatory signaling domain; and (3) an activation domain comprising a CD3ζprimary intracellular signaling domain.

In one aspect, provided herein are chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD19; a transmembrane region; and an intracellular domain comprising an intracellular signaling domain derived from FcRγ.

In one aspect, provided herein are chimeric antigen receptors (CARs) comprising: an extracellular antigen-binding domain that binds to CD20; a transmembrane region; and an intracellular domain comprising an intracellular signaling domain derived from FcRγ.

The extracellular antigen-binding domain that binds to CD19 can be or include the CD19 binding fragment (e.g., FMC63, SJ25C1, or those disclosed in different patents such as WO 2022/012683, etc) . The antigen-binding domain that binds to CD19 may comprise 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 a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.

The antigen-binding domain that binds to CD19 may comprise a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58.

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

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

The antigen-binding domain that binds to CD20 may comprise a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.

The antigen-binding domain that binds to CD20 may comprise a V_HH antibody moiety that comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.

The transmembrane domain of a CAR may comprise a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRT AM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CDIOO (SEMA4D) , SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. The transmembrane domain of the CAR may be derived from a α chain of a T cell receptor, β chain of the T cell receptor, 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 domain.

The transmembrane domain of the CAR may comprise a transmembrane domain of CD8α. The CD8α transmembrane domain may have an amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 11.

The transmembrane domain of the CAR may comprise a transmembrane domain of CD28. The CD28 transmembrane domain may have an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 12.

The extracellular antigen-binding domain may be connected to the transmembrane domain by a hinge region. The hinge region may be derived from CD8α, CD28, IgG1, IgG2, IgG3, or IgG4. The hinge region may comprise a hinge region of CD8α. The CD8α hinge region may have an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 9. The hinge region may comprise a hinge region of CD28. The CD28 hinge region may have an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 10. The hinge region may comprise a hinge region of IgG4. The IgG4 hinge region may have an amino acid sequence set forth in SEQ ID NO: 67 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 67.

The CAR may comprise an intracellular signaling domain derived from FcRγ and a CD28 co-stimulatory signaling domain. The CAR may comprise an intracellular signaling domain derived from FcRγ and a CD137 (4-1BB) co-stimulatory signaling domain. The intracellular signaling domain derived from FcRγ may be located at the C-terminus of the one or more additional signaling domain.

The CAR may comprise a signal peptide (SP) , such as a CD8α signal peptide. The CD8α signal peptide may have an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 8.

The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 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 a sequence set forth in any one of SEQ ID Nos: 33, 36-43 and 68. The CAR may specifically bind to CD19-positive tumor cells.

The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 a sequence set forth in any one of SEQ ID Nos: 69-71 and 74. The CAR may specifically bind to CD20-positive tumor cells.

The dual-CAR system may comprise an amino acid sequence set forth in 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 SEQ ID NO: 19 and 60-66. The dual-CAR system may specifically bind to CD19-positive, CD20-positive and/or CD19-and CD20-positive tumor cells.

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

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

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

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

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

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

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

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

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

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

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

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 24. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 77.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain 2F2 (e.g., SEQ ID NO: 3 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 3) that specifically binds to CD20, a CD8/CD8α hinge region (e.g., SEQ ID NO: 9 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 26.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain 3H7 (e.g., SEQ ID NO: 4 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 27.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 28.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a Dap12 intracellular signaling domain (e.g., SEQ ID NO: 17 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 17) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 29.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 30.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 31. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 78.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and a Dap12 intracellular signaling domain (e.g., SEQ ID NO: 17 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 17) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 32. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 79.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 sdAb (e.g., SEQ ID NO: 6 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 33. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 80.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 34.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD8/CD8αhinge region (e.g., SEQ ID NO: 9 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 35.

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

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

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

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

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

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

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

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

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD8/CD8αhinge region (e.g., SEQ ID NO: 9 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 47. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 89.

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

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 69. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 91.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 70. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 92.

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 71. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 93.

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

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

The engineered receptor (e.g., CAR) provided herein may comprise an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , an intracellular domain comprising an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 74. The engineered receptor (e.g., CAR) may further comprise a signal peptide. The engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 96.

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

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

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

The dual-CAR system may comprise a first engineered receptor (e.g., CAR) wherein the first engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 36. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 81. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) , wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and a CD3ζprimary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 24. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 77. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 19.

The dual-CAR system may comprise a first engineered receptor (e.g., CAR) wherein the first engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, an IgG4 hinge region (e.g., SEQ ID NO: 67 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 68. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 90. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) , wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain sdAb1 (e.g., SEQ ID NO: 1 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and a CD3ζprimary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 24. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 77. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 60.

The dual-CAR system may comprise 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 69. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 91. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD8/CD8α hinge region (e.g., SEQ ID NO: 9 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and a CD3ζprimary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 47. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 89. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 61.

The dual-CAR system may comprise 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 70. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 92. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD8/CD8α hinge region (e.g., SEQ ID NO: 9 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and a CD3ζprimary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 47. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 89. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 62.

The dual-CAR system may comprise 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 9) , a CD8/CD8αtransmembrane domain (e.g., SEQ ID NO: 11 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 71. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 93. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 72. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 94. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 63.

The dual-CAR system may comprise 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 9) , a CD8/CD8αtransmembrane domain (e.g., SEQ ID NO: 11 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 71. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 93. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, an IgG4 hinge region (e.g., SEQ ID NO: 67 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 73. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 95. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 64.

The dual-CAR system may comprise 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 9) , a CD8/CD8αtransmembrane domain (e.g., SEQ ID NO: 11 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , an intracellular domain comprising an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 74. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 96. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 75. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 97. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 65.

The dual-CAR system may comprise 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 9) , a CD8/CD8αtransmembrane domain (e.g., SEQ ID NO: 11 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , an intracellular domain comprising an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 13) and an intracellular signaling domain derived from FcRγ (SEQ ID NO: 16 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 16) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 74. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 96. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, an IgG4 hinge region (e.g., SEQ ID NO: 67 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 76. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 98. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 66.

Also provided herein is a dual-CAR system that comprises 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 a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 24. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 77. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and a Dap12 intracellular signaling domain (SEQ ID NO: 17 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 17) . The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 44.

Also provided herein is a dual-CAR system that comprises 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 a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , and an intracellular domain comprising a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 24. The first engineered receptor (e.g., CAR) may further comprise a signal peptide. The first engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 77. The dual-CAR system may comprise a second engineered receptor (e.g., CAR) wherein the second engineered receptor (e.g., CAR) comprises an extracellular antigen-binding domain CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) that specifically binds to CD19, a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 hinge region (e.g., SEQ ID NO: 10 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 10) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , and an intracellular domain comprising a CD28 co-stimulatory signaling domain (SEQ ID NO: 14 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 14) and a CD3ζ primary intracellular signaling domain (e.g., SEQ ID NO: 15 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 75. The second engineered receptor (e.g., CAR) may further comprise a signal peptide. The second engineered receptor (e.g., CAR) may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 97. The dual-CAR system may comprise an amino acid sequence set forth 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 set forth in SEQ ID NO: 59.

Also provided herein is a dual-CAR system that comprises, from the N-terminus a to the C-terminus, a tandem of a CD20-binding scFv, Leu16 (e.g., SEQ ID NO: 5 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 5) , a CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is 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 a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 11) , a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The dual-CAR system may comprise an amino acid sequence set forth in SEQ ID NO: 45, or an amino acid sequence that is at least 80%, 85%, 90%, 95%or 99%identical to the amino acid sequence set forth in SEQ ID NO: 45.

Also provided herein is a dual-CAR system that comprises, from the N-terminus a to the C-terminus, a tandem of a CD20-binding scFv, 2F2 (e.g., SEQ ID NO: 3 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 3) a CD19 scFv, FMC63 (e.g., SEQ ID NO: 7 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 7) , a IgG4 hinge region (e.g., SEQ ID NO: 67 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 67) , a CD28 transmembrane domain (e.g., SEQ ID NO: 12 or a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 12) , a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 13 or a sequence that is 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 a sequence that is at least 80%, 85%, 90%, or 95%identical to SEQ ID NO: 15) . The dual-CAR system may comprise an amino acid sequence set forth in SEQ ID NO: 46, or an amino acid sequence that is at least 80%, 85%, 90%, 95%or 99%identical to the amino acid sequence set forth in SEQ ID NO: 46.

