CCR4-targeting chimeric antigen receptor cell therapy

Abstract

The present disclosure provides compositions and methods for anti-CCR4 chimeric antigen receptors (CARs), as well as engineered immune cells or their precursors (e.g., engineered T cells) that contain anti-CCR4 CARs. Also provided are methods of using anti-CCR4 CAR-expressing cells to treat cancer, and T cell depletion systems for use in combination with anti-CCR4 CAR T cell therapy. TIFF2024506647000029.tif129166

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 148,489, filed February 11, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] 2. Background of the Invention A challenge in targeting T-cell lymphomas with chimeric antigen receptor (CAR) T-cell therapy is that target antigens are often shared between T cells and tumor cells, resulting in fratricidal loss of normal T cells and CAR T-cell products. CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane domain chemokine receptor that binds the chemokines CCL17 / TARC and CCL22 / MDC1. CCR4 is highly expressed by many mature T-cell malignancies, including most adult T-cell leukemia / lymphoma (ATLL), cutaneous T-cell lymphoma (CTCL) and 30-40% of peripheral T-cell lymphoma (PTCL) cases, and has a unique expression profile on normal T cells. Specifically, CCR4 is predominantly expressed by type 2 and type 17 helper T cells (Th2 and Th17), effector regulatory T cell (Treg) subsets and cutaneous lymphocyte antigen (CLA)+ skin-homing T cells, but is rarely expressed by other helper T cell subsets and CD8+ T cells. All T cell malignancies that express CCR4 show poor prognosis and there are few curative treatments. In addition, CCR4 on tumors is functional and its expression is associated with poorer disease outcome. Thus, CCR4 is a potential target for the treatment of mature T cell malignancies.

[0003] The feasibility of CCR4 targeting therapy has been proposed in the context of monoclonal antibody (mAb) treatment. Mogamulizumab (KW-0761) is a defucosylated humanized anti-CCR4 mAb designed to induce enhanced antibody-dependent cellular cytotoxicity (ADCC). Phase I / II trials of mogamulizumab for CTCL or ATLL showed an acceptable safety profile and some efficacy, and phase III trials demonstrated that mogamulizumab significantly prolonged progression-free survival in patients with CTCL compared with vorinostat, one of the standard treatments for CTCL. Adverse events were comparable to vorinostat, and most of them were manageable, although cases of severe cutaneous hypersensitivity reactions (Stevens-Johnson syndrome) have been reported. Based on these trials, mogamulizumab has been approved by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan for the treatment of refractory ATLL, CTCL and PTCL, and recently by the FDA in the United States for CTCL. However, the majority of patients treated with mogamulizumab experience relapse, and therefore cure is rarely achieved. In addition, previous reports have proposed that functional CCR4-redirected chimeric antigen receptor (CCR4-CAR) T cells can be established (Perera LP, et al. Am J Hematol. 2017;92(9):892-901). However, the exact mechanism of sibling rivalry events and its association with T cell expansion, transduction, function, target sensing, phenotype, and antitumor effects remain unclear. Thus, there is a need in the art for novel anti-CCR4 CAR-based therapies for T cell malignancies that overcome these obstacles and challenges. The present invention addresses this need. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Perera LP, et al. Am J Hematol. 2017;92(9):892-901 Summary of the Invention

[0005] In some aspects, the present invention provides an anti-CC chemokine receptor 4 (CCR4) chimeric antigen receptor (CAR), comprising an anti-CCR4 antigen binding domain, a transmembrane domain, and an intracellular domain.

[0006] In some embodiments, the anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

[0007] In some embodiments, the anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12.

[0008] In some embodiments, the anti-CCR4 antigen binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

[0009] In some embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv).

[0010] In some embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv) having heavy and light chains derived from mogamulizumab.

[0011] In some embodiments, the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:112.

[0012] In some embodiments, the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:114.

[0013] In some embodiments, the anti-CCR4 antigen binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16.

[0014] In some embodiments, the CAR further comprises a CD8 alpha hinge, optionally comprising the amino acid sequence depicted in SEQ ID NO:34.

[0015] In some embodiments, the transmembrane domain comprises an artificial hydrophobic sequence and a transmembrane domain selected from the group consisting of a transmembrane domain of a type I transmembrane protein, a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, a transmembrane domain of CD28, a transmembrane domain of CD3 epsilon, a transmembrane domain of CD45, a transmembrane domain of CD4, a transmembrane domain of CD5, a transmembrane domain of CD8, a transmembrane domain of CD9, a transmembrane domain of CD16, a transmembrane domain of CD22, a transmembrane domain of CD33, a transmembrane domain of CD37, a transmembrane domain of CD64, a transmembrane domain of CD80, a transmembrane domain of CD86, a transmembrane domain of OX40 (CD134), a transmembrane domain of 4-1BB (CD137), a transmembrane domain of ICOS, and a transmembrane domain of CD154, or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0016] In some embodiments, the transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally, the CD8 alpha transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:36.

[0017] In some aspects, the intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

[0018] In some embodiments, the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0019] In some embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:38.

[0020] In some embodiments, the intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof.

[0021] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:40.

[0022] In some embodiments, the CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, an intracellular domain comprising a CD8 alpha hinge, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0023] In some embodiments, the CAR further comprises a CD8 leader sequence, optionally comprising the amino acid sequence depicted in SEQ ID NO:32.

[0024] In some embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102.

[0025] In some embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:29, 53, 61, 69, 81, 89, and 101.

[0026] In some embodiments, the CAR is linked to the safety switch via a self-cleavable linker, and the safety switch binds to a safety switch substance.

[0027] In some embodiments, the safety switch is selected from: (a) truncated EGFR (EGFRt), where the safety switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), where the safety switch agent is AP1903; and (d) herpes simplex virus type 1 thymidine kinase (HSVTK), where the safety switch agent is ganciclovir (GCV).

[0028] In some embodiments, the CAR is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

[0029] In some embodiments, the safety switch is linked to the dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

[0030] In some aspects, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding an anti-CCR4 CAR, wherein the CAR comprises an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0031] In some embodiments, the anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

[0032] In some embodiments, the anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12.

[0033] In some embodiments, the anti-CCR4 antigen binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

[0034] In some embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv).

[0035] In some embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv) having heavy and light chains derived from mogamulizumab.

[0036] In some embodiments, the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:112.

[0037] In some embodiments, the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:114.

[0038] In some embodiments, the anti-CCR4 antigen binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16.

[0039] In some embodiments, the CAR further comprises a CD8 alpha hinge, optionally comprising the amino acid sequence depicted in SEQ ID NO:34.

[0040] In some embodiments, the transmembrane domain comprises an artificial hydrophobic sequence and a transmembrane domain selected from the group consisting of a transmembrane domain of a type I transmembrane protein, a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, a transmembrane domain of CD28, a transmembrane domain of CD3 epsilon, a transmembrane domain of CD45, a transmembrane domain of CD4, a transmembrane domain of CD5, a transmembrane domain of CD8, a transmembrane domain of CD9, a transmembrane domain of CD16, a transmembrane domain of CD22, a transmembrane domain of CD33, a transmembrane domain of CD37, a transmembrane domain of CD64, a transmembrane domain of CD80, a transmembrane domain of CD86, a transmembrane domain of OX40 (CD134), a transmembrane domain of 4-1BB (CD137), a transmembrane domain of ICOS, and a transmembrane domain of CD154, or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0041] In some embodiments, the transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally, the CD8 alpha transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:36.

[0042] In some aspects, the intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

[0043] In some embodiments, the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0044] In some embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:38.

[0045] In some embodiments, the intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof.

[0046] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:40.

[0047] In some embodiments, the CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, an intracellular domain comprising a CD8 alpha hinge, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0048] In some embodiments, the CAR further comprises a CD8 leader sequence, optionally comprising the amino acid sequence depicted in SEQ ID NO:32.

[0049] In some embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102.

[0050] In some embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:29, 53, 61, 69, 81, 89, and 101.

[0051] In some embodiments, the CAR is linked to the safety switch via a self-cleavable linker, and the safety switch binds to a safety switch substance.

[0052] In some embodiments, the safety switch is selected from: (a) truncated EGFR (EGFRt), where the safety switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), where the safety switch agent is AP1903; and (d) herpes simplex virus type 1 thymidine kinase (HSVTK), where the safety switch agent is ganciclovir (GCV).

[0053] In some embodiments, the CAR is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

[0054] In some embodiments, the safety switch is linked to the dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

[0055] In some aspects, the invention provides a vector comprising a nucleic acid described herein.

[0056] In some aspects, the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0057] In some aspects, the viral vector is a lentiviral vector.

[0058] In some aspects, the present invention provides an engineered immune cell or a precursor thereof comprising a CAR described herein, a nucleic acid described herein, and / or a vector described herein.

[0059] In some aspects, the engineered immune cells or precursors thereof include: (a) CCR4 null knockout allele; (b) suppressed CCR4 gene expression; and (c) a fusion protein comprising an anti-CCR4 scFv and a KDEL motif, and / or a nucleic acid encoding said fusion protein. The method further includes one or more of:

[0060] In some embodiments, a CCR4 null knockout allele, or suppressed CCR4 gene expression, is obtained through genetic engineering techniques involving nucleases selected from the group consisting of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases.

[0061] In some embodiments, the modified immune cell or a precursor thereof further comprises an inhibitory RNA molecule that suppresses CCR4 gene expression.

[0062] In some embodiments, the inhibitory RNA molecule is selected from the group consisting of RNA interference (RNAi) RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), microRNA (miRNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), single guide RNA (sgRNA), double-stranded RNA (dsRNA), ribozyme, and any combination thereof.

[0063] In some aspects, the modified cells are autologous cells.

[0064] In some embodiments, the modified immune cells are derived from peripheral blood mononuclear cells (PBMCs).

[0065] In some embodiments, the PBMCs comprise tumor cells.

[0066] In some aspects, the engineered immune cells or progenitors thereof are cells isolated from a human subject.

[0067] In some embodiments, the engineered immune cell is an engineered T cell.

[0068] In some aspects, the invention provides methods for producing an engineered immune cell or a progenitor thereof, comprising introducing into said immune cell or a progenitor thereof a nucleic acid described herein or a vector described herein.

[0069] In some aspects, the vector is introduced via viral transduction.

[0070] In some aspects, the invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a modified immune cell or progenitor cell of any one of aspects 54 to 63, or a modified immune cell or progenitor cell thereof produced by the method of aspect 64 or aspect 65.

[0071] In some embodiments, the modified cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in a subject, thereby treating cancer.

[0072] In some embodiments, the subject has previously received mogamulizumab.

[0073] In some aspects, the cancer is refractory to mogamulizumab.

[0074] In some aspects, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a modified T cell comprising an anti-CCR4 CAR, wherein the CAR comprises an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0075] In some embodiments, the anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

[0076] In some embodiments, the anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12.

[0077] In some embodiments, the anti-CCR4 antigen binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

[0078] In some embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv).

[0079] In some embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv) having heavy and light chains derived from mogamulizumab.

[0080] In some embodiments, the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:112.

[0081] In some embodiments, the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:114.

[0082] In some embodiments, the anti-CCR4 antigen binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16.

[0083] In some embodiments, the CAR further comprises a CD8 alpha hinge, optionally comprising the amino acid sequence depicted in SEQ ID NO:34.

[0084] In some embodiments, the transmembrane domain comprises an artificial hydrophobic sequence and a transmembrane domain selected from the group consisting of a transmembrane domain of a type I transmembrane protein, a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, a transmembrane domain of CD28, a transmembrane domain of CD3 epsilon, a transmembrane domain of CD45, a transmembrane domain of CD4, a transmembrane domain of CD5, a transmembrane domain of CD8, a transmembrane domain of CD9, a transmembrane domain of CD16, a transmembrane domain of CD22, a transmembrane domain of CD33, a transmembrane domain of CD37, a transmembrane domain of CD64, a transmembrane domain of CD80, a transmembrane domain of CD86, a transmembrane domain of OX40 (CD134), a transmembrane domain of 4-1BB (CD137), a transmembrane domain of ICOS, and a transmembrane domain of CD154, or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0085] In some embodiments, the transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally, the CD8 alpha transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:36.

[0086] In some aspects, the intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

[0087] In some embodiments, the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0088] In some embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:38.

[0089] In some embodiments, the intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof.

[0090] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:40.

[0091] In some embodiments, the CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, an intracellular domain comprising a CD8 alpha hinge, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0092] In some embodiments, the CAR further comprises a CD8 leader sequence, optionally comprising the amino acid sequence depicted in SEQ ID NO:32.

[0093] In some embodiments, the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102.

[0094] In some embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:29, 53, 61, 69, 81, 89, and 101.

[0095] In some embodiments, the CAR is linked to the safety switch via a self-cleavable linker, and the safety switch binds to a safety switch substance.

[0096] In some embodiments, the safety switch is selected from: (a) truncated EGFR (EGFRt), where the safety switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), where the safety switch agent is AP1903; and (d) herpes simplex virus type 1 thymidine kinase (HSVTK), where the safety switch agent is ganciclovir (GCV).

[0097] In some embodiments, the CAR is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

[0098] In some embodiments, the safety switch is linked to the dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

[0099] In some embodiments, the method further comprises administering a safety-switch substance to the subject.

[0100] In some embodiments, the modified T cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in a subject, thereby treating cancer.

[0101] In some embodiments, the modified T cells are derived from PBMCs.

[0102] In some embodiments, the PBMCs comprise tumor cells.

[0103] In some embodiments, the modified T cells are human T cells.

[0104] In some embodiments, the modified T cells are autologous.

[0105] In some embodiments, the subject is a human.

[0106] In some embodiments, the subject has previously received mogamulizumab.