Genetically Engineered Cells

The present disclosure provides cells (e.g., genetically engineered immune cells, T cells, NK cells, tumor-infiltrating lymphocytes) that express the engineered receptors (CARs) or the dual-CAR systems described herein. These engineered cells can be used to treat various disorders or disease as described herein (e.g., a cancer) .

In one aspect, provided herein are 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.

In one aspect, provided herein are 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.

The first and/or second engineered receptor can be any one of the engineered receptors (e.g., CARs) described herein.

The cell may be an immune cell. The immune cell may be selected from a group consisting of T cell, NK cell, NKT, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, a macrophage, a monocyte, a neutrophil, an eosinophil and a combination thereof.

The T cell may be a αβT cell or γδT cell. The modified γδT cells may be Vδ1 γδT cells. The modified γδT cells may be Vδ2 γδT cells. The modified γδT cells may be Vδ1-Vδ2-γδT cells. The modified γδT cells may include a polyclonal population of γδT cells including Vδ1, Vδ2 and Vδ1-Vδ2-γδT cells. The modified γδT cells may be panT cells.

The modified cells (e.g., modified γδT cells) may express any one of the engineered receptors (e.g., CARs) or the dual-CAR systems described herein.

The cell that is engineered may be obtained from, e.g., humans and non-human animals. The cell that is engineered may be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species. The cell may be from humans, rats or mice. The cells may be mouse lymphocytes and engineered (e.g., transduced) to express the engineered receptor (CAR) or the dual-CAR system described herein. The cell may be obtained from humans. The cell that is engineered may be a blood cell. The cell may be a CD8+ T cell, a CD4+ T cell, a memory T cell, a Treg cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a B cell, or a macrophage/monocyte.

The preparation of the engineered cells may include one or more culture and/or preparation steps. The cells for introduction of the binding molecule, e.g., CAR, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. The subject from which the cell is isolated may be one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject may be a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

The cell may be an NK cell. Human natural killer cells (NK cells) play an important role in innate immune defense against malignant lymphoma cells, and thus are suitable for adoptive immune therapy (i.e., adoptive cellular immunotherapy) . However, due to difficulties in ex vivo cell expansion and differences in activities of NK cells in individual patients, it is difficult to use the NK cells.

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 the need for prior sensitization and have the ability to interact with, and activate other immune cells. NK cells for 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, as well as cell lines.

NK cells activation and effector function is a complex process as it depends upon the integration of signals from two distinct 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 self-tolerance of NK cells. Cellular stress associated with viral infection or tumor development such as DNA damage, senescence or tumor suppressor genes upregulate ligands for activating receptors. This results in shift of balance to NK cells activation. Transmembrane and cytoplasmic stimulatory/activator molecules in NK cells can affect NK cell differentiation pathways, metabolic cycles, apoptosis as well as activation induced cell death.

The cell may be a T cell. The T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. The cell surface receptor can 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 that is associated with the target cell. T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients) , with a viral vector. The T cells can be CD4+ T cells, CD8+ T cells, or regulatory T cells. TheT cells can be T helper type 1 T cells and T helper type 2 T cells. The T cell expressing this receptor may be an αβT cell. The T cell expressing this receptor may be a γδT cell. The T cells may be central memory T cells. The T cells may be effector memory T cells. The T cells may be T cells.

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

Different cell types can be obtained from appropriate isolation methods. The isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. Any known method for separation based on such markers can be used. The separation can be affinity-or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. Negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

Also provided are populations of cells (e.g., genetically engineered cells) , compositions containing such cells and/or enriched for such cells, such as in which cells expressing the CAR or the dual-CAR system make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as NK cells, T cells, CD8+ or CD4+ cells.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, 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 the engineered receptor, e.g., CAR or the dual-CAR system, described herein) can be activated.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may have a cytotoxicity effect on tumor cells. The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may kill about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%or more tumor cells when contacted with the tumor cells. The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be contacted with tumor cells at a ratio of about 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.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be contacted with the tumor cells for about 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 more.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may induce the production of one or more cytokines after contacted with the target cells. The cytokine may be interferon γ (IFNγ) . The cytokine may be granulocyte-macrophage colony-stimulating factor (GM-CSF) . The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may induce less cytokine production than cells with the expression of other engineered receptor or the dual-CAR system. The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may induce more cytokine production than cells without the expression of the engineered receptor or the dual-CAR system.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may reduce or slow down tumor progression in a subject having cancer. The progression of the cancer may be evaluated every 1, 2, 3, 4, 5, 6, or 7 days. The progression of the cancer may be evaluated every 1, 2, 3, 4 weeks. The progression of the cancer may be evaluated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may retain therapeutic activity, efficacy and persistence after about 1, 2, 3, 4, 5, 6, or 7 days. The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may retain therapeutic activity, efficacy and persistence after about 1, 2, 3, 4 weeks. The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may retain therapeutic activity, efficacy and persistence after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. About 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be retained after about 1, 2, 3, 4, 5, 6, or 7 days, about 1, 2, 3, 4 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may have increased proliferation capability compared to cells that do not express the engineered receptor, e.g., CAR or the dual-CAR system, described herein. The proliferation of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be increased by about 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 the dual-CAR system, described herein. The proliferation of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 folds or more compared to cells that do not express the engineered receptor, e.g., CAR or the dual-CAR system, described herein.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) expressing the CAR that comprises an intracellular signaling domain derived from FcRγ described herein may have increased proliferation capability compared to cells that do not express the CAR that comprises the intracellular signaling domain derived from FcRγ (e.g., a CAR that expresses other intracellular signaling domains) . The proliferation of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be increased by about 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 CAR that comprises the intracellular signaling domain derived from FcRγ (e.g., a CAR that expresses other intracellular signaling domains) . The proliferation of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 folds or more cells that do not express the CAR that comprises the intracellular signaling domain derived from FcRγ (e.g., a CAR that expresses other intracellular signaling domains) .

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may have lower cytokine release (e.g., IFNγ) compared to cells that express other engineered receptor, e.g., CAR or the dual-CAR system, described herein. The cytokine release of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be reduced by about 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 express other engineered receptor, e.g., CAR or the dual-CAR system, described herein. The cytokine release of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 folds or more compared to cells that express other engineered receptor, e.g., CAR or the dual-CAR system, described herein.

The engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) expressing the CAR that comprises an intracellular signaling domain derived from FcRγ described herein may have lower cytokine release (e.g., IFNγ) compared to cells that do not express the CAR that comprises the intracellular signaling domain derived from FcRγ (e.g., a CAR that expresses other intracellular signaling domains) . The cytokine release of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be reduced by about 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 CAR that comprises the intracellular signaling domain derived from FcRγ (e.g., a CAR that expresses other intracellular signaling domains) . The cytokine release of the engineered cells (e.g., cells expressing the engineered receptor, e.g., CAR or the dual-CAR system, described herein) may be reduced by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 folds or more cells that do not express the CAR that comprises the intracellular signaling domain derived from FcRγ (e.g., a CAR that expresses other intracellular signaling domains) .

The engineered cells expressing the dual-CAR system described herein may have increased anti-tumor efficacy (e.g., cytotoxicity towards tumor cells) compared to cells that do not express the engineered receptor, e.g., CAR or the dual-CAR system, or cells that express single-targeting receptors (e.g., CARs) . The anti-tumor efficacy (cytotoxicity towards tumor cells) of the engineered cells expressing the dual-CAR system described herein may be increased by about 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 the dual-CAR system, or cells that express single-targeting receptors (e.g., CARs) . The anti-tumor efficacy (cytotoxicity towards tumor cells) of the engineered cells expressing the dual-CAR system described herein may be increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 folds or more compared to cells that do not express the engineered receptor, e.g., CAR or the dual-CAR system, or cells that express single-targeting receptors (e.g., CARs) .

The engineered cells expressing the dual-CAR system described herein may have increased long-term proliferation compared to cells that do not express the engineered receptor, e.g., CAR or the dual-CAR system, or cells that express single-targeting receptors (e.g., CARs) . The long-term proliferation of the engineered cells expressing the dual-CAR system described herein may be increased by about 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 the dual-CAR system, or cells that express single-targeting receptors (e.g., CARs) . The long-term proliferation of the engineered cells expressing the dual-CAR system described herein may be increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 folds or more compared to cells that do not express the engineered receptor, e.g., CAR or the dual-CAR system, or cells that express single-targeting receptors (e.g., CARs) . The proliferation of the cells may be observed after about 1, 2, 3, 4, 5, 6, or 7 days, about 1, 2, 3, 4 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.

Nucleic Acids (Polynucleotides)

The present disclosure provides nucleic acids comprising a nucleic acid sequence encoding the engineered receptor, e.g., CAR, or the dual-CAR system described herein.