[0107] In some aspects, the cancer is refractory to mogamulizumab. [Brief description of the drawings]

[0108] The foregoing and other features and advantages of this invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Figure 1] FIG. 1 illustrates the finding that subcutaneous T-cell lymphoma cells express high levels of CCR4. CCR4 expression on skin biopsy samples from four patients with CTCL was assessed by immunohistochemistry (IHC). Each scale bar represents 100 um. [Diagram 2] Illustrating the findings that normal tissues express little CCR4. CCR4 expression (in triplicate) on normal tissue microarrays (TMA) for 27 organs was analyzed. The table below represents a map of the TMA. [Diagram 3] Depicts a schematic of the CCR4-CAR construct. CD8 leader, CD8 leader sequence; scFv, single chain variable fragment; TM, transmembrane domain. [Figure 4]Figure 1 illustrates the finding that CCR4-CAR T cells can be expanded while depleting CCR4-expressing T cells. 21-day expansion (left panel) and total doublings at day 10 (right panel) of CCR4-CAR-transduced T cells. The left panel shows a representative T cell expansion curve. T cells were counted and diluted to 0.7x106 cells / ml by adding fresh medium every day to every other day. T cells were harvested for subsequent experiments when the T cells had shrunk in size to 350um3, at which time a portion of the cells was set aside for continued counting. The right panel shows aggregate data of total T cell expansion at day 10 after anti-CD3 / 28 stimulation. Dots and bars represent the mean and standard error of the mean (SEM) (n=5). Data are representative (left panel) or aggregated (right panel) of five experiments from five donors. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (vs. CD19CART group) by one-way ANOVA with Tukey's post-hoc test. [Diagram 5] The final yield of CAR T cell product is depicted. T cells were stimulated with anti-CD3 / CD28 beads and then transduced with CCR4-CAR, control CD19-CAR, or not transduced. Once the cells had shrunk in size to 350um3 (typically 10-14 days after bead stimulation), T cells were harvested and frozen for subsequent experiments. The total number of T cell doublings on the day of harvest is shown. Bars represent the mean and SEM. Data are aggregates of 5 experiments from 5 donors. ns, not significant; *, p<0.05; ***, p<0.001 (vs. 19CART group) by 1-way ANOVA with Tukey's post-hoc test. [Figure 6] Depicts T cell size during CAR T cell expansion. T cell size was analyzed daily to every other day by cell counting device (Coulter counter) (right panel). Size at day 10 from aggregated data is shown in the right panel. Data are representative or aggregated of 5 experiments from 5 donors. [Figure 7]Figure 1 illustrates the finding that CCR4-CAR T cells can be expanded while depleting CCR4-expressing T cells. Time course analysis of T cell viability. Cell viability was analyzed by amine-reactive dye staining (LIVE-DEAD staining) and FCM. Dots and bars represent mean and SEM (n=5). Statistical analysis was applied to data at day 10. Data are aggregates of 5 experiments from 5 donors. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 vs. CD19CART group by 1-way ANOVA with Tukey's post-hoc test. [Figure 8] Depicts T cell memory phenotype by CD45RO and CCR7 expression on day 10. Numbers indicate the percentage of cells in each quadrant. [Figure 9] Figure 1 illustrates the finding that CCR4-CAR T cells can be expanded while depleting CCR4-expressing T cells. Time course analysis of CAR expression in T cells showing CAR-positive cell selection in several CAR T cells. Dots and bars represent mean and SEM (n=5). Statistical analysis was applied to data at day 10. Data are aggregated from 5 experiments from 5 donors. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 vs. CD19CART group by 1-way ANOVA with Tukey's post-hoc test. [Figure 10] FIG. 1 depicts the expression of CAR on the surface of T cells. CAR expression was analyzed by FCM 10 days after stimulation. Gates represent the CAR-positive population and numbers indicate the CAR-positive percentage. [Figure 11]Depict that the anti-CCR4 mAb used in CCR4-CAR construction does not share the epitope of CCR4 with the detection antibody. CCR4 on HH cells was blocked with either purified CCR4 mAb clone KW-0761 or h1567, and then stained with either clone KW-0761 (Ax647 conjugate), h1567 (PE conjugate) or D8SEE (PE conjugate). CCR4 was analyzed by FCM. CCR4 was still detectable after CCR4 blocking with clone KW-0761 and h1567, but KW-0761 slightly reduced the detection efficiency with the detection mAb (D8SEE). [Figure 12] Figure 1 illustrates the finding that CCR4-CAR T cells can be expanded while depleting CCR4-expressing T cells. Representative flow plot showing CAR and CCR4 expression on T cells 6 days after stimulation. Numbers indicate the percentage of cells in each quadrant. Data are representative of 5 experiments from 5 donors. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 vs. CD19CART group by 1-way ANOVA with Tukey's post-hoc test. [Figure 13] Figure 1 illustrates the finding that CCR4-CAR T cells can be expanded while depleting CCR4-expressing T cells. Representative CCR4 expression kinetics (left panel) and aggregated data of CCR4 expression at day 10 (right panel). Bars represent mean and SEM (n=5). Data are representative (left panel) or aggregated (right panel) of five experiments from five donors. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 vs. CD19CART group by one-way ANOVA with Tukey's post-hoc test. [Figure 14]Depicts changes in CD4 / 8 ratios during CAR T cell expansion. CD4 / 8 ratios in CAR-positive (upper panel) and CAR-negative (lower panel) populations at each time point were analyzed by FCM. Data are pooled from five experiments from five donors. ns, not significant; *, p<0.05; **, p<0.01 (vs. 19CART group at day 10) by one-way ANOVA with Tukey's post-hoc test. [Figure 15] Depicts HH cells transduced with two different CCR4-CARs; CCR4-CAR(KW_L2H) and CCR4-CAR(h1567_L2H) (related to FIG. 16). CCR4-CAR expression on HH cells was analyzed by FCM. [Figure 16] Illustrates findings that CCR4-CAR T cells can be expanded while depleting CCR4-expressing T cells. Epitope-specific blocking of CCR4 by CCR4-CAR on HH cells. Shown is CCR4 expression detected with either anti-CCR4 mAb clone h1567 (left panel) or anti-CCR4 mAb clone KW-0761 (right panel) on HH cells transduced with the indicated CCR4 CAR. Data are representative of two experiments. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (vs. CD19CART group) by one-way ANOVA with Tukey's post-hoc test. [Figure 17]Figure 1 illustrates findings that sibling competition alters the proportions of Th subsets, sparing Th1 cytokine production while decreasing Th2, Th17, and Treg cytokine production. Ratios of Th1 and Th2 subsets in CAR T cell products. Th1 subsets were defined as CD4(+), CXCR3(+), CCR4(-), CCR6(-), and CCR10(-). Th2 subsets were defined by FCM as CD4(+), CXCR3(-), CCR4(+), CCR6(-), and CCR10(-). Phenotypes were analyzed by gating on the CAR-positive population excluding the UTD group. Data are tabulated from three experiments from three donors. Bars represent mean and SEM (n=3). ns, not significant; ***, p<0.001; ****, p<0.0001 (vs. CD19-CAR T group) by one-way ANOVA with Tukey's post-hoc test. [Figure 18] Figure 1 illustrates findings that sibling competition alters the proportions of Th subsets, sparing Th1 cytokine production while decreasing Th2, Th17 and Treg cytokine production. Heatmap of Th-associated cytokine production by CCR4-CAR T cells compared to CD19-CAR T cells. CAR T cells were stimulated with PMA / Iono. Cytokine levels in supernatants 24 hours post-stimulation were analyzed by LUMINEX assay. Cytokine levels from CD19-CAR T cells were set to 1. Data are representative of two experiments from two donors. [Figure 19]Figure 1 illustrates the findings that sibling competition alters the proportion of Th subsets, sparing Th1 cytokine production while decreasing Th2, Th17 and Treg cytokine production. Depletion of CCR4-positive subsets by adding CCR4-CAR T cells to CD19-CAR T cells. T cells were stimulated and transduced separately with either CCR4 CAR or CD19 CAR. Five days after stimulation, 10% of the number of CCR4-CAR T cells were added to CD19-CAR T cells. At day 10, CCR4 expression in CD19-CAR positive cells was analyzed. Numbers indicate the percentage of cells in each quadrant. Data are representative of two experiments from two donors. [Figure 20] Figure 1 illustrates the findings that sibling competition alters the proportions of Th subsets, sparing Th1 cytokine production while decreasing Th2, Th17 and Treg cytokine production. Heatmap of Th-associated cytokine production by CD19-CAR T cells pre-treated with CCR4-CAR T cells compared to untreated CD19-CAR T cells. CAR T cells were stimulated with the indicated stimulators. Cytokine levels in supernatants 24 hours post-stimulation were analyzed by LUMINEX assay. Cytokine levels from untreated CD19-CAR T cells were set to 1. [Figure 21] Figure 1 depicts the expression of CCR4 on tumor cell lines by FCM. The numbers in each column indicate the MFI of CCR4-PE. Nalm6, HH, MJ, and HuT78 are cell lines derived from patients with B-ALL, leukemic CTCL, Sezary syndrome, and HTLV-1 positive MF, respectively. [Figure 22] Depicts lytic activity of CAR T cells against tumor cell lines. CAR T cells were incubated with luciferase-expressing tumor cells at the indicated E:T ratios. Specific lysis was determined based on luminescence after 16 h of co-culture. Bars represent mean and SD (triplicates). Data are representative of three experiments from three donors. [Figure 23]Degranulation and cytokine production by CCR4-CAR T cells are depicted. CAR T cells were stimulated with either medium, PMA-Iono or the indicated cells at an R:S ratio of 1:4 in the presence of monensin and CD107a detection antibody. After 6 hours of co-culture, cells were stained for intracellular cytokines using FoxP3 staining buffer set and analyzed by FCM. Bars represent mean and SD (in duplicate). Data are representative of three experiments from three donors. [Figure 24] Illustrates findings that CCR4-CAR (KW_L2H) cures HH cell xenografted mice and induces excellent T cell engraftment. Schematic of the experiment. NSG mice were inoculated intravenously with 1x106 luciferase expressing HH cells (HH-CGB-GFP). Seven days after tumor cell inoculation, engrafted tumors were treated intravenously with either 0.5x106 CCR4-CAR positive T cells or non-transduced T cells. Data are representative of two experiments from two donors. [Diagram 25] Illustrates findings that CCR4-CAR (KW_L2H) cures HH cell xenografted mice and induces excellent T cell engraftment. Overall kinetics of tumor burden by BLI. The dashed line represents background levels of photons determined by imaging tumor-free mice. Dots and bars represent mean and SEM (n=4 for UTD group, n=5 for other groups). Data are representative of two experiments from two donors. [Figure 26] Figure 1 illustrates the findings that CCR4-CAR(KW_L2H) cures HH cell xenografted mice and induces superior T cell engraftment. Kaplan-Meier survival curves. *, p<0.05; **, p<0.01 (vs. KW_L2H group) by log-rank test. Data are representative of two experiments from two donors. [Figure 27]Figure 1 illustrates findings that CCR4-CAR (KW_L2H) cures HH cell xenografted mice and induces excellent T cell engraftment. Peripheral blood T cell counts on days 12 and 19 by FCM. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (vs. KW_L2H group) by 1-way ANOVA with Tukey's post-hoc test. Data are representative of two experiments from two donors. [Figure 28] Depicts BW changes during CCR4-CAR T cell treatment. Dots and bars represent mean and SEM (n=4 for UTD group, n=5 for other groups). Some mice showed rapid BW increase within 30 days after treatment, reflecting hepatosplenomegaly caused by tumor infiltration. All mice from the KW_L2H group maintained CR during treatment, so the gradual BW increase in the KW_L2H group was associated with normal growth of the mice. [Figure 29] Depicts CD19 expression on HH cells transduced with truncated CD19. HH cells were transduced with truncated CD19 and repeatedly sorted to purify the CD19 positive population. Dot plots show 100% purity of CD19 expression in FCM. Gates represent the CD19 positive population and numbers indicate the percentage of positive cells (left panel) or MFI of PE (right panel). [Diagram 30] Figure 1 illustrates the findings that CCR4-CAR (KW_L2H) induces antitumor effects comparable to those of CD19-CAR in NSG mice engrafted with HH expressing CD19 (HH-CBG-GFP-t19). Schematic of the experiment. NSG mice were inoculated intravenously with 1x106 HH cells expressing luciferase and truncated CD19 (HH-CGB-GFP-t19). Seven days after tumor cell inoculation, engrafted tumors were treated intravenously with either 2x106 CCR4-CAR positive T cells or UTD T cells. [Diagram 31]Figure 1 illustrates findings that CCR4-CAR (KW_L2H) induces antitumor effects comparable to those of CD19-CAR in NSG mice engrafted with CD19-expressing HH (HH-CBG-GFP-t19). Overall kinetics of tumor burden by BLI. The dashed line represents background levels of photons determined by imaging tumor-free mice. Dots and bars represent mean and SEM (n=5 for UTD group, n=7 for other groups). Data are representative of two experiments from two donors. [Diagram 32] Figure 1 illustrates findings that CCR4-CAR (KW_L2H) induces antitumor effects comparable to those of CD19-CAR in NSG mice engrafted with CD19-expressing HH (HH-CBG-GFP-t19). Peripheral blood T cell counts on days 7 and 14. On days 7 and 14, peripheral blood was analyzed for T cell counts by FCM using counting beads. Bars represent mean and SEM (n=5 for UTD group, n=7 for other groups). Data are representative of two experiments from two donors. [Diagram 33] Peripheral blood T cell counts on days 7, 14 and 28 are depicted (related to FIG. 32). Peripheral blood was analyzed for total T cell, CD4 and CD8 T cell counts by FCM. Bars represent mean and SEM. Dots and bars represent mean and SEM. Data are representative of two experiments from two donors. [Diagram 34] Figure 1 illustrates the findings that CCR4-CAR (KW_L2H) induces antitumor effects comparable to those of CD19-CAR in NSG mice engrafted with CD19-expressing HH (HH-CBG-GFP-t19). Cytokine levels in serum on day 6. Serum was collected from mouse whole blood on day 6 using the same experimental schedule as in Figures A, B, and C. Cytokines were analyzed by high-sensitivity LUMINEX assay. Bars represent mean and SEM (n=4 for UTD group, 5 for other groups). ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001 by one-way ANOVA with Tukey's post-hoc test. [Diagram 35] Depict the phenotype of primary CTCL cells. Primary CTCL cells were obtained from a patient with Sezary syndrome. The phenotype of tumor cells was analyzed by FCM. Normal T cell populations (CD8 positive or CD4, CD7 and CD26 positive) were almost absent. [Diagram 36] Figure 1 illustrates the findings that CCR4-CAR T cells can lyse and respond to patient-derived primary CTCL cells. Cytokine production by CCR4-CAR T cells upon stimulation of primary CTCL cells. Cytokine production of CAR T cells 6 hours after stimulation was analyzed by intracellular staining and FCM. Gates represent the positive population of the indicated cytokines in CAR-positive cells. Data are representative of two experiments from two donors. [Figure 37] Figure 1 illustrates the findings that CCR4-CAR T cells can lyse and respond to patient-derived primary CTCL cells. Lytic activity of CCR4-CAR T cells against primary CTCL cells. The killing activity of CAR T cells against each cell at 4 hours of co-culture was analyzed by 51Cr release assay. Dots and bars represent the mean and SD (triplicates). Data are representative of two experiments from two donors. [Figure 38] Illustrates the CCR4CAR_KW_L2H_tEGFR vector map. [Figure 39] Illustrates the CCR4CAR_KW_L2H_CD20 vector map. [Diagram 40] Illustrates the CCR4CAR_KW_L2H_iCasp9 vector map. [Diagram 41] Illustrates the CCR4CAR_KW_L2H_HSVTK vector map. [Diagram 42] 1 is a plot illustrating GCV-induced depletion of CAR T cells over 7 days in culture using the HSVTK depletion system. [Diagram 43] Illustrates the dnTGFbR-T2A-CCR4CAR-P2A-HSVTK vector map. [Diagram 44]Illustrates the CCR4CAR-P2A-dnTGFbR-T2A-HSVTK vector map. [Diagram 45] Illustrates the CCR4CAR-P2A-HSVTK-T2A-dnTGFbR vector map. [Diagram 46] 1 is a plot illustrating the cytotoxicity of CCR4CAR-HSVTK-dnTGFbRII. [Figure 47] Data are provided illustrating the findings that shRNA targeting CCR4 (shCCR4) efficiently knocks down CCR4 in the CCR4-expressing T cell lines HH and ATN-1. HH cells (left panel) and ATN-1 cells (right panel) were transduced with sh-CCR4 or sh-Cre (negative control) using lentivirus. CCR4 expression was analyzed by FCM 2 days after sh-RNA. [Figure 48A] The findings illustrate that the sh-CCR4 target sequence can be carefully selected to efficiently generate CCR4 knockdown CCR4CART cells. Figure 48A provides a schedule for the dual transduction of shCCR4 and CCR4-CAR. [Figure 48B] The findings illustrate that CCR4 knockdown CCR4CART cells can be efficiently generated by selecting sh-CCR4 target sequences. Figure 48B provides data showing reduced CCR4 expression on T cells 3 days after anti-CD3 / 28 stimulation (2 days after sh-RNA introduction). [Figure 48C] The findings are illustrated in Figure 48C, which shows that CCR4 knockdown CCR4CART cells can be efficiently generated by selecting sh-CCR4 target sequences. Figure 48C shows the expansion of CART cells during CCR4 knockdown CCR4-CAR T cell preparation. Sh-CCR4#3, which provides the lowest knockdown efficiency, induced greater T cell expansion. [Figure 48D-1]The findings illustrate that CCR4 knockdown CCR4CART cells can be efficiently generated by selecting sh-CCR4 target sequences. Figure 48D shows CCR4-CAR expression 5 and 10 days after T cell stimulation. Sh-CCR4#3, which has the lowest CCR4 knockdown efficiency, induced greater T cell expansion and CAR transduction efficiency. [Figure 48D-2] See description of Figure 48D-1. [Figure 49A] Figure 49A illustrates the findings that CCR4-CART cells can cure mogamulizumab-resistant tumors. Figure 49A is a schematic diagram of a mogamulizumab-resistant HH tumor xenograft mouse model. [Figure 49B] Figure 49B provides data illustrating the findings that CCR4-CAR T cells can cure mogamulizumab-resistant tumors. Figure 49B provides data illustrating the findings that CCR4-CAR T cells can cure mogamulizumab-refractory HH tumors. [Figure 50A] The findings illustrate that CCR4-CAR T cells can be efficiently and safely established from patient-derived PBMCs. Figure 50A shows the expression of CCR4-CAR and CD19-CAR on T cells 7, 14 and 24 days after T cell stimulation. [Figure 50B] Figure 50B illustrates the findings that CCR4-CAR T cells can be efficiently and safely established from patient-derived PBMCs. Figure 50B shows a significant reduction in contaminating ATLL cells in CCR4-CAR T cell products but not in CD19-CAR T cell products. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] Detailed Description The present invention is based on the discovery that CCR4-CAR T cells, containing anti-CCR4 CARs derived from mogamulizumab, are highly effective at killing tumor cells, including mogamulizumab-refractory tumor cells. Efficacy was shown to be associated with depletion of Th2, Treg and Th17 subsets (all CCR4 positive) by peer combat while sparing Th1 subsets (CCR4 negative).