A polynucleotide of the present disclosure may comprise a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequence can be separated by a linker. A linker for use in the present disclosure allows for multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multicistronic or bicistronic sequence) , which are translated as a polyprotein that is dissociated into separate protein components. The polynucleotide may comprise from 5’ to 3’ the first polynucleotide sequence, the linker, and the second polynucleotide sequence. The polynucleotide comprises from 5' to 3' the second polynucleotide sequence, the linker, and the first polynucleotide sequence. The first polynucleotide sequence may encode a first engineered receptor (e.g., a first CAR) described herein and the second polynucleotide sequence may encode a second engineered receptor (e.g., a second CAR) described herein. The first polynucleotide sequence may encode a second engineered receptor (e.g., a second CAR) described herein and the second polynucleotide sequence may encode a first engineered receptor (e.g., a first CAR) described herein.

The linker may comprise a nucleic acid sequence that encodes for an internal ribosome entry site (IRES) . As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunogloublin heavy-chain binding protein (BiP) ; vascular endothelial growth factor (VEGF) ; fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV) , and Moloney murine leukemia virus (MoMLV) . Those of skill in the art would be able to select the appropriate IRES.

The linker may comprise a nucleic acid sequence that encodes for a self-cleaving peptide. As used herein, a “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. 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 of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV) , and porcine tescho virus-1 (PTV-1) ; and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A, ” “E2A, ” “P2A, ” and “T2A, ” respectively. Those of skill in the art would be able to select the appropriate self-cleaving peptide.

The linker can comprise a spacer sequence. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers (also known as GS linkers) 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 of skill in the art would be able to select the appropriate spacer sequence.

A polynucleotide of the present disclosure can be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

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

Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences can also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

An expression vector (e.g., a lentiviral vector) can be used to introduce the engineered receptor (e.g., CAR) or the dual-CAR system into an immune cell or precursor thereof (e.g., a T cell) . Accordingly, an expression vector (e.g., a lentiviral vector) of the present disclosure can comprise a polynucleotide encoding for an engineered receptor (e.g., CAR) or a dual-CAR system. The expression vector (e.g., lentiviral vector) can comprise additional elements that will aid in the functional expression of the engineered receptor (e.g., CAR) or the dual-CAR system encoded therein. An expression vector comprising a polynucleotide encoding for the engineered receptor (e.g., CAR) or the dual-CAR system may further comprise a mammalian promoter. The vector may comprise an elongation-factor-1-alpha promoter (EF-1α promoter) . The use of an EF-1αpromoter can increase the efficiency in expression of downstream transgenes (e.g., an engineered receptor (e.g., CAR) or a dual-CAR system) . Physiologic promoters (e.g., an EF-1α promoter) can be less likely to induce integration mediated genotoxicity, and can abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and can be incorporated into a vector of the present disclosure. The vector (e.g., lentiviral vector) may further comprise a non-requisite cis acting sequence that can improve titers and gene expression.

The polynucleotide may encode an amino acid sequence set forth in any one of SEQ ID NOs: 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 the amino acid sequence set forth in any one of SEQ ID NOs: 19, 24, 26-47, 59-66 and 68-98.

The disclosure also provides a nucleic acid sequence that is 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%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is 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%, 99%identical to any amino acid sequence as described herein. In some cases, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. The nucleic acid sequence may be less 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 may 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.

The amino acid sequence may (i) comprise an amino acid sequence; or (ii) consist of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

The nucleic acid sequence may (i) comprise a nucleic acid sequence; or (ii) consist of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. 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, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Introduction of Polynucleotides into Host Cells

The polynucleotides (e.g., vectors) described herein can be introduced as one or more polynucleotides or constructs, optionally comprising a marker that will allow for selection of host cells that contain the construct (s) . The genes and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing. Particularly, using PCR, individual fragments including all or portions of a functional unit can be isolated, where one or more mutations can be introduced using "primer repair" , ligation, in vitro mutagensis, etc. as appropriate. The polynucleotides obtained and demonstrated to have the appropriate sequences can then be introduced into the host cell by any convenient means. The polynucleotides can be integrated and packaged into non-replicating, defective viral genomes like lentivirus, Adenovirus, Adeno-associated virus (AAV) , or Herpes simplex virus (HSV) or others, including retroviral vectors, for infection or transduction into cells. The polynucleotides can include viral sequences for transfection, if desired. Alternatively, the polynucleotides can be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cells can be grown and expanded in culture before introduction of the construct (s) , followed by the appropriate treatment for introduction of the construct (s) and integration of the construct (s) . The cells are then expanded and screened by virtue of a marker present in the construct. Various markers that can be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

The engineered receptor (e.g., CAR) or the dual-CAR system can be introduced into the modified cells as an RNA for transient expression. RNA can be delivered to the immune cells of the disclosure by various means including microinjection, electroporation, and lipid-mediated transfection, for example. Introduction of constructs into the cell's genome can occur via transposons. An example of a synthetic transposon for use is the Sleeping Beauty transposon that comprises an expression cassette including the appropriate gene of active fragment thereof. The construct can be integrated at a particular locus in the genome of the host cell. An endogenous gene can be replaced with the gene encoded for by the construct using homologous recombination.

A construct encoding an engineered receptor (e.g., a CAR) or a dual-CAR system can be introduced into the host cell using a lentiviral delivery system. A construct encoding an engineered receptor (e.g., a CAR) or a dual-CAR system can be introduced into the host cell using a retroviral delivery system.

The host cells can be human cells. The host cells can be human T cells. The human T cells can be purified from commercialized PBMCs. The host cells can be αβT cells. The host cells can be γδT cells. The host cells can be NK cells. The host cells can be panT cells.

Methods of Treatment

The engineered receptor (CAR) or the 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.

In one aspect, the disclosure provides a pharmaceutical composition, comprising the engineered receptor (e.g., a CAR) or the dual-CAR system described herein, the cell described herein, or the nucleic acid described herein and a pharmaceutically acceptable carrier.

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

In one aspect, the disclosure provides a method of modulating an immune response comprising administering an effective amount of modified cells described herein to a subject in need thereof.

The disease or disorder may be a cancer, an inflammatory or autoimmune disease.

The cancer may be solid cancer or hematologic cancer. The cancer may be liver cancer, gastric cancer, colon cancer, lymphoma, acute myeloid leukemia (AML) or chronic myelogenous leukemia (CML) .

The term “effective amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results.

Examples of cancer that can be treated include, but are not limited to, leukemias including chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, and T cell and B cell leukemias, lymphomas (Hodgkin's and non-Hodgkins) , lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, carcinomas of solid tissues, hypoxic tumors, squamous cell carcinomas, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, head and neck cancers, and nervous system cancers.

The disclosure further includes the use of the modified cells described herein in the manufacture of a medicament or pharmaceutical composition to treat a disease or disorder or modulate an immune response, to treat an infection or to treat cancer as described hereinabove.

The modified cells can also be used in experimental models, for example, to further study and elucidate the function of the cells.

One or more of the modified cells described herein can be administered to a subject in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions or injections, or subcutaneous injections. In some cases, the modified cells can expand within a subject's body, in vivo, after administration to a subject. The modified cells can be frozen to provide cells for multiple treatments with the same cell preparation. The modified cells of the disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit can include instructions (e.g., written instructions) on the use of the modified cells and compositions comprising the same.

In one aspect, the present disclosure provides a method of treatment that comprises administering to a subject a therapeutically-effective amount of the modified cells. The therapeutically-effective amount of the 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 the modified cells may be administered for at least one week. The therapeutically-effective amount of the modified cells may be administered for at least two weeks.

The modified cells described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the modified cells can vary. For example, the modified cells can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The modified cells can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the modified cells can be initiated immediately within the onset of symptoms, within the first 3 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within 48 hours of the onset of the symptoms, or within any period of time from the onset of symptoms. The initial administration can be via any route practical (e.g., intravenous infusions or injections) , such as by any route described herein using any formulation described herein. In some examples, the administration of the modified cells of the disclosure is an intravenous administration. One or multiple dosages of the modified cells can be administered as soon as is practicable after the onset of a cancer or an infectious disease, and for a length of time necessary for the treatment of the disease, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. For the treatment of cancer, one or multiple dosages of the modified cells can be administered years after onset of the cancer and before or after other treatments. In some examples, the modified cells can be administered for at least about 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 length of treatment can vary for each subject.