[0110] The present invention provides compositions and methods for modified immune cells or their precursors (e.g., modified T cells) that contain a chimeric antigen receptor (CAR) that can bind to CCR4 (anti-CCR4 CAR). Also provided are methods of using anti-CCR4 CAR-expressing cells to treat cancer, and T cell depletion systems for use in combination with anti-CCR4 CAR cell therapy. In one aspect, the present invention provides a method of treating cancer, comprising administering modified cells that contain anti-CCR4 CAR to a subject with cancer, wherein the modified cells deplete CCR4-positive T cells and do not deplete CCR4-negative T cells in the subject, thereby treating cancer.

[0111] Optimization of the CAR construct, especially the scFv, can enhance CAR potency. Thus, in one aspect, the present disclosure identifies highly effective CCR4-CAR T cells for the treatment of T cell malignancies and explores the impact of potential CCR4-CAR-mediated sibling competition between normal T cells and CAR T cell products. In another aspect, the present disclosure provides a T cell depletion system for use as a safety switch in combination with anti-CCR4 CAR cell therapy to shut down CAR T cells when off-tumor toxicity is observed.

[0112] It is to be understood that the methods described in this disclosure are not limited to the particular methods and / or experimental conditions disclosed herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0113] Furthermore, the experiments described herein use conventional molecular cell biology and immunological techniques within the skill of those skilled in the art, unless otherwise indicated.Such techniques are well known to skilled workers and are fully described in the literature.See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0114] A. definition Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any potential meaning uncertainty, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Unless otherwise stated, the use of "or" means "and / or." The use of the term "including" and other forms such as "includes" and "included" are non-limiting.

[0115] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and widely used in the art.The methods and techniques provided herein are generally performed according to conventional methods well known in the art, unless otherwise indicated, as described in various general and more specific references cited and discussed throughout this specification.Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as generally accomplished in the art or as described herein.The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, and the test procedures and techniques thereof are well known and widely used in the art.Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0116] In order that this disclosure may be more readily understood, selected terms are defined below.

[0117] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0118] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are reasonable in practicing the disclosed methods.

[0119] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation.Activation can also be associated with induced cytokine production and detectable effector function.The term "activated T cells" refers specifically to T cells that undergo cell division.

[0120] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0121] The term "antigen" as used herein is defined as a molecule that provokes an immune response. This immune response may include either or both antibody production or activation of specific immunologically competent cells. Those skilled in the art will appreciate that virtually any macromolecule can act as an antigen, including any protein or peptide.

[0122] Furthermore, the antigen can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0123] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into that individual.

[0124] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0125] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0126] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

[0127] The term "downregulation," as used herein, refers to a decrease or elimination of gene expression of one or more genes.

[0128] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the present invention. The immune response can be readily assessed by a myriad of art-recognized methods. Those skilled in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on a number of factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0129] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and which is usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0130] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue or system.

[0131] The term "epitope" as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response and induce a B-cell response and / or a T-cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. In general, an epitope is approximately the size of about 10 amino acids and / or sugars. Preferably, an epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. In general, one skilled in the art will understand that the overall three-dimensional structure of the molecule, rather than the specific linear sequence, is the primary criterion for the specificity of an antigen, and thus, this distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0132] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0133] The term "expanded" as used herein refers to an increase in number, such as an increase in the number of T cells. In one embodiment, ex vivo expanded T cells are increased in number relative to the number originally present in the culture. In another embodiment, ex vivo expanded T cells are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0134] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0135] "Expression vector" refers to a vector that contains a recombinant polynucleotide that contains an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) that incorporate a recombinant polynucleotide.

[0136] "Identity" as used herein refers to the identity of subunit sequences between two amino acid molecules, such as between two polymer molecules, particularly between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that are matched or identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0137] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0138] The term "immunosuppression" is used herein to refer to the overall reduction of the immune response.

[0139] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0140] "Lentivirus" as used herein refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors.HIV, SIV and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0141] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0142] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of response in a subject, compared to the level of response in the subject in the absence of treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0143] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0144] The term "oligonucleotide" typically refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it is understood that this nucleotide sequence also includes an RNA sequence (i.e., A, U, C, G) (where "U" is replaced by "T").

[0145] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding said protein may, in some version, contain introns.

[0146] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0147] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and they can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0148] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, also commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0149] The term "specifically binds" as used herein with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in and of itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in and of itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in relation to the interaction of an antibody, protein or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure rather than the entire protein. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0150] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures.

[0151] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0152] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0153] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, farm animals and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as monkeys and non-human primate mammals. Preferably, the subject is a human.

[0154] "Target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0155] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of antigen. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (a) and beta (β) chains, although in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0156] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0157] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0158] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder from which a subject suffers.

[0159] A "vector" is a composition of material that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0160] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0161] B. Chimeric Antigen Receptor The present invention provides compositions and methods comprising a chimeric antigen receptor (CAR) capable of binding to CCR4. The CAR of the present invention comprises an anti-CCR4 antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the anti-CCR4 antigen binding domain is an anti-CCR4 scFv. In some embodiments, the CAR comprises an anti-CCR4 scFv, a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. In some embodiments, the CAR further comprises a leader sequence and a hinge domain. In a preferred embodiment, the anti-CCR4 scFv has a heavy chain and a light chain derived from mogamulizumab.

[0162] Also provided are compositions and methods for engineered immune cells or precursors thereof, e.g. engineered T cells, comprising CAR. Thus, in some embodiments, immune cells are genetically engineered to express CAR. Also provided are nucleic acids encoding CARs, vectors comprising said nucleic acids, and engineered cells (e.g. engineered T cells) comprising said CARs, vectors, or nucleic acids. In certain embodiments, the nucleic acids encode CARs comprising an anti-CCR4 antigen binding domain, a transmembrane domain, and an intracellular domain, and further encode a safety switch for inducing CAR T cell exhaustion in a subject when the safety switch substance is administered. The CAR can be linked to the safety switch via a 2A self-cleaving sequence as described herein, such as a P2A or T2A sequence. In one embodiment, the safety switch is a truncated EGFR (EGFRt) and the safety switch substance is an anti-EGFR antibody, such as cetuximab. In one embodiment, the safety switch is CD20 and the safety switch substance is an anti-CD20 antibody, such as rituximab. In one embodiment, the safety switch is inducible caspase 9 (iCasp9) and the safety switch agent is a dimerizing drug such as AP1903. In one embodiment, the safety switch is herpes simplex virus type 1 thymidine kinase (HSVTK) and the safety switch agent is ganciclovir (GCV).

[0163] In certain embodiments, the CAR-linked safety switch further comprises a dominant-negative TGFb receptor type II (dnTGFbRII). In some embodiments, the CAR of the CAR-linked safety switch is linked to the dnTGFbRII via a 2A self-cleaving sequence, e.g., as described herein, such as a P2A or T2A sequence. In some embodiments, the safety switch of the CAR-linked safety switch is linked to the dnTGFbRII via a 2A self-cleaving sequence, e.g., as described herein, such as a P2A or T2A sequence. In some embodiments, the CAR and the safety switch of the CAR-linked safety switch are each linked to the dnTGFbRII via a 2A self-cleaving sequence, e.g., as described herein, such as a P2A or T2A sequence. In some embodiments, the CAR-linked safety switch comprises an anti-CCR4 CAR, an HSVTK safety switch, and a dnTGFbRII, and the anti-CCR4 CAR, HSVTK, and dnTGFbRII can be present in any order from the N-terminus to the C-terminus of the CAR-linked safety switch.

[0164] The antigen binding domain of the CAR is operably linked to another domain of the CAR, such as a hinge, transmembrane domain, or intracellular domain, each of which is described elsewhere herein, for expression in a cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a hinge and / or transmembrane domain, and is further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0165] The antigen-binding domain described herein can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that can be included in the CAR of the present invention.The CAR of the present invention can also include a leader sequence as described herein.The CAR of the present invention can also include a hinge domain as described herein.The CAR of the present invention can also include one or more spacer domains or linkers as described herein that can serve to link one domain of the CAR to the next domain.

[0166] Antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids. The CAR of the present invention comprises an antigen-binding domain that can bind to CCR4.

[0167] The antigen-binding domain can include any domain that binds to an antigen, including, but not limited to, a monoclonal antibody (mAb), a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single domain antibody, a full-length antibody or any antigen-binding fragment thereof, a Fab, and a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises a deglycosylated antibody or a fragment thereof or a scFv thereof. In certain embodiments, the antigen-binding domain comprises a defucosylated humanized anti-CCR4 mAb. In certain embodiments, the antigen-binding domain is an scFv with heavy and light chains derived from mogamulizumab.

[0168] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The variable heavy chain (VH) and the variable light chain (VL) are either directly joined or joined by a peptide linker connecting the N-terminus of the VH to the C-terminus of the VL or connecting the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., the CCR4-binding domain) comprises an scFv with the following configuration from N-terminus to C-terminus: VH-linker-VL. In some embodiments, the antigen-binding domain comprises an scFv with the following configuration from N-terminus to C-terminus: VL-linker-VH. Those skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0169] The linker is usually glycine-rich for flexibility, but also serine or threonine-rich for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers, e.g., (GS) n , (SG) n , (GSGGS) n (SEQ ID NO:115), (GGGS) n (SEQ ID NO:116), and (GGGGS) n(SEQ ID NO:117), where n represents an integer of at least 1. Exemplary linker sequences can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:118), GGSGG (SEQ ID NO:119), GSGSG (SEQ ID NO:120), GSGGG (SEQ ID NO:121), GGGSG (SEQ ID NO:122), GSSSG (SEQ ID NO:123), GGGGS (SEQ ID NO:124), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:125), and the like. One of skill in the art would be able to select an appropriate linker sequence for use in the present invention. In one aspect, the antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are linked to a nucleic acid sequence TIFF2024506647000002.tif12161.

[0170] Despite the removal of constant regions and the introduction of linkers, scFv proteins retain the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0171] As used herein, "Fab" refers to the fragment of an antibody structure that binds an antigen but is monovalent and does not have an Fc portion; for example, digestion of an antibody with the enzyme papain produces two Fab fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind antigen).

[0172] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, where each (ab') region contains two separate amino acid chains, a portion of a heavy chain and a light (L) chain, linked by an S-S bond for binding to an antigen, and the remaining heavy chain portions are linked together. The "F(ab')2" fragment can be split into two separate Fab' fragments.

[0173] In some embodiments, the antigen binding domain can be derived from the same species as the species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR can comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain can be derived from a different species than the species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR can comprise a mouse antibody or a fragment thereof, or a humanized mouse antibody or a fragment thereof.

[0174] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO:2, and / or HCDR2 comprises the amino acid sequence of SEQ ID NO:4, and / or HCDR3 comprises the amino acid sequence of SEQ ID NO:6, and / or LCDR1 comprises the amino acid sequence of SEQ ID NO:8, and / or LCDR2 comprises the amino acid sequence of SEQ ID NO:10, and / or LCDR3 comprises the amino acid sequence of SEQ ID NO:12.

[0175] In certain embodiments, the heavy chain variable region (VH) of the antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:112, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:114.

[0176] In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16.

[0177] In certain embodiments, the antigen binding domain comprises a linker. In certain embodiments, the linker comprises SEQ ID NO:125.

[0178] Acceptable variations of antigen-binding domain sequences will be known to those of skill in the art. For example, in some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 16, 112, 114, and 125.

[0179] Transmembrane domain The CAR of the present invention may comprise a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen-binding domain and the intracellular domain of the CAR.

[0180] In some embodiments, the transmembrane domain naturally associates with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid such domains binding to the transmembrane domain of the same or a different surface membrane protein in order to minimize interactions with other members of the receptor complex.

[0181] The transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein, for example, type I transmembrane protein.When the source is synthetic, the transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane domains that are particularly useful in the present invention include, but are not limited to, a transmembrane domain (i.e., comprising at least the transmembrane region thereof) derived from the alpha, beta, or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), ICOS, CD278, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0182] In certain embodiments, the transmembrane domain comprises the transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is the transmembrane domain of CD8α. In certain embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:36.

[0183] In some embodiments, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of a synthetic transmembrane domain.

[0184] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that can be included in a CAR.

[0185] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that may be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0186] In some embodiments, the CAR of the present disclosure comprises a hinge region that connects the antigen binding domain to the transmembrane domain, which in turn connects it to the intracellular domain. The hinge region can preferably assist the antigen binding domain to recognize and bind to the target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows it to adopt many different conformations.

[0187] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region). In certain embodiments, the hinge region is a CD8α hinge. In certain embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO:34.

[0188] The hinge region can have a length of about 4 amino acids to about 50 amino acids, e.g., about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0189] Suitable hinge regions can be readily selected and can be any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of more than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0190] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 115) and (GGGS) n (SEQ ID NO:116), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are less structured and therefore can act as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the phi-psi space than the equivalent alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). An exemplary hinge region is (GGGGS) n(SEQ ID NO:117), GGSG (SEQ ID NO:118), GGSGG (SEQ ID NO:119), GSGSG (SEQ ID NO:120), GSGGG (SEQ ID NO:121), GGGSG (SEQ ID NO:122), GSSSG (SEQ ID NO:123), GGGGS (SEQ ID NO:124), and the like.

[0191] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, an immunoglobulin hinge region may have the following amino acid sequence: TIFF2024506647000003.tif48169, etc.

[0192] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, the hinge region can comprise the sequence His229 of the human IgG1 hinge can be substituted with Tyr, to include TIFF2024506647000004.tif4128; see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can include an amino acid sequence derived from human CD8, or a variant thereof.

[0193] Intracellular signaling domains The CAR of the present invention also comprises an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for the activation of at least one of the effector functions of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular signaling domain transmits effector function signals and directs the cell (e.g., immune cell) to carry out its special function, such as damaging and / or destroying target cells.