Methods for administration of modified cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8 (10) : 577-85) . See, e.g., 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. The cell therapy, e.g., adoptive T cell therapy may be carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

The cell therapy (e.g., adoptive T cell therapy) may be carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such cases, the cells then are administered to a different subject, e.g., a second subject, of the same species. The first and second subjects may be genetically identical. The first and second subjects may be genetically similar. The second subject may express the same HLA class or supertype as the first subject.

The subject may have been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. The subject may have persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT. The administration may effectively treat the subject despite the subject having become resistant to another therapy.

The subject may be responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. The subject may not have relapsed. The subject may be determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

The subject may have persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT. The administration may effectively treat the subject despite the subject having become resistant to another therapy.

The modified cells described herein can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the modified cells can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell (s) , where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers can be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors/cancers and pediatric tumors/cancers are also included. The cancer can be a solid tumor or a hematological tumor. The cancer can be a carcinoma. The cancer can be a sarcoma. The cancer can be a leukemia. The cancer can be a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas) . Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas) , glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases) .

Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (aform of skin cancer) , squamous cell carcinoma (various tissues) , bladder carcinoma, including transitional cell carcinoma (amalignant neoplasm of the bladder) , bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

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

The modified cells can be immediately used in the above therapeutic, experimental or commercial applications or the cells can be cryopreserved for use at a later date. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The modified cells disclosed herein can be formulated in unit dosage forms suitable for single administration of precise dosages. In some cases, the unit dosage forms comprise additional lymphocytes. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities 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-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative or without a preservative. In some examples, the pharmaceutical composition does not comprise a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

EXAMPLES

The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

Example 1: Generation of CD20 CAR constructs

CD20 sdAb1 (SEQ ID NO: 1) is an anti-CD20 V_HH that has been disclosed in WO 2022/012680A1.2F2 scFv (SEQ ID NO: 3) is an anti-CD20 binder derived from anti-CD20 antibody, ofatumumab. 3H7 scFv (SEQ ID NO: 4) is an anti-CD20 binder used in ADI-001, which is a CD20 CAR-γδT cell product developed by Adicet. Leu16 scFv (SEQ ID NO: 5) is anti-CD20 binder that is widely used in clinic with CD20 CAR-T cell therapy. Exemplary CD20 CAR constructs were designed in the format as shown in Table 1 and the schematic representation of a CD20 CAR construct is shown in FIG. 1. The CAR fragment was then cloned into a retroviral vector or a lentiviral vector to create CD20 CAR construct in a single coding frame, using human EF1 alpha promoter for expression.

Table 1. Exemplary CD20 CAR Constructs

Example 2: Generation of CD19 CAR constructs

CD19 sdAb (SEQ ID NO: 6) is anti-CD19 V_HH that has been disclosed in WO 2022/012683A1. FMC63 scFv (SEQ ID NO: 7) is an anti-CD19 binder that is widely used in clinic 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 with natural cytotoxicity receptors (NCRs) , such as NKp46, NKp44, and NKp30. Following the ligation of NCRs with their putative ligands, these adaptor molecules allow for intracellular activating signaling through immune-receptor tyrosine-based activating motifs (ITAMs) (Kelly Hudspeth, et al., Frontiers in Immunology. 2013) . Exemplary CD19 CAR constructs were designed in the format as shown in Table 2 and schematic representation of a CD19 CAR construct is shown in FIG. 2. The CAR fragment was then cloned into a retroviral vector or a lentiviral vector to create CD19 CAR construct in a single coding frame, using human EF1 alpha promoter for expression.

Table 2. Exemplary CD19 CAR constructs

Example 3: Generation of CD20/CD19 dual CAR constructs

The CD20/CD19 dual CAR construct was generated by bicistronic vector that CD19 CAR was linked to CD20 CAR via a P2A self-cleavage element (SEQ ID NO: 18) at N-terminus or C-terminus. Schematic representation of a CD20/CD19 dual CAR construct is shown in FIG. 3. TanCAR7 construct (SEQ ID NO: 45) and C-CAR039 construct (SEQ ID NO: 46) , that targeting CD20 and CD19 by making anti-CD20 scFv Leu16 or 2F2 in tandem with anti-CD19 scFv FMC63 were generated as reference controls as well. Exemplary CD20/CD19 dual CAR constructs were designed in the format as shown in Table 3. The CD20/CD19 dual CAR fragment was then cloned into a retroviral vector or a lentiviral vector to create CD20/CD19 dual CAR construct in a single coding frame, using human EF1 alpha promoter for expression.

Table 3. Exemplary CD20/CD19 dual CAR constructs

Example 4: Preparation of virus and CAR-T cells

Preparation of retrovirus

After thawing the packaging cells and expansion, the packaging cells was transient transfected with transfer plasmid at a pre-optimized ratio with polyethylenimine, and then medium exchange, after harvest, move to concentration step, followed by ultra-centrifugation. The virus pellets were rinsed with formulation buffer. The virus was aliquoted properly, then stored at -80℃immediately. The virus titer was determined by measurement of transduction efficiency to Jurkat cell line via flow cytometry.

Preparation of lentivirus

The lentivirus packaging plasmid mixture including pCMV-△R-8.47 and pMD2. G (Addgene, Cat#12259) was pre-mixed with the CAR gene bearing vectors at a pre-optimized ratio with polyethylenimine, followed by added to the HEK-293 cells. The supernatants were collected after overnight incubation. The virus-containing supernatants were filtered through a 0.45 μm PES filter, followed by ultra-centrifugation for lentivirus concentration. The virus pellets were rinsed with pre-chilled DPBS. The virus was aliquoted properly, then stored at -80℃ immediately. The virus titer was determined by measurement of transduction efficiency to supT1 cell line via flow cytometry.

Collection and transduction of αβ T lymphocyte

Leukocytes were collected from healthy donors by apheresis. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque^TM PLUS Media according to manufacturer’s protocol. Human T cells were purified from PMBCs using Pan T cell isolation kit (Miltenyi, Cat#130-096-535) , following manufacturer’s protocol. The purified T cells were subsequently pre-activated for 48 hours with human T cell activation/expansion kit (Miltenyi, Cat#130-091-441) according to manufacturer’s protocol in which anti-CD3/CD28 MACSiBead particles were added at a bead-to-cell ratio of 1: 2. The pre-activated T cells were transduced with lentivirus stock in the presence of 7 μg/mL polybrene. The transduced cells were then transferred to the cell culture incubator for transgene expression under suitable conditions.

Collection and transduction of γδ T lymphocyte

Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using a Ficoll-Paque-based density gradient centrifugation protocol. The 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 culture medium on day 0. 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) were included in the medium as well. After 24-96 hours post-activation, cells were transduced with lentiviral at an MOI of 5. Cells were harvested 10 days post-transduction and the total number, purity and transduction efficiency were determined. Cells were further enriched with a negative TCRγ/δ+ T cell isolation kit (Miltenyi Biotec) before future applications or cryopreserved.

Example 5: CD20 and CD19 expression in tumor cell lines

Expression of CD20 and CD19 on tumor cell lines was determined by flow cytometry. As shown in FIG. 4, both CD20 and CD19 are expressed in Raji cells, Z-138 cells, and NALM6-CD20 cells. CD20 is expressed in Raji-CD19 KO cells, while CD19 is absent. CD19 is expressed in NALM6 cells, while CD20 is absent.

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

Different anti-CD20 binders were integrated with CD8 hinge, CD8 transmembrane domain, 4-1BB and CD3ζ signaling, respectively, to generate CD20 CAR constructs bearing different anti-CD20 binders in 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 CD20 positive NHL cell line Raji cells at the E: T (effector-to-target) ratios of 3: 1 and 1: 1 for 20-24 hours. Untransduced γδT cells (UnT) were used as a negative control. The cytotoxicity was assessed by ONE-Glo^TM Luciferase Assay System from Promega. As shown in FIG. 5A, all CAR constructs exhibited potent cytotoxicity against Raji cells.

Additionally, co-culture supernatants were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIGs. 5B-5C, increased secretions of IFNγ and TNFα were detected in the supernatants collected from co-cultures of CAR-T cells with Raji cells, no increased cytokine release was observed in the supernatants collected from co-cultures of UnT cells with Raji cells. Taken together, CD20 CAR-T cells demonstrated cytotoxicity and cytokine release against CD20 positive Raji cells in vitro.