[0194] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecules that act in concert to initiate signaling in a T cell, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capability.

[0195] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling, such as CD2, CD5, and CD28. In one embodiment, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0196] In one embodiment, the intracellular signaling domain of the CAR comprises at least one signaling domain from any portion of one or more costimulatory molecules, such as CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capacity, and any combination thereof.

[0197] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma Rila, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0198] Additional examples of intracellular domains include the intracellular signaling domains of several different other immune signaling receptors, including but not limited to, first, second and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory receptors and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0199] Intracellular signaling domains suitable for use in the CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cellular behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but is instead diffused in the cytoplasm.

[0200] Intracellular signaling domains suitable for use in the CARs of the invention comprise an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, where the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises three ITAM motifs.

[0201] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0202] Suitable intracellular signaling domains can be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains can be ITAM motif-containing domains derived from any ITAM motif-containing protein. Therefore, suitable intracellular signaling domains do not need to contain the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0203] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0204] Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0205] The intracellular domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0206] In certain embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB. In certain embodiments, the costimulatory domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:38. In certain embodiments, the intracellular domain comprises the intracellular domain of CD3ζ or a variant thereof. In certain embodiments, the intracellular domain of CD3ζ comprises the amino acid sequence set forth in SEQ ID NO:40. In certain embodiments, the intracellular domain comprises 4-1BB and CD3ζ. In certain embodiments, the intracellular domain comprises the amino acid sequences set forth in SEQ ID NO:38 and SEQ ID NO:40.

[0207] Acceptable variations of individual CAR domain sequences (leader, antigen binding domain, hinge, transmembrane, and intracellular domains) will be known to one of skill in the art. For example, in certain embodiments, the CAR domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:16, 32, 34, 36, 38, and 40.

[0208] In one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an anti-CCR4 antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 30, 54, 62, 70, 82, 90, and 102. In some embodiments, the CAR is linked to a safety switch via a self-cleavable linker, and the CAR-linked safety switch comprises any one of the amino acid sequences as set forth in SEQ ID NOs: 42, 52, 60, 68, 76, 88, and 100.

[0209] Acceptable variations of CAR sequences and CAR-linked safety switch sequences will be known to those of skill in the art. For example, in certain embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102. In certain embodiments, the CAR-linked safety switch comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:42, 52, 60, 68, 76, 88, and 100.

[0210] Table 1 Nucleotide and amino acid sequences TIFF2024506647000005.tif167140TIFF2024506647000006.tif226140TIFF20245066470000 07.tif225140TIFF2024506647000008.tif227140TIFF2024506647000009.tif227140TIFF202 4506647000010.tif225140TIFF2024506647000011.tif226140TIFF2024506647000012.tif22 4140TIFF2024506647000013.tif225140TIFF2024506647000014.tif224140TIFF20245066470 00015.tif226140TIFF2024506647000016.tif227140TIFF2024506647000017.tif225140TIF F2024506647000018.tif227140TIFF2024506647000019.tif227140TIFF2024506647000020.t if225140TIFF2024506647000021.tif227140TIFF2024506647000022.tif224140TIFF2024506 647000023.tif225140TIFF2024506647000024.tif227140TIFF2024506647000025.tif162140

[0211] C. Nucleic acids and expression vectors The present disclosure provides a nucleic acid encoding a CAR. The nucleic acid of the present disclosure can include a polynucleotide sequence encoding any one of the CARs disclosed herein.

[0212] In certain aspects, the present invention includes nucleic acids comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an anti-CCR4 antigen binding domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs), where HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6; and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), where LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:10, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12.

[0213] In certain embodiments, the antigen binding domain comprises a heavy chain variable region encoded by a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:111, and / or a light chain variable region encoded by a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:113.

[0214] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) encoded by a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15.

[0215] In certain embodiments, the nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker. The linker for use in the present disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., multicistronic or bicistronic sequences), which are translated as a polyprotein that is dissociated into separate protein components. In certain embodiments, the nucleic acid comprises, from 5' to 3', a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, the nucleic acid comprises, from 5' to 3', a second polynucleotide sequence, a linker, and a first polynucleotide sequence.

[0216] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the initiation codon, such as ATG, of a protein coding region, leading to cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRES available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES available from, for example, cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select the appropriate IRES for use in the present invention.

[0217] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein, which dissociates into its constituent proteins after translation. The use of the term "self-cleavage" is not intended to imply a proteolytic cleavage reaction. A variety of self-cleaving peptides or 2A peptides are known to those skilled in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1); and cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A", "E2A", "P2A" and "T2A", respectively. One of skill in the art would be able to select an appropriate self-cleaving peptide for use in the present invention.

[0218] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors prior to secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. A variety of furin consensus recognition sequences (or "furin cleavage sites") are known to those of skill in the art and include, but are not limited to, Arg-X1-Lys-Arg (SEQ ID NO:127) or Arg-X1-Arg-Arg (SEQ ID NO:128), X2-Arg-X1-X3-Arg (SEQ ID NO:129) and Arg-X1-X1-Arg (SEQ ID NO:130), such as Arg-Gln-Lys-Arg (SEQ ID NO:131), where X1 is any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those of skill in the art will be able to select an appropriate furin cleavage site for use in the present invention.

[0219] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding a furin cleavage site and F2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and E2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and P2A, and a linker comprising a nucleic acid sequence encoding a furin cleavage site and T2A. Those skilled in the art will be able to select the appropriate combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the furin cleavage site and the 2A peptide. In some embodiments, the linker comprises a furin cleavage site 5' of the 2A peptide. In some embodiments, the linker comprises a 2A peptide 5' of the furin cleavage site. A variety of spacer sequences are known in the art and include, but are not limited to, glycine serine (GS) spacers (also referred to as GS linkers), such as (GS)n, (SG)n, (GSGGS)n (SEQ ID NO:115) and (GGGS)n (SEQ ID NO:116), where n represents an integer of at least 1. Exemplary spacer sequences can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:118), GGSGG (SEQ ID NO:119), GSGSG (SEQ ID NO:120), GSGGG (SEQ ID NO:121), GGGSG (SEQ ID NO:122), GSSSG (SEQ ID NO:123), and the like. One of skill in the art will be able to select an appropriate spacer sequence for use in the present invention.

[0220] In some aspects, the nucleic acids of the disclosure may be operably linked to transcriptional regulatory elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0221] In certain embodiments, the nucleic acid encoding the exogenous CAR is in operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0222] Suitable promoters for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. Suitable promoters for expression in eukaryotic cells include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoter; promoters present in long terminal repeats from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulated promoters, (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0223] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565.

[0224] Suitable promoters for expression in yeast cells are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of one of ordinary skill in the art. Promoters suitable for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, for example, Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma70 promoters, e.g., the consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, e.g., the dps promoter, the spv promoter, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035). Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0225] 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 operably linked to it. Other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0226] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched via induction of an inducible system. Suitable systems for induction of irreversible switches are well known in the art, for example, induction of irreversible switches may utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art may be used to generate irreversibly switchable promoters. Methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly switched promoters and are well known in the art. See, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0227] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducing expression cassette. In one embodiment, the CAR-inducing expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine that is operably linked to a T cell activation response promoter. In some embodiments, the cytokine that is operably linked to a T cell activation response promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0228] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example, but should not be construed as limiting in any way: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Prokaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0229] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated viruses (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0230] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0231] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0232] In some embodiments, an expression vector (e.g., lentiviral vector) may be used to introduce a CAR into immune cells or their precursor cells (e.g., T cells). Thus, an expression vector (e.g., lentiviral vector) of the present invention may comprise a nucleic acid encoding a CAR. In some embodiments, an expression vector (e.g., lentiviral vector) will comprise additional elements that aid in the functional expression of the CAR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding a CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency in the expression of downstream transgenes (e.g., nucleic acid sequences encoding a CAR). Physiological promoters (e.g., EF-1α promoters) may be less likely to induce integration-mediated genotoxicity and may negate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and may be incorporated into the vectors of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises non-essential cis-acting sequences that may improve titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear transport. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element may improve the translation of RNA, improve the expression of the transgene, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence. A variety of post-transcriptional regulator elements are known to those skilled in the art and may be incorporated into the vector (e.g., lentiviral vector) of the present invention.The vector of the present invention may further comprise additional elements such as a rev response element (RRE) for transport of RNA, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeats" or "LTRs" refers to a domain of base pairs located at the end of retroviral DNA, including the U3, R and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vector of the present invention (e.g., lentiviral vector) comprises a 3' U3 deleted LTR. Thus, the vector of the present invention (e.g., lentiviral vector) may comprise any combination of elements described herein to increase the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., lentiviral vector) may comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3' U3 deleted LTR' in addition to the nucleic acid encoding CAR.

[0233] The vector of the present invention can be a self-inactivating vector. The term "self-inactivating vector" as used herein refers to a vector in which the 3' LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, a self-inactivating vector can infect and then be integrated into the host genome (e.g., mammalian genome) only once, but cannot be further passaged. Therefore, a self-inactivating vector can greatly reduce the risk of generating replication-competent virus.

[0234] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into a host cell can be performed in vitro or ex vivo or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising a nucleotide sequence encoding a CAR of the present disclosure in vitro or ex vivo.

[0235] To evaluate the expression of a polypeptide or a portion thereof, the expression vector to be introduced into cells may also contain one or both of a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected by the viral vector.In some embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure.Both the selection marker and the reporter gene may be adjacent to the appropriate regulatory sequence to allow expression in the host cell.Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0236] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and code for polypeptides whose expression is revealed by some easily detectable properties, such as enzymatic activity. The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes may include, but are not limited to, luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes that code for secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0237] In some aspects, the nucleic acids of the disclosure provide for the production of a CAR as described herein, e.g., in a mammalian cell. In some aspects, the nucleic acids of the disclosure provide for the amplification of the nucleic acid encoding the CAR.

[0238] D. Engineered immune cells The present invention provides modified immune cells or precursors thereof (e.g., T cells) comprising a CAR as described herein. Also provided are modified immune cells or precursors thereof comprising a nucleic acid encoding a CAR. Thus, such modified cells possess the specificity directed by the CAR expressed therein. For example, modified cells of the present disclosure comprising a CAR possess specificity for CCR4 on target cells (e.g., cancer cells). In one aspect, the present invention provides a method of treating cancer, comprising administering modified cells comprising an anti-CCR4 CAR to a subject having cancer, wherein the modified cells deplete CCR4-positive T cells and do not deplete CCR4-negative T cells in the subject, thereby treating the cancer.

[0239] Also provided is an engineered immune cell or a precursor thereof comprising a nucleic acid encoding a CAR and further encoding a safety switch, wherein the safety switch induces CAR T cell depletion in a subject when a safety switch substance is administered. The CAR can be linked to the safety switch via a 2A self-cleaving sequence as described herein, such as a P2A or T2A sequence. Thus, the safety switch depletion system comprises an anti-CCR4 CAR (i.e., a CAR-linked safety switch) linked to a safety switch via a self-cleavable linker, and further comprises a safety switch substance. In one embodiment, the safety switch is a truncated EGFR (EGFRt) and the safety switch substance is an anti-EGFR antibody, such as cetuximab. In one embodiment, the safety switch is CD20 and the safety switch substance is an anti-CD20 antibody, such as rituximab. In one embodiment, the safety switch is an inducible caspase 9 and the safety switch substance is a dimerization drug, such as AP1903. In one embodiment, the safety switch is herpes simplex virus type 1 thymidine kinase (HSVTK) and the safety switch agent is ganciclovir (GCV).

[0240] In certain embodiments, the CAR-linked safety switch further comprises a dominant-negative TGFb receptor type II (dnTGFbRII). In some embodiments, the CAR of the CAR-linked safety switch is linked to the dnTGFbRII via a 2A self-cleaving sequence, e.g., as described herein, such as a P2A or T2A sequence. In some embodiments, the safety switch of the CAR-linked safety switch is linked to the dnTGFbRII via a 2A self-cleaving sequence, e.g., as described herein, such as a P2A or T2A sequence. In some embodiments, the CAR and the safety switch of the CAR-linked safety switch are each linked to the dnTGFbRII via a 2A self-cleaving sequence, e.g., as a P2A or T2A sequence. In some embodiments, the CAR-linked safety switch comprises an anti-CCR4 CAR, an HSVTK safety switch, and a dnTGFbRII, and the anti-CCR4 CAR, HSVTK, and dnTGFbRII can be present in any order from the N-terminus to the C-terminus of the CAR-linked safety switch.

[0241] In some embodiments, the CAR-linked safety switch comprises any one of the amino acid sequences set forth in SEQ ID NOs: 42, 52, 60, 68, 76, 88, and 100. Acceptable variations of CAR sequences and CAR-linked safety switch sequences will be known to those of skill in the art. For example, in certain embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 30, 54, 62, 70, 82, 90, and 102. In certain embodiments, the CAR-linked safety switch comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:42, 52, 60, 68, 76, 88, and 100.

[0242] In one aspect, the present invention includes an engineered immune cell or a precursor thereof comprising a CAR comprising an anti-CCR4 antigen binding domain, a transmembrane domain, and an intracellular domain. In another aspect, the present invention includes an engineered immune cell or a precursor thereof comprising a nucleic acid encoding a CAR comprising an anti-CCR4 antigen binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the nucleic acid further encodes a safety switch, and the safety switch induces CAR T cell depletion in the subject when the safety switch substance is administered.

[0243] In certain embodiments, the modified cell is a modified immune cell. In certain embodiments, the modified cell is an autologous cell. In certain embodiments, the modified cell is an autologous cell obtained from a human subject. In certain embodiments, the modified cell is a T cell.

[0244] In certain embodiments, the modified immune cells or precursor cells thereof are further modified to reduce or eliminate CCR4 expression compared to the CCR4 expression level in unmodified immune cells. The knockdown (k / d) or knockout (k / o) of CCR4 in cells can be achieved before, simultaneously with, or after CCR4-CAR transduction. In some embodiments, the CCR4 k / d or k / o can be achieved by any method known in the art for gene k / d or k / o, such as RNAi (shRNA or siRNA), genetic engineering (e.g., via CRISPR / Cas9), or modification of cells with a protein-based system (e.g., anti-CCR4 scFv with one or more C-terminal KDEL motifs). In some embodiments, the modified immune cells or precursor cells thereof further comprise one or more of: (a) a CCR4 null knockout allele; (b) suppressed CCR4 gene expression; and (c) a fusion protein comprising an anti-CCR4 scFv and a KDEL motif, and / or a nucleic acid encoding said fusion protein. In certain embodiments, the CCR4 null knockout allele or suppressed CCR4 gene expression is obtained through genetic engineering techniques that include nucleases selected from the group consisting of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases. In some embodiments, the modified immune cells or their precursors further include an inhibitory RNA molecule that suppresses CCR4 gene expression. Examples of inhibitory RNA molecules include, but are not limited to, RNA interference (RNAi) RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), microRNA (miRNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), single guide RNA (sgRNA), double-stranded RNA (dsRNA), ribozyme, and any combination thereof.

[0245] In some embodiments, the modified immune cells or precursor cells thereof are further modified with a CCR4 targeting RNA (e.g., shRNA) or with a nucleic acid encoding said shRNA. In some embodiments, the cells are further modified with a CCR4 targeting RNA (e.g., guide RNA) and a Cas nuclease, or with a CCR4 targeting RNA and a nucleic acid encoding a Cas nuclease. In some embodiments, the cells are further modified with an anti-CCR4 scFv-KDEL motif fusion protein, or with a nucleic acid encoding an anti-CCR4 scFv-KDEL motif fusion protein.

[0246] In some embodiments, the modified immune cells further comprise a CCR4 targeting shRNA (sh-CCR4). Exemplary sh-CCR4 sequences are shown in Table 4, as is a control shRNA (i.e., a Cre recombinase targeting shRNA sequence (sh-Cre)). In some embodiments, the CCR4 is human CCR4 having an mRNA sequence of NCBI Accession No. NM_005508.4. In some embodiments, the Cre gene sequence is the Cre gene sequence of NCBI Accession No. NC_005856.1. In some embodiments, the modified immune cells further comprise a CCR4 targeting shRNA comprising any one of SEQ ID NOs: 144-147, or a nucleic acid encoding a CCR4 targeting shRNA comprising any one of SEQ ID NOs: 144-147. In some embodiments, the modified immune cells further comprise a nucleic acid encoding a CCR4-targeting shRNA, wherein the nucleic acid comprises any one of SEQ ID NOs: 149-152.

[0247] Table 4: shRNA sequences TIFF2024506647000026.tif202170

[0248] In certain embodiments, the modified immune cells are derived from the peripheral blood mononuclear cells (PBMCs) of the patient.Thus, in some embodiments, the PBMCs comprise tumor cells.In some embodiments, the subject has previously been administered mogamulizumab.In some embodiments, the cancer is refractory to mogamulizumab.

[0249] E. Treatment methods The modified cells (e.g., T cells) described herein can be included in a composition for immunotherapy. The composition can include a pharmaceutical composition and can further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells can be administered.

[0250] In one aspect, the invention includes a method for adoptive cell transfer therapy, comprising administering a modified T cell of the invention to a subject in need thereof. In another aspect, the invention includes a method for treating a disease or condition in a subject, comprising administering a population of modified T cells to a subject in need thereof. In one aspect, the invention provides a method for treating cancer, comprising administering modified cells comprising an anti-CCR4 CAR to a subject having cancer, wherein the modified cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in the subject, thereby treating the cancer.

[0251] Methods for administration of immune cells for adoptive cell therapy are known and may be used with the provided methods and compositions.For example, adoptive T cell therapy is described in, for example, Gruenberg et al., U.S. Patent Application Publication No. 2003 / 0170238; Rosenberg, U.S. Patent No. 4,690,915; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85).See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, where cells are isolated and / or otherwise prepared from the subject who will receive cell therapy or a sample derived from such a subject.Thus, in some aspects, cells are derived from a subject, e.g., a patient, in need of treatment, and the cells are administered to the same subject after isolation and processing.

[0252] In some embodiments, cell therapy, such as adoptive T cell therapy, is carried out by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject that will or will eventually receive cell therapy, such as the first subject.In such embodiments, the cells are then administered to a different subject of the same species, such as the second subject.In some embodiments, the first and second subjects are genetically identical.In some embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0253] In some embodiments, the subject has been treated with a disease or condition, e.g., a tumor-targeting therapeutic agent, prior to administration of the cells or cell-containing composition. In some aspects, the subject is refractory or non-responsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, e.g., chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, the subject has previously been administered mogamulizumab. In some embodiments, the cancer is refractory to mogamulizumab. In some embodiments, administration effectively treats the subject, even though the subject has become resistant to another therapy (mogamulizumab).

[0254] In some embodiments, the subject is responsive to another therapeutic agent, and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits recurrence of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, e.g., at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0255] In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including, for example, chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject having become refractory to another therapy.

[0256] The modified immune cells of the present invention can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to cancer, particularly cell-mediated immune response against tumor cells, when it is desirable to treat or alleviate the disease. The types of cancer to be treated by the modified cells or pharmaceutical compositions of the present invention include certain leukemias or lymphoid tumors, benign and malignant tumors, and malignant diseases, such as sarcomas, carcinomas, and melanomas. Exemplary cancers include, but are not limited to, mature T-cell malignancies, such as adult T-cell leukemia / lymphoma (ATLL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and the like, as well as colon cancer, breast cancer, ovarian cancer, renal cancer, non-small cell lung cancer, melanoma, lymphoma, and hepatocellular carcinoma. The cancer can be a non-solid tumor (such as a hematological tumor) or a solid tumor. Also included are adult tumors / cancers and pediatric tumors / cancers. In one embodiment, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is leukemia and / or lymphoma. In certain embodiments, the cancer cells express CCR4.

[0257] The cells of the invention to be administered can be autologous to the subject undergoing treatment.

[0258] Administration of the cells of the present invention may be performed in any convenient manner known to those skilled in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In other examples, the cells of the present invention are directly injected into a site of inflammation in a subject, a site of local disease in a subject, a lymph node, an organ, a tumor, etc.

[0259] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, the dosage of cells is based in some embodiments on the total number of cells (or number per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg body weight) in an individual population or of an individual cell type. In some embodiments, the dosage is based on a combination of such features, such as the desired total number of cells, the desired ratio, and the desired total number of cells in an individual population.

[0260] In some embodiments, CD8 + T cells and CD4 + A population or subtype of cells, such as T cells, is administered at or within an acceptable variance of a desired dose of total cells, e.g., a desired dose of T cells. In some aspects, the desired dose is a desired number of cells, or a desired number of cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or exceeds a minimum number of cells or a minimum number of cells per unit body weight. In some aspects, among the total cells administered at a desired dose, individual populations or subtypes are administered at or above a desired output ratio (CD4 + vs. CD8 + (e.g., a ratio of 0.1 to 0.5), e.g., within a certain allowed variation or error of such ratio.

[0261] In some embodiments, the cells are administered at or within a tolerable difference of the desired dose of one or more of the individual populations or subtypes of cells, e.g., the desired dose of CD4+ cells and / or the desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is or exceeds the minimum number of cells of a population or subtype, or the minimum number of cells of a population or subtype per unit body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose and a desired ratio of total cells, and / or based on one or more, e.g., a desired fixed dose of each, of the individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or minimum dose of T cells and a desired number of CD4+ cells per unit body weight. + Cells vs. CD8 + Based on the desired ratio of cells and / or CD4 + Cells and / or CD8 + Based on a desired fixed or minimum dose of cells.

[0262] In certain embodiments, a distinct population of cells, or subtypes of cells, is administered to a subject in the range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values). , about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges.

[0263] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is greater than or equal to 1×10 cells. 5 pcs or approx. 1×10 5 pieces / kg ~ approx. 1×10 11 cells / kg, 10 cells 4 ~10 pieces 11 Pieces or about 10 11 pieces / kilogram (kg) body weight, e.g., 10 cells 5 ~10 6 In the range of 1 x 10 cells / kg body weight, for example, 5 pcs or approx. 1×10 5 cells / kg, cells 1.5×10 5 cells / kg, cells 2×10 5 cells / kg, or 1 x 10 cells 6 For example, in some embodiments, the cells are 10 T cells / kg body weight. 4 Pieces or about 10 4 ~10 pieces 9 Pieces or about 109 cells / kilogram (kg) body weight, e.g., 10 T cells 5 ~10 pieces 6 / kg body weight or within a certain margin of error, e.g., 1 x 10 T cells 5 pcs or approx. 1×10 5 cells / kg, T cells 1.5×10 5 cells / kg, T cells 2×10 5 cells / kg, or 1 x 10 T cells 6 In other exemplary embodiments, the dosage range of modified cells suitable for use in the methods of the present disclosure is about 1×10 cells / kg body weight. 5 cells / kg~cells approx. 1×10 6 cells / kg, approximately 1×10 cells 6 cells / kg~cells approx. 1×10 7 cells / kg, approximately 1×10 cells 7 cells / kg~cells approx. 1×10 8 cells / kg, approximately 1×10 cells 8 cells / kg~cells approx. 1×10 9 cells / kg, approximately 1×10 cells 9 cells / kg~cells approx. 1×10 10 cells / kg, approximately 1×10 cells 10 cells / kg~cells approx. 1×10 11 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1×10 cells / kg. 8 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1×10 cells / kg. 7 In other embodiments, a suitable dosage is about 1×10 total cells / kg. 7 ~ Total cells: approx. 5 x 10 7 In some embodiments, a suitable dosage is about 1×10 total cells. 8 ~ Total cells: approx. 5 x 10 8 In some embodiments, a suitable dosage is about 1.4 x 10 total cells. 7 Total cells: approx. 1.1 x 10 9 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 7×10 total cells. 9 There are 10.

[0264] In some embodiments, the cells are 4 or about 10 4 ~10 9 or about 10 9 CD4 count / kilogram (kg) of body weight + and / or CD8 + Cells, e.g., 10 5 ~10 6 CD4 count / kg body weight + and / or CD8 + in cells, or within a certain margin of error, e.g., 1 x 10 5 Or about 1 x 10 5 CD4 cells / kg + and / or CD8 + cells, 1.5 x 10 5 CD4 cells / kg + and / or CD8 + cells, 2 x 10 5 CD4 cells / kg + and / or CD8 + cells, or 1 x 10 6 CD4 count / kg body weight + and / or CD8 + In some embodiments, the cells are administered at a concentration of about 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 More than 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 CD4 + cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 CD8 + cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×106 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 In some embodiments, the cells are administered at about 10 T cells or within a certain margin of error. 8 ~10 pieces 12 Pieces or about 10 10 ~10 pieces 11 T cells, approximately 10 8 ~10 pieces 12 Pieces or about 10 10 ~10 pieces 11 CD4 + cells, and / or about 10 8 ~10 pieces 12 Pieces or about 10 10 ~10 pieces 11 CD8 + The dose is administered at or within a certain range of error in cells.

[0265] In some embodiments, the cells are administered at a desired output ratio or within a tolerated range of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, e.g., in some embodiments, the desired ratio (e.g., CD4 + Cells vs. CD8 +The ratio of cells is 5:1 or about 5:1 to 5:1 or about 5:1 (or greater than about 1:5 and less than about 5:1), or 1:3 or about 1:3 to 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), for example, 2:1 or about 2:1 to 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1, for example, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 , or 1:5, or about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, or about 1:5. In some aspects, tolerated differences are within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[0266] In some embodiments, the dose of modified cells is administered to the subject in need thereof in a single dose or multiple doses.In some embodiments, the dose of modified cells is administered in multiple doses, for example, once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof by rapid intravenous infusion.

[0267] The dosage appropriate for preventing or treating a disease may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, prior therapy, the subject's clinical history and response to the cells, and the judgment of the attending physician. The compositions and cells are, in some embodiments, administered to a subject at one time or over a series of treatments, as appropriate.

[0268] In some embodiments, the cells are administered as part of a combination treatment, e.g., simultaneously with another therapeutic intervention, e.g., an antibody or engineered cell or receptor, or an agent, e.g., a cytotoxic or therapeutic agent, or sequentially in any order. The cells are co-administered in some embodiments simultaneously or sequentially in any order with one or more additional therapeutic agents or in association with another therapeutic intervention. In some situations, the cells are co-administered with another therapy close enough in time that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0269] In certain embodiments, the modified cells of the present invention (e.g., modified cells comprising a CAR) may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PD1 antibody, an anti-CTLA-4 antibody, or an anti-PDL1 antibody). For example, the modified cells may be administered in combination with, for example, an antibody or antibody fragment that targets PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®), or an antigen-binding fragment thereof. In certain embodiments, the modified cells may be administered in combination with an anti-PD-L1 antibody, or an antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi). In certain embodiments, the modified cells may be administered in combination with an anti-CTLA-4 antibody or an antigen-binding fragment thereof. An example of an anti-CTLA-4 antibody includes, but is not limited to, ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may be used, including but not limited to small molecules, siRNA, miRNA, and CRISPR systems. The immune checkpoint modulator may be administered before, after, or simultaneously with the modified cells comprising the CAR. In certain embodiments, combination treatments including immune checkpoint modulators may enhance the therapeutic efficacy of therapies including the modified cells of the present invention.

[0270] Following administration of the cells, in some embodiments, the biological activity of the engineered cell population is measured, for example, by any of a number of known methods. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to antigens, for example, in vivo by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as, for example, cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by evaluating clinical outcomes, such as reduction in tumor burden or tumor burden.

[0271] In certain embodiments, the subject is provided with a secondary treatment, including, but not limited to, chemotherapy, radiation, surgery, and drug therapy.

[0272] In some embodiments, conditioning therapy can be performed on the subject before CAR T cell therapy. In some embodiments, conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, conditioning therapy comprises administering an effective amount of fludarabine to the subject. In a preferred embodiment, conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. The implementation of conditioning therapy before CAR T cell therapy can enhance the efficacy of CAR T cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0273] In some embodiments, certain dosing regimens of the present disclosure include a lymphodepletion step prior to administration of the modified T cells. In exemplary embodiments, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0274] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m 2 In some embodiments, the lymphodepletion step is at about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m 2 / day.

[0275] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, the lymphodepletion step comprises administration of fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and approximately 30 mg / m 2 The treatment involves administration of fludarabine at a dose of 100 mg / day.

[0276] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m for 3 days. 2 / day and fludarabine dosing was 30 mg / m for 3 days. 2 / day.

[0277] Dosing of lymphodepleting chemotherapy may be scheduled for days -6 to -4 (with a -1 day window, i.e., dosing on days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, modified T cells, etc.) infusion on day 0.

[0278] In an exemplary embodiment, for subjects with cancer, the subject is administered 300 mg / m 2 of erythropoietin by intravenous infusion 3 days prior to administration of the engineered T cells. 2 In an exemplary embodiment, for subjects with cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at 300 mg / m by intravenous infusion for 3 days prior to administration of the modified T cells. 2 will undergo lymphodepleting chemotherapy, including cyclophosphamide.

[0279] In an exemplary embodiment, for a subject with cancer, the subject receives about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing a dose of fludarabine for three days.

[0280] In an exemplary embodiment, for a subject with cancer, the subject receives about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing a dose of fludarabine for three days.

[0281] The cells of the present invention can be administered at dosages and routes and times to be determined in appropriate preclinical and clinical experiments and tests. The cell composition can be administered multiple times at dosages within these ranges. The administration of the cells of the present invention can be combined with other methods useful for treating the desired disease or condition as determined by those skilled in the art.

[0282] It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation known as cytokine release syndrome (CRS). CRS is immune activation leading to elevated inflammatory cytokines. CRS is a known on-target toxicity and its occurrence is likely to correlate with efficacy. Clinical and laboratory measures range from mild CRS (systemic symptoms and / or grade 2 organ toxicity) to severe CRS (sCRS; grade ≧3 organ toxicity, aggressive clinical intervention and / or potentially life-threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal dysfunction, liver failure, and disseminated intravascular coagulation. Dramatic elevation of cytokines including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6 has been shown following CAR T cell infusion. One sign of CRS is elevated cytokines including IL-6 (severely elevated), IFN-gamma, TNF-alpha (moderately elevated), and IL-2 (mildly elevated). Elevations of clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with CRS syndrome. The presence of CRS generally correlates with expansion of adoptively transferred cells and progressive immune activation. It has been demonstrated that the severity of CRS is determined by the disease burden at the time of infusion, as patients with high tumor burden experience greater sCRS.

[0283] Therefore, the present invention provides a CRS management strategy suitable for alleviating the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS without compromising the anti-tumor efficacy of engineered cells (e.g., CAR T cells).CRS management strategies are known in the art.For example, systemic corticosteroids may be administered to rapidly reverse the symptoms of sCRS (e.g., grade 3 CRS) without compromising the initial anti-tumor response.

[0284] In some embodiments, anti-IL-6R antibody may be administered.An example of anti-IL-6R antibody is the monoclonal antibody tocilizumab, also known as atlizumab, approved by the US Food and Drug Administration (commercially available as Actemra or RoActemra).Tocilizumab is a humanized monoclonal antibody against interleukin-6 receptor (IL-6R).Administration of tocilizumab has demonstrated almost immediate regression of CRS.