Example 7: In vivo evaluation of CD20 CAR constructs

Anti-tumor activity of CD20 CAR-γδT cells was assessed in vivo in an Raji xenograft model. 2×10⁶ Raji cells were implanted subcutaneously on day 0 in NCG mice. Once tumors reaches 100-150 mm³ (12 days after tumor implantation) , mice were randomized into treatment groups. 5×10⁶ CAR positive T cells (CD20 sdAb1 BBZ, 2F2 BBZ, 3H7 BBZ) were suspended in a 400 μL HBSS medium before administration into mice via tail vein. Untransduced γδT cells (UnT) were used as a negative control. Tumor volume and body weight were measured twice a week. Percentage of human CD3⁺ T cells in live cells of peripheral blood was determined by flow cytometry once a week. As shown in FIGs. 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. CD20 sdAb1 BBZ CAR-T cells treated mice achieved tumor free with no significant change in body weight comparing to cohorts treated with UnT. Moreover, proliferation of CD3⁺ T cells was greater in cohorts treated with CD20 sdAb1 BBZ CAR-T cells comparing to cohorts treated with 2F2 BBZ CAR-T cells and 3H7 BBZ CAR-T cells. Human CD3⁺ T cells contracted 3 weeks after infusion of CAR-T cells. No tumor inhibitory effect was observed by infusion of UnT. The results demonstrated that CD20 sdAb1 bearing CD20 CAR-T cells have better anti-tumor efficacy than CD20 CAR-T cells bearing other anti-CD20 binders in xenograft mouse model.

Example 8: In vitro cytotoxicity and cytokine release of CD19 CAR constructs in αβT cells

Three different ITAM (Immunoreceptor tyrosine-based activation motif) bearing domains, CD3ζ cytoplasmic domain, Dap12 cytoplasmic domain, and FcRγ cytoplasmic domain, were integrated with CD19 sdAb, CD8 hinge and CD8 transmembrane domain, respectively, with or without (2^nd generation CAR or 1^st generation CAR) membrane proximal insertion of 4-1BB intracellular domain. These CD19 CAR 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 CD19 positive NHL cell line Raji cells at the E: T ratio of 5: 1 for 20-24 hours. Untransduced αβT cells (UnT) were used as a negative control. The cytotoxicity was assessed by ONE-Glo^TM Luciferase Assay System from Promega. As shown in FIG. 7A, CD3ζ, Dap12, and FcRγ bearing CAR-T cells exhibited cytotoxicity against Raji cells, proximal insertion of 4-1BB domain dampened the killing potency of Dap12 bearing CAR and FcRγ bearing CAR, whereas CD3ζbearing CAR demonstrated comparable cytotoxicity with or without 4-1BB.

Additionally, co-culture supernatants were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIG. 7B, increased secretions of IFNγ were detected in the supernatants collected from co-cultures of CAR-T cells with Raji cells, no increased IFNγ release was observed in the supernatants collected from co-cultures of UnT cells with Raji cells. FcRγ bearing CAR-T cells secreted less IFNγ than CD3ζbearing CAR-T cells and Dap12 bearing CAR-T cells, which is in line with the cytotoxicity data.

Example 9: In vitro cytotoxicity of CD19 CAR constructs in γδT cells

FMC63 scFv was integrated with CD8 or CD28 hinge, CD8 or CD28 transmembrane domain, 4-1BB, CD28, Dap10 (SEQ ID NO: 20) , or OX40 (SEQ ID NO: 21) intracellular domain, and FcRγ cytoplasmic domain to generate a panel of CAR constructs targeting CD19 as shown in Table 4. To confirm the effect of distance of FcRγ cytoplasmic domain to the cell membrane, 4-1BB inserted at the upstream or downstream of FcRγ were generated in parallel. FMC63 BBZ constructs with traditional CD8 hinge, CD8 transmembrane domain and CD3ζ cytoplasmic domain in the presence or absence of 4-1BB intracellular domain were used as positive controls.

Table 4. CAR constructs targeting CD19

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 CD19 positive NHL cell line Raji cells at the E: T ratios of 5: 1 and 1: 1 for 20-24 hours. Untransduced γδT cells (UnT) were used as a negative control. The cytotoxicity was assessed by ONE-Glo^TM Luciferase Assay System from Promega. As shown in FIG. 8, each type of CD19 CAR-T cells exhibited dose-dependent cytotoxicity against Raji cells. CAR-T cells bearing CD28 hinge and transmembrane showed superior cytotoxicity to CAR-T cells bearing CD8 hinge and transmembrane domain, suggesting that CD28 hinge and transmembrane domain is more beneficial to the cytotoxicity effect of FcRγthan CD8 hinge and transmembrane domain. Moreover, FMC63 FcRγBB (4-1BB at the downstream of FcRγ) CAR-T cells outperformed FMC63 BBFcRγ (4-1BB at the upstream of FcRγ) CAR-T cells, indicating that membrane proximal location facilitates the cytotoxicity of FcRγ. That is in line with that FMC63 FcRγBB, FMC63 FcRγDap10, FMC63 FcRγOX40 showed comparable cytotoxicity to FMC63 FcRγ. Interestingly, the cytotoxicity was well maintained while CD28 intracellular domain was inserted at the upstream of FcRγ. In other words, FcRγ can withstand the membrane insertion of CD28 intracellular domain with minimal reduction in cytotoxicity.

Example 10: In vitro proliferation of CD19 CAR constructs in αβT cells

CD19 sdAb was integrated 4-1BB and CD3ζ cytoplasmic domain, Dap12 cytoplasmic domain, or FcRγ cytoplasmic domain to generate 2^nd generation CAR constructs targeting CD19. Constructs including CD19 sdAb BBZ (SEQ ID NO. 31) , CD19 sdAb BBDap12 (SEQ ID NO. 32) , CD19 sdAb BBFcRγ (SEQ ID NO. 33) .

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 CD19 positive NHL cell line Raji cells at the E: T ratios 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 the co-culture, the cells were collected, and the number of CAR-T cells was determined by cell counting and flow cytometry. Then the collected CAR-T cells from the previous cycle of co-culture were added to freshly prepared Raji cells already plated in a new 24-well plate for next cycle of co-culture. Each cycle lasted 2-3 days, total 4 cycles (9 days of co-culture in total) were performed. The cumulative proliferation fold of CAR-T cells is determined by multiplying the proliferation fold in each co-culture cycle. As shown in FIGS. 9A-9B, CD19 CAR-T cells bearing FcRγ cytoplasmic domain exhibited superior proliferation to CAR-T cells bearing CD3ζ and Dap12 cytoplasmic domains at both E: T ratios of 1: 1 and 1: 3. At the E: T ratio of 1: 3, only CD19 CAR-T cells bearing FcRγ demonstrated proliferative capability at such a low E: T ratio.

Additionally, supernatants from the first cycle of co-culture were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIG. 9C, CD19 CAR-T cells bearing FcRγ cytoplasmic domain secreted much less IFNγ than increased secretions of IFNγ CAR-T cells bearing CD3ζ and Dap12 cytoplasmic domains at the E:T ratio of 1: 1. At the E: T ratio of 1: 3, comparable level of IFNγ release was detected for these three types of CD19 CAR-T cells. No increased IFNγ release was observed in the supernatants collected from co-cultures of UnT cells with Raji cells. Taken together, CD19 CAR-T cells bearing FcRγ cytoplasmic domain demonstrated greater proliferation with lower cytokine release comparing to CD19 CAR-T cells bearing CD3ζ and Dap12 cytoplasmic domains, suggestion the potential advantages of using FcRγ cytoplasmic domain in CAR design for clinical applications.

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

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

Table 5. CAR constructs targeting CD19

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 CD19 positive NHL cell line Raji cells at the E: T ratio of 1: 1 for 4 days, as the first co-culture cycle. After the co-culture, the cells were collected, and the number of CAR-T cells was determined by cell counting and flow cytometry. Then the collected CAR-T cells from the previous cycle of co-culture were added to freshly prepared Raji cells already plated in a new 24-well plate additional 4 days of co-culture, as the second co-culture cycle. Two cycles (8 days of co-culture in total) were performed. The cumulative proliferation fold of CAR-T cells is determined by multiplying the proliferation fold in each co-culture cycle. As shown in FIG. 10, CD19 CAR-T cells bearing CD28 hinge, CD28 transmembrane domain, CD28 intracellular domain, and FcRγ cytoplasmic domain exhibited greater proliferation than CD19 CAR-T cells with other modalities.

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

Table 6. Constructs used for the generation of dual CAR system

The CAR constructs shown in Table 6 were transduced into γδT cells to prepare CAR-γδT cells. 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 cell line Raji cells, CD20 positive CD19 negative cell line Raji-CD19 KO cells, CD20 negative CD19 positive cell line NALM6 cells, respectively, at the E: T ratios of 2: 1 and 1: 2 for 20-24 hours. The cytotoxicity was assessed by ONE-Glo^TM Luciferase Assay System from Promega. As shown in FIGs. 11A-11C, CD20/CD19 dual CAR-T cells exhibited potent cytotoxicity against all three types of cell lines, whereas CD20 sdAb1 BBZ CAR-T cells only killed CD20 positive cell lines, FMC63 28FcRγ and FMC63 BBZ CAR-T cells only killed CD19 positive cell lines.

Additionally, co-culture supernatants were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIGs. 11D-11F, increased secretion of INFγ 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 secretion of INFγ was detected for CD20/CD19 dual CAR with CD20 negative CD19 positive NALM6 cells. This is in line with low cytokine release of CD19 CAR bearing FcRγ cytoplasmic domain. No increased INFγ release was observed in the supernatants collected from co-cultures of UnT cells with each type of cell lines. Taken together, CD20/CD19 dual CAR-T cells demonstrated anti-tumor activity against CD20 and/or CD19 positive tumor cells.

Example 13: CD20/CD19 dual CAR-γδT cells induced no cytotoxicity against CD20/CD19 double negative cells

To confirm the killing specificity of CD20/CD19 dual CAR construct, CD20/CD19 dual CAR-γδT cells from two individual donors were co-cultured with three types of tumor cell lines (A-371, A549, H929) that do 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, H929, respectively, at the E: T ratios of 10: 1, 3: 1, and 1: 1 for 20-24 hours. The cytotoxicity was assessed by ONE-Glo^TM Luciferase Assay System from Promega. As shown in FIGs. 12A-12C, CD20/CD19 dual CAR-T cells exhibited no cytotoxicity against all three types of cell lines.

Additionally, co-culture supernatants were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIGs. 12D-12F, no increased secretion of INFγ was observed in the supernatants collected from co-cultures of CD20/CD19 dual CAR-T cells with each type of cell lines. Taken together, CD20/CD19 dual CAR construct does not exert cytotoxicity or cytokine release in co-culture with CD20 and CD19 double negative cells.

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

CD20/CD19 dual CAR construct, CD20 sdAb1 BBZ construct, FMC63 28FcRγconstruct, and FMC63 BBZ construct were transduced into γδT cells to prepare CAR-γδT cells. 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 positive cell line NALM6-CD20 cells at the E: T ratio of 1: 1 for 3 days, as the first co-culture cycle. After the co-culture, the cells were collected, and the percentage of CD3⁺ T cells in total live cells was determined by flow cytometry, the number of CAR-T cells was determined by cell counting and flow cytometry. Then the collected CAR-T cells from the previous cycle of co-culture were added to freshly prepared NALM6-CD20 cells already plated in a new 24-well plate for next cycle of co-culture. Seven cycles (21 days of co-culture in total) were performed. The cumulative proliferation fold of CAR-T cells is determined by multiplying the proliferation fold in each co-culture cycle. As shown in FIGs. 13A-13B, CD20/CD19 dual CAR-T cells showed the most durable killing capability and the greatest proliferation. It is demonstrated that CD20/CD19 dual CAR-T cells are superior to single targeting CAR-T counterparts in durability and long-term proliferation.

Example 15: In vivo evaluation of CD20/CD19 dual CAR in γδT cells

CD20/CD19 dual CAR construct, CD20 sdAb1 BBZ construct were transduced into γδT cells from two individual donors, to prepare CAR-γδT cells cells. FMC63 BBZ construct was transduced into αβT cells to prepare CAR-αβT cells, as a reference control. Untransduced γδT cells (Un-γδT) were used as a negative control. Anti-tumor activity of these CAR-T cells was assessed in vivo in a Z-138 xenograft model. 3×10⁶ Z-138 cells were implanted subcutaneously on day 0 in NCG mice. Once tumor volume reaches 100-150 mm³ (14 days after tumor implantation) , mice were randomized into treatment groups. 2×10⁶ CAR positive T cells were suspended in a 400 μL HBSS medium before administration into mice via tail vein. HBSS medium without T cells was administered as a vehicle control. Tumor volume was measured twice a week. Percentage of CAR-T cells in live cells of peripheral blood was determined by flow cytometry once a week. As shown in FIGs. 14A-14B, for CAR-γδT cells from donor 1, treatment of CD20 sdAb1 BBZ CAR-T cells resulted in tumor free in 3 out of 4 mice, while failed to inhibit the tumor growth in 1 mouse. However, tumor relapsed after 30 days post CAR-T infusion in all 3 mice that achieved tumor free. In contrast, CD20/CD19 dual CAR-T cells achieved tumor free in all 4 mice, tumor relapsed only in 2 out of 4 mice, the other 2 mice remained over 60 days post CAR-T infusion. Additionally, proliferation of CAR-T cells was greater in cohorts treated with CD20/CD19 dual CAR-T cells comparing to cohorts treated with CD20 sdAb1 BBZ CAR-T cells. As shown in FIGs. 14C-14D, for CAR-γδT cells from donor 2, treatment of CD20 sdAb1 BBZ CAR-T cells resulted in tumor free in all 4 mice. However, tumor relapsed after 40 days post CAR-T infusion in all 4 mice. In contrast, CD20/CD19 dual CAR-T cells achieved and remained tumor free in all 4 mice over 60 days post CAR-T infusion. Additionally, proliferation of CAR-T cells was greater in cohorts treated with CD20/CD19 dual CAR-T cells comparing to cohorts treated with CD20 sdAb1 BBZ CAR-T cells. As shown in FIG. 14C, treatment of FMC63 BBZ CAR-αβT cells failed to effectively inhibit the tumor growth in 1 out of 3 mice, while resulted tumor free in other 2 mice. No tumor inhibitory effect was observed by infusion of vehicle control, Un-γδT from 2 donors. Taken together, it is demonstrated that CD20/CD19 dual CAR-T cells have superior efficacy and proliferation to CD20 single CAR counterpart in γδT cells. CD20/CD19 dual CAR-T cells provide long-term protection from tumor relapse in xenograft mouse model, which is accordance with long PFS achieved by treatment of CD20/CD19 dual CAR-T cell therapy in clinical trial.

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

CD20/CD19 dual CAR construct, CD20 sdAb1 BBZ construct, and FMC63 28FcRγwere transduced into γδT cells, to prepare CAR-γδT cells cells. Untransduced γδT cells (Un-γδT) were used as a negative control. Anti-tumor activity of these CAR-T cells was assessed in vivo in a Z-138 xenograft model. 3×10⁶ Z-138 cells were implanted subcutaneously on day 0 in NCG mice. Once tumor volume reaches 100-150 mm³ (14 days after tumor implantation) , mice were randomized into treatment groups. 2×10⁶ CAR positive T cells were suspended in a 400 μL HBSS medium before administration into mice via tail vein. Tumor volume was measured twice a week. Percentage of CAR-T cells in live cells of peripheral blood was determined by flow cytometry once a week. As shown in FIGs. 15A-15B, CD20/CD19 dual CAR-T cells achieved better tumor inhibition effect than CD20 sdAb1 BBZ CAR-T cells and FMC63 28FcRγ CAR-T cells. Additionally, proliferation of CAR-T cells was greater in cohorts treated with CD20/CD19 dual CAR-T cells comparing to cohorts treated with CD20 sdAb1 BBZ CAR-T cells and FMC63 28FcRγ CAR-T cells.

Example 17: CD20/CD19 dual CAR has better durability and proliferation than tandem CAR in γδT cells

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

Example 18: CD20/CD19 dual CAR has better durability and proliferation than tandem CAR in αβT cells

CD20/CD19 dual CAR construct, and two tandem CAR constructs (TanCAR7 and C-CAR039) were transduced into αβT cells from two individual donors, to prepare CAR-γδT cells. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post transduction, CAR-T cells were co-cultured with CD20 and CD19 double positive cell line Raji cells at the E: T ratio of 1: 1 for 3 days, as the first co-culture cycle. After the co-culture, the cells were collected, and the percentage of CD3⁺ T cells in total live cells was determined by flow cytometry, the number of CAR-T cells was determined by cell counting and flow cytometry. Then the collected CAR-T cells from the previous cycle of co-culture were added to freshly prepared NALM6-CD20 cells already plated in a new 24-well plate for next cycle of co-culture. Seven cycles (16 days of co-culture in total, 2-3 days per cycle) were performed. The cumulative proliferation fold of CAR-T cells is determined by multiplying the proliferation fold in each co-culture cycle. As shown in FIGs. 17A-17B, CD20/CD19 dual CAR-T cells showed more durable killing capability and better proliferation than TanCAR7 CAR-T cells and C-CAR039 CAR-T cells. It is demonstrated that CD20/CD19 dual CAR-T cells are superior to tandem CAR-T cells in durability and long-term proliferation in αβT cells.