[0285] CRS is generally managed based on the severity of the syndrome observed, and interventions are individualized accordingly. Decisions for management of CRS can be based on clinical signs and symptoms, not just laboratory values, and response to interventions.

[0286] Mild to moderate cases are generally treated with symptom management using fluid therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) and antihistamines as needed for adequate relief of symptoms. More severe cases include patients with any degree of hemodynamic instability; in any hemodynamic instability, administration of tocilizumab is recommended. First-line management of CRS may be tocilizumab IV over 60 minutes at a labelled dose of 8 mg / kg (not to exceed 800 mg / dose) in some embodiments; tocilizumab may be repeated Q8 hours. If the response to the first dose of tocilizumab is suboptimal, additional doses of tocilizumab may be considered. Tocilizumab may be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms with inadequate clinical improvement or poor response to tocilizumab within 12-18 hours may be treated with high-dose corticosteroid therapy, typically hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of CRS. Guidance for CRS management may be based on published criteria (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0287] Concurrent with the clinical signs of CRS, features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007). MAS appears to be a response to immune activation resulting from CRS and should therefore be considered a manifestation of CRS. MAS is similar to HLH, also a response to immune stimulation. The clinical syndrome of MAS is characterized by high-grade non-releasing fever, cytopenias affecting at least two of the three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, as well as reduced circulating natural killer (NK) activity.

[0288] In one aspect, the invention includes a method of treating cancer in a subject in need thereof comprising administering to the subject any one of the modified immune cells or progenitor cells disclosed herein. Yet another aspect of the invention includes a method of treating cancer in a subject in need thereof comprising administering to the subject modified immune cells or progenitor cells produced by any one of the methods disclosed herein.

[0289] F. Source of immune cells In certain embodiments, the source of immune cells (e.g., T cells) is obtained from a subject for ex vivo manipulation. The source of target cells for ex vivo manipulation can also include, for example, autologous or heterologous donor blood, umbilical cord blood or bone marrow. For example, the source of immune cells can be from the subject that is to be treated with the modified immune cells of the present invention, for example, the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human.

[0290] Immune cells can be obtained from several sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, such as lymphocytes, typically myeloid or lymphoid cells, including T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as cells directly isolated from the subject and / or isolated and frozen from the subject.

[0291] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or a granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In certain embodiments, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject, engineered to alter (e.g., induce mutations in) or manipulate expression of one or more target genes, and differentiated, e.g., into a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0292] In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, e.g., those defined by function, activation state, maturation, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art may be used.

[0293] In some embodiments, the method includes isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, preparing the engineered cells includes one or more culturing and / or preparation steps. Cells for manipulation as described can be isolated from a sample, e.g., a biological sample, e.g., obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of cell therapy, or a subject for whom cell therapy will be performed. The subject is, in some embodiments, a human in need of a specific therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells are, in some embodiments, primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or samples that have been processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0294] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy. In certain embodiments, the modified immune cells are derived from the peripheral blood mononuclear cells (PBMCs) of a patient. Thus, in some embodiments, the PBMCs include tumor cells.

[0295] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a heterologous source, e.g., mouse, rat, non-human primate, and pig. In some embodiments, the isolation of cells includes one or more preparative and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, to enrich for desired components, to lyse or remove cells sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0296] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, in some aspects, and in some aspects, cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium. In some embodiments, the methods include density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0297] In one embodiment, the immune cells are cells obtained from the circulating blood of an individual obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps, for example, phosphate-buffered saline (PBS), or the washing solution may be calcium-free, magnesium-free, or many, but not all, divalent cations. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0298] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in cells. In some embodiments, any known method for such marker-based separation can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, the isolation comprises in some aspects the separation of cells and cell populations based on the cellular 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 marker, typically followed by a washing step, and separation of cells that are bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0299] Such separation steps can be based on positive selection, where cells that are bound to the reagent are retained for further use, and / or negative selection, where cells that are not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when an antibody that specifically identifies a certain cell type in a heterogeneous population is not available, whereby separation is best performed based on a marker expressed by cells other than the desired population. Separation does not need to result in 100% enrichment or removal of a particular cell population or cells that express a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells that express a marker, refers to increasing the number or percentage of such cells, but does not need to result in a complete lack of cells that do not express the marker. Similarly, negative selection, removal or depletion of a particular type of cell, such as cells that express a marker, refers to decreasing the number or percentage of such cells, but does not need to result in a complete removal of all such cells.

[0300] In some examples, multiple rounds of separation steps are performed, with the positively or negatively selected fraction from one step being subjected to another separation step, such as a subsequent positive or negative selection. In some examples, cells expressing multiple markers can be simultaneously depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific for a marker that is the target of negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners that are expressed on different cell types.

[0301] In some embodiments, one or more of the T cell populations are positive for (marker+) or express high levels of one or more particular markers, e.g., surface markers (marker+). hlgh ) or cells that are negative for one or more markers (marker-) or express them at relatively low levels (marker low) cells are enriched or depleted. For example, in some aspects, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (such as CD8+ cells or T cells, e.g., CD3+ cells) are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and / or CD62L (i.e., positively selected), and / or depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negatively selected). In some embodiments, cells are enriched or depleted for cells that are positive for or express high surface levels of CD 122, CD95, CD25, CD27, and / or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched for cells that are positive for CD45RO (negative for CD45RA) and positive for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0302] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells such as B cells, on monocytes, or on other white blood cells such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection of markers expressed or expressed to a relatively high degree on one or more naive, memory and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, for example, to improve long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells and CD4+ T cells.

[0303] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies. In some embodiments, CD4+ T cell populations and CD8+ T cell subpopulations, such as subpopulations enriched for central memory (TCM) cells. In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and / or CD 127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, the isolation of CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, the enrichment of central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some aspects, the same CD4 expression-based selection step used to prepare the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, so that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0304] CD4+ T helper cells are sorted into naive, central memory and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0305] In some embodiments, the cells are incubated and / or cultured prior to or in association with genetic manipulation. The incubation step can include culturing, cultivating, stimulating, activating, and / or propagating. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimuli. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of the cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic manipulation, such as the introduction of a recombinant antigen receptor. The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents, such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulatory conditions or agents include one or more agents, such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, e.g., specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28 (e.g., bound to a solid support such as beads), and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies (e.g., at a concentration of at least about 0.5 ng / ml) to the culture medium. In some embodiments, the stimulator includes IL-2 and / or IL-15, e.g., IL-2 at a concentration of at least about 10 units / mL.

[0306] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In either event, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0307] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including but not limited to CD34, CD8, CD14, CD19 and CD56. Depletion of these cells can be accomplished using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to a physical support, and cell-bound antibodies.

[0308] Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, CD4 + To enrich cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

[0309] For the isolation of a desired population of cells by positive or negative selection, the concentration and surface of cells (e.g., particles such as beads) can be altered. In certain embodiments, it may be desirable to greatly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to maximize cell-bead contact. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml is used. Using higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0310] T cells can also be frozen after a washing step that does not require the removal of monocytes. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and would be useful in this context, in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0311] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified populations of T cells, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0312] In certain embodiments, T regulatory cells (Tregs) can be isolated from the sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, Tregs are isolated by flow cytometry sorting. The sample can be enriched for Tregs before isolation by any means known in the art. The isolated Tregs can be cryopreserved and / or expanded before use. Methods for isolating Tregs are described in U.S. Patent Nos. 7,754,482, 8,722,400 and 9,555,105, and U.S. Patent Application No. 13 / 639,927, the contents of which are incorporated herein in their entirety.

[0313] G. Increase in immune cells The cells, either before or after modification of the cells to express a CAR, can be modified with any of the methods described in, e.g., U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; The T cells of the present invention can be activated and expanded in number using methods such as those described in US Patent Publication Nos. 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Publication No. 20060121005. For example, the T cells of the present invention can be expanded by contact with a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface are attached. In particular, the T cell population can be stimulated by contact with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For costimulation of accessory molecules on the T cell surface, a ligand that binds to the accessory molecule is used. For example, T cells can be contacted with anti-CD3 and anti-CD28 antibodies under suitable conditions to stimulate T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention, but other methods and reagents known in the art can be used as well (see, for example, ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0314] The expansion of T cells by the methods disclosed herein can be about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold or more, and any and all whole integers and partial integers therebetween. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold.

[0315] Following culture, the T cells can be incubated in the cell medium in the culture device for a period of time or until the cells reach confluence or a high cell density for optimal passaging before passaging the cells to another culture device. The culture device can be any culture device commonly used for in vitro culture of cells. Preferably, the level of confluence is 70% or more before passaging the cells to another culture device. More preferably, the level of confluence is 90% or more. The period of time can be any time suitable for in vitro cell culture. The T cell medium can be replaced at any time during the culture of the T cells. Preferably, the T cell medium is replaced about every 2-3 days. The T cells are then harvested from the culture device, after which the T cells can be used immediately or cryopreserved and stored for later use. In one aspect, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing the nucleic acid into the T cells.

[0316] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding the chimeric membrane protein.

[0317] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 can be an alternative or an addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors or other factors such as those described in U.S. Patent No. 5,199,942, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding T cells includes culturing T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0318] The culturing step as described herein (following contact with an agent as described herein or electroporation) can be very short, e.g., less than 24 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as further described herein (contact with an agent as described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0319] Various terms are used to describe cultured cells. Cell culture generally refers to cells taken from a living organism and grown under controlled conditions. Primary cell culture is a culture of cells, tissues or organs taken directly from an organism, before the first subculture. Cells are expanded in culture when the cells are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger cell population. When cells are expanded in culture, the proliferation rate of the cells is typically measured by the time required for the number of cells to double, otherwise known as the doubling time.

[0320] Each round of subculture is called a passage. When cells are subcultured, they are said to be passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of times they have been subcultured. For example, a cultured cell population that has been subcultured 10 times may be called a P10 culture. A primary culture, i.e., the first culture after isolating cells from tissue, is called P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there may be many population doublings during the subculture period; therefore, the population doubling number of a culture is greater than the number of passages. The expansion of cells between subcultures (i.e., the number of population doublings) depends on many factors, including but not limited to the seeding density, substrate, medium, and subculture interval.

[0321] In one embodiment, cells may be cultured for a few hours (about 3 hours) to about 14 days or any integer value of time therebetween. Conditions suitable for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Media 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta and TNF-α or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, either serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for T cell growth and expansion, with addition of amino acids, sodium pyruvate and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be infused into subjects. Target cells are grown under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2). 2 ) is maintained below

[0322] The medium used to culture T cells can include an agent that can costimulate T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. The cells isolated by the method disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold, or more. In one embodiment, human regulatory T cells are expanded via KT64.86 artificial antigen presenting cells (aAPCs) coated with anti-CD3 antibodies. Methods for expanding and activating T cells can be found in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entireties.

[0323] In one embodiment, the method of expanding T cells can further include isolating the expanded T cells for further application. In another embodiment, the method of expanding can further include subsequent electroporation of the expanded T cells before culturing. Subsequent electroporation can include introducing a nucleic acid encoding an agent into the expanded T cell population, such as transducing the expanded T cells with a nucleic acid, transfecting the expanded T cells, or electroporating the expanded T cells, and the agent further stimulates the T cells. The agent can stimulate the T cells, such as by further expanding, stimulating effector function or another T cell function.

[0324] H. Pharmaceutical Compositions and Formulations Also provided are immune cell populations of the present invention, compositions containing such cells and / or enriched with such cells, for example, compositions in which the cells expressing CAR account for at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells or a certain type of cells, such as T cells or CD8+ cells or CD4+ cells, in the composition.The compositions are, among others, pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.Also provided are therapeutic methods for administering the cells and compositions to subjects, for example, patients.

[0325] Also provided are compositions that contain cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions that contain the number of cells for administration at a given dose or fraction thereof.Pharmaceutical compositions and formulations generally contain one or more optional pharmaceutically acceptable carriers or excipients.In some embodiments, the composition contains at least one additional therapeutic agent.

[0326] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined, in part, by the particular cell and / or by the method of administration. Thus, there is a wide variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0327] In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0328] The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition treated by the cells, preferably with complementary activities to the cells, each of which does not adversely affect the other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmacologic active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition, in some embodiments, contains the cells in an amount effective to treat or prevent the disease or condition, such as a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodically evaluating the subject being treated. The desired dosage can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by administration of continuous infusions of cells.

[0329] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The composition is provided in some embodiments as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially for administration by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0330] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, in a solvent mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard textbooks may be consulted for preparing appropriate preparations.

[0331] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical dosage forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0332] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0333] The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0334] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications and equivalent substitutions may be made without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. Although specific embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0335] Experimental Examples The invention will now be described with reference to the following examples, which are provided for purposes of illustration only, and the invention is not limited to these examples, but includes all variations that are evident as a result of the teachings provided herein.

[0336] material and method Generation of CCR4-CAR T cells: Eight anti-CCR4 scFvs were constructed from four anti-CCR4 mAbs. Each heavy chain variable region (H) and light chain variable region (L) were fused via a four-repeat linker in either H-to-L or L-to-H orientation (Table 2). The scFvs were subcloned into a second-generation scaffold CAR containing 4-1BB and CD3ζ (Supplementary Figure S1A). A control CD19-CAR containing 4-1BB and CD3ζ was constructed as previously described (Milone MC, et al. Mol Ther. 2009;17(8):1453-1464.). T cells from normal donors were stimulated with anti-CD3 / 28 beads and transduced with CAR using lentivirus at a multiplicity of infection (MOI) of 4 and expanded without any cytokine supplementation.

[0337] Table 2. List of CCR4-CARs and the mAbs from which they are derived TIFF2024506647000027.tif81165NT, N-terminus; ECL, extracellular loop; ND, not determined; Kd values ​​are from data as antibody, not as scFv.

[0338] Cell lines: T-cell lymphoma-derived cell lines HH (ATCC CRL-2105), MJ (ATCC CRL-8294) and HuT78 (ATCC TIB-161) were obtained from the American Type Culture Collection (ATCC). B-ALL-derived cell line Nalm6 was a gift from Joseph A Fraietta, University of Pennsylvania. Cell lines were transduced with lentivirus to express click beetle luciferase green and GFP (CBG-GFP). HH cells were further transduced to express truncated CD19 (HH-CBG-GFP-t19) and then sorted by INFLUX cell sorter (BD Biosciences) for purification. To mimic CCR4 and CAR double positive T cell populations, HH cells were transduced with either CCR4(KW_L2H)-CAR or CCR4(h1567_L2H)-CAR using the same lentivirus used for regular T cell transduction; HH-CCR4(KW_L2H)CAR and HHCCR4(h1567_L2H)CAR, respectively. All cell lines were authenticated by the Genetics Core at the University of Arizona and tested for the presence of mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza). HH and Nalm6 were maintained in culture in RPMI (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Seradigm), and MJ and HuT78 were maintained in culture in IMDM (Gibco, Life Technologies) supplemented with 20% FBS.

[0339] Luciferase-based killing assay: CAR T cells and 1 × 10 4were co-cultured with target cells at the indicated effector-target ratios (E:T ratios) in round-bottom 96-well plates (Coster). Triton X-100 was added for total lysis control. After 16 h of co-culture, cells were washed with PBS and lysed with 100ul of luciferase cell culture lysis buffer (Promega), followed by one cycle of freeze-thaw to obtain complete lysis. Lysates were analyzed for luciferase activity on a Synergy H4 plate reader (BioTek) using the Luciferase Assay System (Promega). Percent specific lysis was calculated using the formula; % specific lysis = [(experimental lysis - spontaneous lysis) / (maximum lysis - spontaneous lysis)] x 100.

[0340] 51 Cr release assay: target cells 51 Cr (PerkinElmer) for 2 hours and washed twice with RPMI medium. CAR T cells and 1 × 10 4 Labeled cells were co-cultured for 4 h at the indicated E:T ratios in 96-well round-bottom plates. Sodium dodecyl sulfate (SDS) was added for total cell lysis control. Supernatants from each well were transferred to a LumaPlate-96 (PerkinElmer) and analyzed using a TopCount plate reader (PerkinElmer). 51 The amount of radiation emitted by Cr was determined as counts per minute (CPM). Percent specific lysis was calculated using the formula; % specific lysis = [(experimental cpm - native cpm) / (max cpm - native cpm)] x 100.

[0341] Patient Sample Collection: Clinical samples (tumors) were obtained under an Institutional Review Board (IRB) approved protocol in the clinical practice of the University of Pennsylvania. All subjects provided written informed consent in accordance with the Declaration of Helsinki.

[0342] Mouse experiments and bioluminescence imaging (BLI): 5- to 7-week-old NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice (Jackson Laboratories) were inoculated intravenously with HH-CBG-GFP or HH-CBG-GFP-t19, and 7 days after tumor inoculation, 0.5 or 2 × 10 6 Mice were treated with either 10 CAR-positive T cells or untransduced (UTD) T cells. Tumor burden was tracked by BLI with a Xenogen IVIS-200 Spectrum camera (Caliper Life Sciences) and data were analyzed with LivingImage software (PerkinElmer). The Institutional Animal Care and Use Committee of the University of Pennsylvania approved all animal studies, and all animal procedures were performed in the University of Pennsylvania animal facility in accordance with federal and Institutional Animal Care and Use Committee requirements.

[0343] Analysis of mouse peripheral blood: Peripheral blood was obtained by retro-orbital bleeding or cardiac puncture, and cell numbers of each T cell subset (total T cells, CD4, CD8) were quantified by FCM using TruCount tubes (BD Biosciences). Cytokines in serum were analyzed using a high-sensitivity LUMINEX assay as per the manufacturer's instructions (Merck Millipore).

[0344] Immunohistochemical staining (IHC): IHC was performed on paraformaldehyde-fixed paraffin-embedded samples. Antibody specific for CCR4 (clone: ​​polyclonal, catalog number HPA031613) was purchased from Sigma-Aldrich. Antibodies specific for CCL17 (EPR15861, ab195044) and CCL22 (polyclonal, ab9847) were purchased from abcm. Stained slides were scanned at ×20 magnification.

[0345] Flow cytometry (FCM) analysis and antibodies: Antibodies used for FCM analysis are listed in Table 3. Dead cells were gated out by staining with the violet amine-reactive viability dye LIVE-DEAD Violet (Invitrogen). Intracellular staining was performed using the Foxp3 / Transcription Factor Staining Kit (Affymetrix) as per the manufacturer's instructions. Expression of CCR4-CAR and CD19-CAR was detected by biotinylated anti-human Fab and anti-mouse Fab antibodies (Jackson ImmunoResearch), respectively. Purified anti-CCR4 mAb (clone KW-0761) was conjugated with Alexa Fluor™ 680 using the Alexa Fluor™ 680 Antibody Labeling Kit (Thermo Fisher Scientific). Data were collected by either a Fortessa LSRII or LSR II cytometer (BD Biosciences) and analyzed using FlowJo version 10 software (Tree Star).

[0346] Table 3. List of mAbs for FCM and blocking assays TIFF2024506647000028.tif176170

[0347] Degranulation and intracellular cytokine production assays: T cells were stimulated with either control medium, phorbol 12-myristate 13-acetate and ionomycin (PMA-Iono) (Sigma-Aldrich) or tumor cells at a responder:stimulator (R:S) ratio of 1:4 in the presence of monensin (BD biosciences) and CD107a detection antibody. At 6 hours of co-culture, cells were stained for LIVEDEAD violet, surface antigens, and then intracellular cytokines as described in the FCM and antibody sections.

[0348] Statistics: Statistical analysis was performed with Prism 8 (GraphPad Software). Two-tailed Student's t-test was used to compare two groups, and one-way ANOVA with Tukey's post-hoc test was used to compare three or more groups. Survival curves were depicted using the Kaplan-Meier method, and differences between two curves were compared with the log-rank test. A P value <0.05 was considered significant.

[0349] Example 1: CCR4 is a rational target for CART cell therapy To confirm whether CCR4 is a reasonable target for CART cell therapy, we analyzed CCR4 expression on skin lesions from patients with CTCL and normal tissue arrays (TMA) from 27 organs (Figures 1 and 2). Skin tumor cells from all patients highly expressed CCR4, while normal tissues barely expressed CCR4. This is compatible with the acceptable safety profile of anti-CCR4 mAb (mogamulizumab) therapy in the clinic.

[0350] Example 2: Establishment of eight anti-CCR4-CARs with different scFvs To generate CCR4-targeted CARs for adoptive cell therapy of T-cell lymphomas and other cancers, we engineered a panel of eight anti-CCR4-CARs (CCR4-CARs), each with different spatial combinations of heavy (H) and light (L) binding domains derived from humanized anti-CCR4 monoclonal antibodies; KW-0761 (mogamulizumab) (Ishii T, et al. Clin Cancer Res. 2010;16(5):1520-1531), h1567, and its affinity-tuned derivatives (Chang DK, et al. Mol Cancer Ther. 2012;11(11):2451-2461). The constructed scFvs were cloned into a CAR backbone plasmid carrying the 4-1BB costimulatory domain and the CD3ζ signaling domain (Table 2 and Figure 3). CAR transduction efficiency, T cell expansion and phenotype within a panel of CCR4-CARs were compared to CD19-directed CAR (CD19-CAR) controls. All versions of CCR4-CAR T cells expanded with anti-CD3 / 28 bead stimulation, but CCR4(KW_H2L / L2H)-CAR T cells, T cells expressing KW-0761-derived scFv, showed superior expansion and resulted in the highest CAR T cell product yield (Figure 4 and Figure 5). CCR4(KW_L2H)-CAR T cells were the lowest among the eight CCR4-CAR T cells (Figure 6), suggesting a lower activation state. Superior T cell expansion was associated with high T cell survival (Figure 7). All CCR4-CAR T cells maintained a less differentiated central memory phenotype compared to CD19-CAR T cells and UTD cells (Figure 8). Interestingly, autoselection of CAR-positive cells was observed to different degrees in six of the eight CCR4-CAR T cell products, notably CCR4(KW_H2L / L2H)-CAR T cells, which showed comparable or lower transduction efficiency than the other CCR4-CAR T cells on day 2, but showed nearly 100% CAR expression on day 10 (Figures 9 and 10).

[0351] Example 3: CCR4 (KW_H2L and KW_L2H)-CAR T cells induce complete depletion of CCR4-positive cells To assess whether T cell sibling competition contributes to the enrichment of CCR4-CAR T cell expression in the CCR4-CAR T cell product, CCR4 expression levels on CAR positive and negative populations after CAR T cell expansion were measured by flow cytometry using the anti-CCR4 monoclonal antibody D8SEE, which does not bind to the same epitope as the monoclonal antibody used for CCR4-CAR construction (Figure 11). Rapid loss of CCR4 expression and corresponding decrease in CD4 / 8 ratio occurred in all CAR negative T cell populations (Figures 12, 13 and 14). Similarly, CCR4(KW_H2L / L2H)-CAR T cells rapidly lost CCR4 expression (Figures 12 and 13), suggesting that T cell sibling competition contributed to the enrichment of CCR4-CAR positive T cells in the product. Surprisingly, the CCR4(h1567,1-49)-CAR-positive cell population was relatively resistant to sib competition; abundant CCR4-positive cells remained in the CAR-positive population at days 6 and 10 (Figure 12 and Figure 13), suggesting that the degree of sib competition in the CAR-positive population was anti-CCR4 scFv dependent. To understand why some CCR4-CAR T cells expressing surface detectable CCR4 did not kill neighboring CCR4-expressing CCR4-CAR T cells, in contrast to other versions of CCR4-expressing CCR4-CAR T cells susceptible to sibling competition, and to investigate the mechanism of resistance to sibling competition of CAR-positive T cell populations, a CCR4-positive T cell line (HH) was transduced with either CCR4(KW_L2H)-CAR or CCR4(h1567_L2H)-CAR to mimic a CCR4-CAR and CCR4 double positive T cell population (Figure 15). We hypothesized that CCR4(h1567_L2H)-CAR is unable to kill CCR4-expressing CCR4(h1567_L2H)-CAR T cells due to blocked recognition of the CCR4 epitope. Therefore, the binding of anti-CCR4 clone h1567 and clone KW-0761 monoclonal antibodies to HH cells expressing either CCR4(h1567_L2H)-CAR or CCR4(KW_L2H)-CAR was tested.When HH cells expressed CCR4(h1567_L2H)-CAR, anti-CCR4 clone h1567 monoclonal antibody failed to recognize CCR4 expressed on the surface of CAR T cells, whereas anti-CCR4 clone KW-0761 detected CCR4 surface expression. In HH cells expressing CCR4(KW_L2H)-CAR, CCR4 detection was reduced when CCR4 expression levels were assessed using anti-CCR4 clone KW monoclonal antibody (Figure 17). These results suggest that CCR4-CAR specifically cis-blocks the CCR4 epitope, thereby causing resistance to sibling competition, and show that the degree of cis-binding of anti-CCR4 scFv to CCR4 is scFv-dependent. This phenomenon is consistent with previous findings that expression of CD19-CAR on CD19-positive cells can cis-block the CD19 epitope, resulting in resistance to CAR-mediated killing.

[0352] Example 4: Sibling competition alters the proportions of helper T cell subsets with reduced Th2-, Th17-, and Treg-associated cytokine production To investigate how sibling competition of CCR4-CAR T cells affects the distribution of CD4+ T cell subsets in the CCR4-CAR T cell product, the levels of Th1, Th2, Th17 and Treg subsets as well as the cytokine profile were measured. It was observed that compared to CD19-CAR T cells, CCR4-CAR T cell products contained a significantly reduced percentage of CD4+ Th2 subsets as defined by the following flow cytometry signature; CD4(+) CCR4(-) CXCR3(+) CCR6(-) CCR10(-), but maintained the levels of CD4+ Th1 subsets; CD4(+) CCR4(-) CXCR3(+) CCR6(-) and CCR10(-) (Figure 17). Treg subsets, as identified by FoxP3 and CD25, were largely absent and did not differ between CD19-CAR T cell products and CCR4-CAR T cell products (data not shown). In addition, upon stimulation with PMA-ionomycin, CCR4-CAR T cells produced reduced levels of Th2, Th17 and T-reg associated cytokines while maintaining production of Th1 associated cytokines, INF-γ and proinflammatory cytokines (TNF-α and IL-2) (Figure 18). These results suggested that transduction of T cells with CCR4-CAR resulted in depletion of CCR4 positive Th2 and Th17 and Treg subsets by sibling competition, while maintaining the CCR4 negative Th1 CD4+ subset. This depletion may also be beneficial for other CAR T cell products by altering the Th subset profile. To address this, we utilized CCR4-CAR T cells to create a Th1-enriched CD19-CAR T cell product. Addition of only 10% of CCR4-CAR T cells depleted the total CCR4 positive T cell subsets in CD19-CAR T cells (Figure 19), resulting in a relatively Th1 dominated CD19-CAR T cell product (Figure 20).

[0353] Example 5: CCR4-CAR T cells respond to and lyse CCR4-positive T cell tumor cell lines in vitro As target antigen density influences CAR T cell responses, the lytic activity of a panel of CCR4-CAR T cells was tested against three cell lines derived from patients with T cell malignancies that display a wide range of CCR4 expression (Figure 21). All of the CCR4-CAR T cells lysed the CCR4 expressing cell lines. No difference in lytic activity was observed between the CCR4-CAR T cells against HH, the cell line with the highest CCR4 expression. For both intermediate and low CCR4 expressing cell lines, MJ and HuT78, respectively, CCR4(KW_H2L / L2H)-CAR T cells showed the highest killing capacity and CCR4(1-49_H2L / L2H)-CAR T cells had the lowest killing capacity (Figure 22). All CCR4-CAR T cells degranulated upon CCR4 stimulation as reflected by CD107a upregulation, whereas anti-CCR4(1-49_H2L / L2H)-CAR T cells and CCR4(h1567_H2L / L2H)-CAR T cells showed target cell-independent degranulation at baseline (Figure 23). All CCR4-CAR T cells secreted IFN-γ, IL-2 and TNF-α upon antigen-independent stimulation with PMA / ionomycin.

[0354] Example 6: CCR4-CAR T cells eradicate tumor cells in a HH cell xenograft mouse model To compare the in vivo antitumor efficacy of CCR4-CAR, HH cell xenograft mice were treated with a low dose (0.5 × 10 6) of CAR-positive T cells or control UTD T cells to reveal differences in efficacy (Figure 24). Tumor burden was followed over 6 months by BLI to assess long-term remission. CCR4(KW_H2L / L2H)-CAR T cells showed superior anti-tumor efficacy, but only CCR4(KW_L2H)-CAR T cells cured all mice (Figure 25) with 100% survival efficacy throughout the experiment (Figure 26). Superior anti-tumor efficacy was associated with better T cell engraftment; anti-CCR4(KW_H2L / L2H)-CAR T cells induced robust T cell engraftment (Figure 27). Additionally, CCR4(h1567_L2H)-CAR T cells showed mild anti-tumor efficacy and prolonged survival efficacy. No obvious non-tumor-related toxicity, including GVHD and weight loss, was observed throughout the experiment (Figure 28). Based on these results, CCR4(KW_L2H)-CAR T cells and CCR4(h1567_L2H)-CAR T cells were selected for further evaluation.

[0355] Example 7: Antitumor efficacy and T cell engraftment of CCR4-CAR T cells is comparable or superior to CD19-CAR T cells To further evaluate the in vivo efficacy of CCR4-CAR T cells, their ability to clear tumors expressing both CCR4 and CD19 was compared to that of validated anti-CD19-CAR T cells. HH cells were transduced with CD19 to obtain CCR4 and CD19 double positive cells. Cells were sorted to achieve 100% expression of CD19 by FCM (HH-CBG-GFP-t19) (Figure 29). NSG mice were injected with 1x10 6 HH-CBG-GFP-t19 cells were inoculated, and 7 days after tumor inoculation, 2 × 10 6CAR-positive T cells or UTD cells (Figure 30). CD19-CAR T cells and CCR4(h1567_L2H)-CAR T cells showed similar antitumor effects against CCR4- and CD19-expressing tumors, whereas CCR4(KW_L2H)-CAR T cells induced complete remission (Figure 31). In addition, CCR4(KW_L2H)-CAR T cells showed higher levels of T cell engraftment than CD19-CAR T cells at an earlier stage (day 7) and comparable T cell engraftment at a later stage (day 14; Figures 32 and 33). Very few T cells were detectable in the peripheral blood at day 28, except for one mouse in the CD19-CAR T cell group that developed GVHD and showed a rapid increase in T cells in the peripheral blood (Figure 33). CCR4(KW_L2H)-CAR T cells and CD19-CAR T cells produced comparable levels of IFN-γ and TNF-α, whereas CCR4(h1567_L2H)-CAR T cells secreted lower levels of cytokines (Figure 34). These results establish the in vitro and in vivo efficacy of primary CCR4(KW_L2H)-CAR T cells against CCR4-expressing tumor lines.