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

The CD20/CD19 dual CAR constructs with different modalities were generated by bicistronic vector that CD19 CAR was linked to CD20 CAR via a P2A self-cleavage element (SEQ ID NO: 18) at N-terminus or C-terminus. Schematic representation of CD20/CD19 dual CAR constructs with different modalities are shown in FIG. 18. Exemplary CD20/CD19 dual CAR constructs with different modalities were designed in the format as shown in Table 7. The CD20/CD19 dual CAR fragment was then cloned into a retroviral vector or a lentiviral vector to create CD20/CD19 dual CAR construct in a single coding frame, using human EF1 alpha promoter for expression.

Table 7. Examples of dual CAR constructs

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

CD20/CD19 dual CAR constructs with different modalities were transduced into αβT cells to prepare CAR-αβT cells. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post transduction, CAR-T cells were co-cultured with CD20 and CD19 double positive cell line Raji cells, CD20 positive CD19 negative cell line Raji-CD19 KO cells, CD20 negative CD19 positive cell line K-562-CD19 cells, respectively, at the E: T ratios of 8: 1 and 2: 1 for 20-24 hours. The cytotoxicity was assessed by ONE-Glo^TM Luciferase Assay System from Promega. As shown in FIGs. 19A-19C, CD20/CD19 dual CAR-T cells different modalities exhibited potent cytotoxicity against all three types of cell lines, whereas CD20/CD19 dual CAR with modality of CD20 sdAb1 CD8HTM BB FcRγ /FMC63 IgG4 hinge CD28TM CD28Z exhibited superior cytotoxicity.

Additionally, co-culture supernatants were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIGs. 19D-19F, increased secretion of INFγ was detected in the supernatants collected from co-cultures of CD20/CD19 dual CAR-T cells with all three types of cell lines. No increased INFγ release was observed in the supernatants collected from co-cultures of UnT cells with each type of cell lines. It is noteworthy that the CD20/CD19 dual CAR modalities using FcRγ domain incorporating with CD20 targeting CAR secreted significantly lower level of INFγ in co-culture with Raji cells and Raji-CD19 KO cell, comparing to other CD20/CD19 dual CAR modalities. Taken together, CD20/CD19 dual CAR-T cells with different modalities demonstrated anti-tumor activity against CD20 and/or CD19 positive tumor cells.

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

CD20/CD19 dual CAR constructs with different modalities were transduced into αβT cells to prepare CAR-αβT cells. Untransduced αβT cells (UnT) were used as a negative control. On day 7 post transduction, CAR-T cells were co-cultured with CD20 and CD19 double positive cell line Raji cells at the E: T ratio of 1: 1 for 3 days, as the first co-culture cycle. After the co-culture, the cells were collected, and the percentage of CD3⁺ T cells in total live cells was determined by flow cytometry, the number of CAR-T cells was determined by cell counting and flow cytometry. Then the collected CAR-T cells from the previous cycle of co-culture were added to freshly prepared NALM6-CD20 cells already plated in a new 24-well plate for next cycle of co-culture. Ten cycles (25 days of co-culture in total, 2-3 days per cycle) were performed. The cumulative proliferation fold of CAR-T cells is determined by multiplying the proliferation fold in each co-culture cycle. As shown in FIGs. 20A-20B, CD20/CD19 dual CAR-T cells with different modalities showed durable killing capability and great proliferation. It is noteworthy that CD20/CD19 dual CAR having the modalities of CD20 sdAb1 BBZ /FMC63 CD28HTM CD28Z, which is a widely used parallel CAR format with two CD3ζ domains, showed inferior proliferation than other CD20/CD19 dual CAR modalities with the combination of one CD3ζ domain and one FcRγ domain or one Dap 12 domain.

Example 22: Co-culture CD20/CD19 dual CAR with PBMCs

B cells play an important role in pathogenesis and development of a wide spectrum of autoimmune diseases. Emerging data have demonstrated that CD19 or CD20 targeted depletion of B cells could be an efficacious strategy to treat autoimmune diseases, such as systemic lupus erythematosus. To explore the opportunity of CD20/CD19 dual CAR in autoimmune diseases, in vitro studies were performed to investigate the capability of CD20/CD19 dual CAR in depleting 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. TanCAR7 construct (SEQ ID NO: 45) , C-CAR039 construct (SEQ ID NO: 46) that targeting CD20 and CD19, and FMC63 BBZ construct (SEQ ID NO: 47) that targeting 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 the 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 antibody and anti-CD20 antibody. As shown in FIG. 21, B cells account of 16.89%of PBMCs before co-culture. After co-culture with CAR-T cells for 72 hours, as shown in FIG. 22, B cells were depleted by CAR-T cells with different extents. CD20 sdAb1 BBZ /FMC63 CD28HTM CD28 FcRγ and CD20 sdAb1 CD8HTM BB FcRγ /FMC63 IgG4 hinge CD28TM CD28Z exhibited superior capability of B cell depletion to C-CAR039 and FMC63 BBZ construct at the ratio of 1: 50.

Additionally, co-culture supernatants were collected to measure cytokines using detection kits from Cisbio according to manufacturer’s instructions. As shown in FIG. 23, increased secretion of INFγ was detected in the supernatants collected from co-cultures of CAR-T cells with PBMCs. No increased INFγ release was observed in the supernatants collected from co-cultures of UnT cells with PBMCs or PBMC only. It is noteworthy that the CD20 sdAb1 BBZ /FMC63 CD28HTM CD28 FcRγ and CD20 sdAb1 CD8HTM BB FcRγ /FMC63 IgG4 hinge CD28TM CD28Z secreted lower level of INFγ in co-culture with PBMCs, comparing to FMC63 BBZ construct, suggesting potentially lower CRS risk for CD20/CD19 dual CAR in clinic.

To confirm the conclusion of this study, CD20 sdAb1 BBZ /FMC63 CD28HTM CD28 FcRγ, CD20 sdAb1 CD8HTM BB FcRγ /FMC63 IgG4 hinge CD28TM CD28Z, TanCAR7, C-CAR039, and UnT were generated in parallel to co-culture with human PBMCs from another donor at the E: T ratios of 1: 50 for 72 hours. As shown in FIG. 24, CD20 sdAb1 BBZ /FMC63 CD28HTM CD28 FcRγ and CD20 sdAb1 CD8HTM BB FcRγ /FMC63 IgG4 hinge CD28TM CD28Z exhibited superior capability of B cell depletion to TanCAR7 and C-CAR039. Taken together, CD20/CD19 dual CAR-T cells can efficiently eliminate B cells from PBMCs with low cytokine release.

Table 8. Examples of dual CAR constructs

OTHER EMBODIMENTS

It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the 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

An engineered receptor, 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 domain selected from a CD28 co-stimulatory signaling domain, a Dap10 intracellular signaling domain, an OX40 intracellular signaling domain, a CD27 intracellular signaling domain, a CD137 (4-1BB) co-stimulatory signaling domain and an ICOS intracellular signaling domain.
The engineered receptor of claim 1, wherein the additional signaling domain is CD28 co-stimulatory signaling domain.
The engineered receptor of claim 2, wherein the CD28 co-stimulatory signaling domain is located at the N-terminus of the intracellular signaling domain derived from FcRγ.
The engineered receptor of claim 1, wherein the additional signaling domain is CD137 (4-1BB) co-stimulatory signaling domain.
The engineered receptor of claim 4, wherein the CD137 (4-1BB) co-stimulatory signaling domain is located at the N-terminus of the intracellular signaling domain derived from FcRγ.
The engineered receptor of any one of claims 1-5, wherein the transmembrane domain is a transmembrane domain derived from: α chain of a T cell receptor, β chain of the T cell receptor, ζchain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154.
The engineered receptor of any one of claims 1-6, further comprising a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.
The engineered receptor of any one of claims 1-7, wherein the hinge region is a hinge region derived from: CD8α, CD28, IgG1, IgG2, IgG3, or IgG4.
The engineered receptor of any one of claims 1-8, wherein the engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain, and an intracellular signaling domain derived from FcRγ.
The engineered receptor of any one of claims 1-8, wherein the engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain, and an intracellular signaling domain derived from FcRγ.
The engineered receptor of any one of claims 1-8, wherein the engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signaling domain, and an intracellular signaling domain derived from FcRγ.
The engineered receptor of any one of claims 1-8, wherein the engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and an intracellular signaling domain derived from FcRγ.
The engineered receptor of any one of claims 1-12, wherein the extracellular antigen-binding domain comprises a sdAb, scFv, a (scFv) ₂, a V_HH domain, or a VNAR domain.
The engineered receptor of the any one of claims 1-13, wherein the extracellular antigen-binding domain binds to a tumor antigen.
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-l, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP) , BAFF, B-lymphoma cell, 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 extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-l receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-l, VEGFR2, and vimentin.
The engineered receptor of claim 15, wherein the extracellular antigen-binding domain binds to CD19.
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 a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.
The engineered receptor of claim 17, wherein the extracellular antigen-binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58.
The engineered receptor of claim 18, wherein the extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 7.
The engineered receptor of any one of claims 1-19, wherein the engineered receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33, 36-43 and 68 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 33, 36-43 and 68.
The engineered receptor of any one of claims 1-20, wherein the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 36.
The engineered receptor of claim 15, wherein the extracellular antigen-binding domain binds to CD20.
The engineered receptor of claim 22, wherein the extracellular antigen-binding domain comprises a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.
The engineered receptor of claim 23, wherein the extracellular antigen-binding domain comprises a V_HH antibody moiety comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.
The engineered receptor of any one of claims 1-15 and 22-24, wherein the engineered receptor comprises an amino acid sequence set forth in any one of SEQ ID NOs: 69-71, and 74 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 69-71, and 74.
The engineered receptor of any one of claims 1-25, wherein the engineered receptor further comprises a signal peptide.
The engineered receptor of claim 26, wherein the signal peptide is located at the N-terminus of the engineered receptor.
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.
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 signaling domain derived from FcRγ; and a CD28 co-stimulatory signaling domain or a CD137 (4-1BB) co-stimulatory signaling domain; and

a second engineered receptor comprising:

a second extracellular antigen-binding domain;

a second transmembrane domain; and

a second intracellular domain.
The dual-CAR system of claim 29, wherein the second intracellular domain comprises a CD3ζ primary intracellular signaling domain.
The dual-CAR system of claim 29 or 30, wherein the second engineered receptor further comprises a co-stimulatory signaling domain located between the C-terminus of the transmembrane domain and the N-terminus of the CD3ζ primary intracellular signaling domain.
The dual-CAR system of claim 31, wherein the co-stimulatory signaling domain is CD137 (4-1BB) co-stimulatory signaling domain or CD28 co-stimulatory signaling domain.
The dual-CAR system of any one of claims 29-32, wherein the first and/or second transmembrane domain is a transmembrane domain derived from: α chain of a T cell receptor, β chain of the T cell receptor, ζchain of the T cell receptor, CD8α, CD28, CD3s, CD35, CD3y, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD86, or CD154.
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.
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.
The dual-CAR system of any one of claims 29-35, further comprising 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.
The dual-CAR system of any one of claims 29-36, wherein the first and/or second hinge region is a hinge region derived from: CD8α, CD28, IgG1, IgG2, IgG3, or IgG4.
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 as following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain; or

(2) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain; a hinge region; a t2ansmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a hinge region; a transmembrane domain; a CD28 co-stimulatory signaling domain, and a CD3ζprimary intracellular signaling domain.
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 as following: a first extracellular antigen-binding domain binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain;

(2) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain binds to CD20; a hinge region; a transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain binds to CD19; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain; or

(3) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain binds to CD20; a hinge region; a transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain binds to CD19; a hinge region; a transmembrane domain; a CD28 co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain.
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 as following: a first extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain;

(2) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain; a IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain;

(3) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain;

(4) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD28 co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a IgG4 hinge region; a CD28 transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain.

(5) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a CD28 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain, and a CD3ζ primary intracellular signaling domain; or

(6) the first engineered receptor comprises from the N-terminus to the C-terminus as following: a first extracellular antigen-binding domain; a CD8α hinge region; a CD8α transmembrane domain; a CD137 (4-1BB) co-stimulatory signaling domain; and an intracellular signaling domain derived from FcRγ; and

the second engineered receptor comprises from the N-terminus to the C-terminus as following: an extracellular antigen-binding domain; a IgG4 hinge region; a CD28 transmembrane domain; a CD28 co-stimulatory signaling domain, and a CD3ζprimary intracellular signaling domain.
The dual-CAR system of any one of claims 29-40, wherein the first engineered receptor and the second engineered receptor is connected via a linker.
The dual-CAR system of the any one of claims 29-41, wherein the first and/or second extracellular antigen-binding domain each binds to a tumor antigen.
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-l, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP) , BAFF, B-lymphoma cell, 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 extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-l receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin a5b1, integrin anb3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-l, VEGFR2, and vimentin.
The dual-CAR system of claim 43, wherein the first extracellular antigen-binding domain binds to CD19.
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 a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.
The dual-CAR system of claim 45, wherein the first extracellular antigen-binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58.
The dual-CAR system of claim 46, wherein the first extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 7.
The dual-CAR system of any one of claims 43-47, wherein the second extracellular antigen-binding domain binds to CD20.
The dual-CAR system of claim 48, wherein the second extracellular antigen-binding domain comprises a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.
The dual-CAR system of claim 49, wherein the V_HH antibody moiety comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.
The dual-CAR system of any one of claims 44-50, wherein

(1) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 36; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 24; or

(2) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 68, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 68; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 24.
The dual-CAR system of any one of claims 44-51, comprising an amino acid sequence set forth in SEQ ID NO: 19 or 60, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 19 or 60.
The dual-CAR system of claim 43, wherein the first extracellular antigen-binding domain binds to CD20.
The dual-CAR system of claim 53, wherein the first extracellular antigen-binding domain comprises a V_HH antibody moiety that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected CDR3 amino acid sequence; wherein the selected CDR1 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 48, the selected CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49, and the selected CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 50.
The dual-CAR system of claim 54, wherein the V_HH antibody moiety comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.
The dual-CAR system of any one of claims 53-55, wherein the second extracellular antigen-binding domain binds to CD19.
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 a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a 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 a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, 53, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, 56, respectively.
The dual-CAR system of claim 57, wherein the second extracellular antigen-binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL sequence, wherein the selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58.
The dual-CAR system of claim 58, wherein the second extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 7.
The dual-CAR system of any one of claims 53-59, wherein

(1) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 69; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 47;

(2) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 70, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 70; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 47;

(3) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 71; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 72;

(4) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 71; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 73;

(5) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 74; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 75; or

(6) the first engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 74; and

the second engineered receptor comprising an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 76.
The dual-CAR system of any one of claims 53-60, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 61-66, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in any one of SEQ ID NOs: 61-66.
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.
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.
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.
A nucleic acid comprising a nucleotide sequence encoding the engineered receptor of any one of claims 1-28, or the dual-CAR system of any one of claims 29-64.
A cell comprising the engineered receptor of any one of claims 1-28, the dual-CAR system of any one of claims 29-64, or the nucleic acid of claim 65.
The cell of claim 66, wherein the cell is an immune cell.
The cell of claim 67, wherein the immune cell is selected from the group consisting of T cell, NK cell, NKT, peripheral blood mononuclear cell (PBMC) , hematopoietic stem cell, pluripotent stem cell, an embryonic stem cell, a macrophage, a monocyte, a neutrophil, an eosinophil and a combination thereof.
The cell of claim 68, wherein the T cell is a αβT cell, γδT cell or panT cells.
A modified γδT cell, 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.
The modified γδT cell of claim 70, wherein the first extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 7, and the second extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1.
A modified αβT cell, 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.
The modified αβT cell of claim 72, wherein the first extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 1, and the second extracellular antigen-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, an amino acid sequence having at least 90%, 95%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 7.
A pharmaceutical composition comprising the cell of any one of claims 66-73, and a pharmaceutical acceptable carrier.
A method for treating and/or preventing cancer comprising administering an effective amount of the cell of any one of claims 66-73 or pharmaceutical composition of claim 74 to a subject in need thereof.
[Corrected under Rule 26, 23.10.2024]
A method for treating and/or preventing autoimmune disease comprising administering an effective amount of the cell of any one of claims 66-73 or pharmaceutical composition of claim 74 to a subject in need thereof.