[0356] Example 8: CCR4-CAR T cells respond to and lyse primary CTCL cells To verify that CCR4(KW_L2H) CAR T cells could target and kill T cell lymphoma patient samples, their lytic activity against patient-derived primary CTCL cells was tested. First, it was confirmed that the isolated tumor cells expressed phenotypic markers characteristic of CTCL; CD4(+), CCR4(+), CD7(-), CD25(-), CD157(-), and also expressed CCR4 by flow cytometry32 (Figure 35). In addition, it was also confirmed that the patient samples contained a low frequency of normal cells (less than 5%). When co-cultured with CTCL patient samples, CCR4(KW_L2H)-CAR T cells were activated as demonstrated by CD107a upregulation and secreted IFN-γ, IL-2 and TNF-α cytokines (Figure 36). Importantly, CCR4(KW_L2H)-CAR T cells specifically and efficiently lysed CTCL patient tumor cells (Figure 37).

[0357] Example 9: Anti-CCR4 chimeric antigen receptor T cells provide a T cell depletion system Anti-CCR4-CAR T cells (CCR4CART cells) effectively lysed target tumor cells as well as normal CCR4-expressing T cells, including regulatory T cells (Tregs) and type 2 helper T cells (Th2). While depletion of these cell fractions could “boost” the CCR4CART cell product by relatively increasing Th1 and CD8 T cells, complete loss of normal Tregs could potentially cause severe autoimmune reactions upon clinical conversion. Reported cases of Stevens-Johnson syndrome with high infiltration of CD8 T cells after mogamulizumab therapy may be associated with Treg depletion. Furthermore, CCR4 expression has been reported in normal organs, including kidney, lung, and pancreas in several databases, but fortunately, the expression level is low and no severe organ-specific adverse effects other than manageable thrombocytopenia have been reported in clinical studies of mogamulizumab. Nonetheless, the enhanced activity of the CCR4-CAR T cell therapy described herein may induce such toxicity. Therefore, modulation of CCR4-CAR via suicide systems, strategies of depletion of CAR T cells with chemotherapy or antibiotics, or inducible CAR expression are strategies envisaged herein.

[0358] As safety switches to shut down CAR T cells when anti-CCR4 CAR T cell-mediated toxicity is observed and / or detected, several studies have been conducted to cleave truncated EGFR (EGFRt) via a P2A self-cleaving sequence (Michael C. Jensen et al. US Patent No. US8802374B2), CD20 (Sarah K. Tasian et al. Blood. 2017 Apr 27; 129(17): 2395-2407), inducible caspase 9 (Karin C. Straathof et al. Blood. 2005 Jun 1; 105(11): 4247-4254), or herpes simplex virus type 1 thymidine kinase (HSVTK) (Science.1997 Jun 1; 105(11): 4247-4254). The CCR4CAR(KW_L2H) lentiviral vector was further modified to have a safety switch containing the EGFR-binding domain (KW_L2H) (Figure 38-Figure 41). Each of these systems is expected to induce CART cell depletion upon administration of the corresponding safety switch agent, i.e., anti-EGFR monoclonal antibody (mAb) (e.g., cetuximab), anti-CD20 mAb (e.g., rituximab), dimerizing agent (AP1903), and ganciclovir (GCV). Co-expression of tEGFR and CCR4CAR on T cells has been confirmed (data not shown). Additionally, using the HSVTK depletion system, CCR4CART cells are depleted in a dose-dependent manner by GCV (Figure 42).

[0359] Some tumors, including ATLL, generate a high TGFb tumor microenvironment (Blood (2011) 118 (7): 1865-1876.), which may induce immune cell dysfunction. In addition, TGFb induces CCR4 expression on T cells after anti-CD3 or anti-CD3 / 28 expression, which may result in unwanted killing of CART cells by self-combat. Therefore, we further modified the anti-CCR4CAR(KW-L2H)-HSVTK plasmid vector by inserting a dominant-negative TGFb receptor type II (dnTGFbRII) that can block TGFb signaling (Proc Natl Acad Sci US A. 1997 Mar 18;94(6):2386-91.). Three CCR4CAR(KWL2H)-HSVTK-dnTGFbRII constructs were generated with different orders of CAR, HSVTK and dnTGFbRII; dnTGFbRII-CCR4CAR(KW_L2H)-HSVTK (front), CCR4CAR(KW_L2H)-dnTGFbRII-HSVTK (middle) and CCR4CAR(KW_L2H)-HSVTK-dnTGFbRII (rear) (Figures 43-45). These vectors produced cytokines similarly (data not shown), with dnTGFbRII-CCR4CAR(KW_L2H)-HSVTK (front) tending to induce the highest killing activity (Figure 46).

[0360] Combining these systems with CCR4-CAR can overcome the most important problem for CCR4-CAR T cell therapy, i.e., extratumoral toxicity, and increase its feasibility for clinical use. Also, ATLL, the target tumor of CCR4CART cells, produces high levels of TGFb. Blocking TGFbRII can rescue CART cells from dysfunction in the tumor microenvironment. In addition, blocking TGFb signaling can also abrogate CCR4 overexpression, avoiding the loss of effective CART cells due to undesirable sibling competition.

[0361] Example 10: Knockdown of CCR4 to prepare a CCR4-CAR T cell product free of sibling-phase competition events (CCR4 k / d CCR4-CAR T cells) As demonstrated herein, CCR4-CAR T cells cross-kill and ultimately eradicate CCR4-positive T cells in CAR T cell products, leading to enhanced CCR4-CAR T cell production efficiency and function. A further approach envisioned herein to reduce or eliminate sibling rivalry of CCR4-expressing T cells involves knocking down (k / d) or knocking out (k / o) CCR4 in cells prior to CCR4-CAR transduction. This can be achieved by any method known in the art, for example, by further modification of T cells using RNAi (shRNA or siRNA), genetic engineering (e.g., via CRISPR / Cas9), or protein-based systems (e.g., anti-CCR4 scFv with C-terminal KDEL motif fusion). To this end, four CCR4-targeting shRNAs (sh-CCR4) were constructed, as well as a Cre-targeting shRNA (sh-Cre) as a control (Table 4). sh-Cre or sh-CCR4 shRNA was cloned into pLVSIN-mU6-MCS-PGK-Neo plasmid using EcaRI and BamHI sites. shRNA was tested in CCR4 expressing cell lines HH and ATN-1 (Figure 47). All sh-CCR4 could induce CCR4 knockdown with various levels of efficiency; sh-CCR4#1 and #2 similarly induced efficient CCR4 k / d, while sh-CCR4#4 induced moderate CCR4 k / d and CCR4#3 showed mild CCR4 k / d.

[0362] To establish CCR4-CAR T cells with minimal or no sibling-phase competition events, primary T cells were transduced with sh-CCR4 for CCR4 k / d on day 1 after anti-CD3 / 28 bead stimulation, and then transduced with CCR4CAR(KW_L2H) on days 3 and 4 (Figure 48A). Similar to the efficiency on the cell lines, both shCCR4 induced CCR4 k / d with varying efficiency; sh-CCR4#1 and #2 similarly induced the most efficient CCR4 k / d, while sh-CCR4#4 induced a moderate CCR4 k / d and CCR4#3 showed a mild CCR4 k / d (Figure 48B).

[0363] Importantly, the most efficient T cell proliferation and CAR transduction were achieved in CAR T cells that were not transduced with shCCR4, and similar efficiencies were achieved for CAR T cells transduced with the least efficient shCCR4 (shCCR4#3) (Figure 48C and Figure 48D). In addition, the most efficient sh-CCR4 (sh-CCR4#1) for CCR4 k / d rather exacerbated efficient T cell proliferation and CAR transduction.

[0364] These results confirm that sibling competition events (mutual killing of CCR4-positive cells in the CCR4-CAR T cell product) are beneficial for CCR4-CAR T production. Nevertheless, by curating sh-CCR4 constructs, we can achieve CAR transduction and T cell expansion efficiency levels comparable to those of the original CCR4-CAR T cell product.

[0365] Example 11: CCR4-CAR T cells (KW_L2H) can eradicate mogamulizumab-resistant T cell tumors in vivo In clinical settings, CCR4-CAR T cells are administered to patients with refractory or relapsed disease after mogamulizumab treatment. A concern is epitope blocking with prior mAb therapy, since CCR4-CAR T cells and mogamulizumab share their epitopes on the CCR4 antigen. To address this, CCR4-CAR T cells were tested in a mogamulizumab-refractory T cell lymphoma mouse model (Figure 49A). Two doses of mogamulizumab were used in combination with PBMC to treat HH tumors in NSC mice, resulting in refractory disease. The refractory tumors were then treated with CCR4-CAR(KW_L2H) T cells. CCR4-CAR T cells effectively suppressed and eradicated tumors even in tumors that had not received prior mogamulizumab treatment, suggesting the feasibility of CCR4-CAR T cell therapy for mogamulizumab-pretreated and refractory tumors (Figure 49B).

[0366] Example 12: CCR4-CAR T cells can be safely and efficiently established from patient-derived PBMCs highly contaminated with tumor cells ATLL patients are typically undergoing a variety of treatments, and PBMCs from these patients are contaminated with tumor cells. To investigate whether CCR4-CAR T cells can be generated from such patients, PBMCs were collected from patients with ATLL (70% of blood cells were tumor cells) and CART production was tested. PBMCs from patients and normal donors were transduced with either CD19CAR or CCR4CAR (KWL2H) on day 1 after anti-CD3 / 28 stimulation. Patient-derived PBMCs showed lower transduction efficiency compared to those from normal donors, but finally, the percentage of CCR4-CAR positive cells increased in both patient- and normal donor-derived products, reaching more than 90% (Figure 50A). In addition, contaminating CD7(-), CADM1(+) and CCR4(+) tumor cells were mostly eradicated in the CCR4-CAR T cell product but not in the CD19-CAR T cell product (3.03% vs. 40.2% in the CAR-positive T cell population, and 0% vs. 24.5% in the CAR-negative T cell population) (Figure 50B). This result supports the safe and efficient establishment of CCR4-CAR T cells from patient-derived PBMCs that are highly contaminated with tumor cells.

[0367] Example 13: CCR4-CAR T cells for the treatment of T cell malignancies and solid tumors in vivo Mature T-cell lymphomas, including CTCL, ATLL, and PTCL, are generally associated with poor prognosis. New therapies, such as anti-CCR4 mAb (mogamulizumab), can delay tumor progression, but curative treatment has rarely been achieved. This study demonstrates that mogamulizumab-derived CCR4-CAR T cells can effectively target both CCR4-positive tumor cell lines and primary CTCL cells and eradicate established tumors in preclinical models, which was superior to the previously reported h1567- and mAb2-3-derived CAR T cells (Perera LP, et al. Am J Hematol. 2017;92(9):892-901). This study also demonstrates the mechanism of sibling-phase competition events and its association with CAR T cell function; the superior antitumor effect of CCR4-CAR T cells is associated with the depletion of CCR4-positive T cells by rapid and complete sibling-phase competition. Furthermore, the data presented herein reveal that the degree of sibling competition is scFv-dependent and affects the degree to which CCR4-CAR can mask the target CCR4 in cis. KW-0761 only partially binds to CCR4 in cis, allowing sibling competition to occur without interference, whereas CARs derived from h1567 and its derivatives almost completely block the epitope in cis, allowing CCR4-CAR-positive cells to persist. This observation is consistent with previous reports that CD19-CARs unintentionally introduced into leukemic B cells induced leukemia resistance by binding to the CD19 epitope on the surface of leukemic cells and masking it from recognition by CD19-CAR T cells.

[0368] Since Th1 subset plays a central role in anti-cancer immunity, whereas Th2 and Treg likely act in opposition to it, it is assumed herein that a Th1-dominant CAR T cell product compared to Th2 and Treg will enhance the anti-tumor activity of CAR T cells. CD19-CAR T cells achieved such an "enhanced" CAR T cell product by mixing with a small amount of CCR4 CAR T cells, indicating that CCR4-CAR T cells can be utilized as a tool to improve other CAR T cell products. This result also indicates that the host's normal CCR4-positive T cells can be depleted by CCR4-CAR T cells, as shown in mogamulizumab treatment. Th2-mediated inflammation is often beneficial for tumor growth and cancer immune evasion, and higher Th2 infiltration is associated with poor treatment outcomes. Treg also has an important role in tumor growth and immune evasion. Tregs can suppress effector T cell-mediated tumor killing, and high Treg infiltration into the TME is associated with poor outcomes in many types of tumors, including colon, breast, ovarian, renal, non-small cell lung, melanoma, lymphoma, and hepatocellular carcinoma. Even temporary depletion of Tregs can enhance the efficacy of tumor vaccine therapy in animal models. Therefore, it is assumed herein that depletion of CCR4-positive Tregs and Th2 by CCR4-CAR T cells generally contributes to anti-tumor immunity against cancer in addition to direct killing of CCR4-positive tumor cells. Indeed, mogamulizumab has been tested as a Treg depletion therapy in patients with solid tumors. Significant reductions in CCR4-positive T cells and immune responses against cancer / testis (CT) antigens and autoantibody responses against thyroid peroxidase were observed in several patients after mogamulizumab.

[0369] In summary, this study identifies highly active CCR4-CAR T cells derived from KW-0761 (mogamulizumab). Efficacy was shown to be associated with depletion of Th2, Treg and Th17 subsets (all CCR4 positive) by self-combat while sparing Th1 subsets (CCR4 negative). Furthermore, it is envisioned herein that the CCR4-CAR T cell format will be beneficial for the treatment of patients with T cell malignancies as well as for Th2 and Treg depletion therapy to treat other cancers, including solid tumors.

[0370] Enumerated Aspects The following enumerated aspects are provided, but the numbering should not be construed as designating a level of importance. Aspect 1 provides: An anti-CC chemokine receptor 4 (CCR4) chimeric antigen receptor (CAR), comprising an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain. Aspect 2 provides: The CAR of embodiment 1, wherein said anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Aspect 3 provides: The CAR of embodiment 1 or embodiment 2, wherein said anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. Aspect 4 provides: The CAR of any of aspects 1-3, wherein said anti-CCR4 antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody. Aspect 5 provides the following: The CAR of any of aspects 1-4, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single chain variable fragment (scFv). Aspect 6 provides the following: The CAR of any of aspects 1 to 5, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single chain variable fragment (scFv) having a heavy chain and a light chain derived from mogamulizumab. Aspect 7 provides the following: The CAR of any of embodiments 1-6, wherein said anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:112. Aspect 8 provides the following: The CAR of any of embodiments 1-7, wherein said anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:114. Aspect 9 provides the following: The CAR of any of embodiments 1-8, wherein the anti-CCR4 antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16. Aspect 10 provides the following: The CAR of any of the preceding embodiments, wherein said CAR further comprises a CD8 alpha hinge, optionally wherein said CD8 alpha hinge comprises the amino acid sequence depicted in SEQ ID NO:34. Aspect 11 provides the following: The transmembrane domain is Artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, the transmembrane domain of CD28, the transmembrane domain of CD3 epsilon, the transmembrane domain of CD45, the transmembrane domain of CD4, the transmembrane domain of CD5, the transmembrane domain of CD8, the transmembrane domain of CD9, the transmembrane domain of CD16, the transmembrane domain of CD22, the transmembrane domain of CD33, the transmembrane domain of CD37, the transmembrane domain of CD64, the transmembrane domain of CD80, the transmembrane domain of CD86, the transmembrane domain of OX40 (CD134), the transmembrane domain of 4-1BB (CD137), the transmembrane domain of ICOS, and the transmembrane domain of CD154. or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 12 provides the following:

[0023] The CAR of any preceding embodiment, wherein said transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally said CD8 alpha transmembrane domain comprises the amino acid sequence depicted in SEQ ID NO:36. Aspect 13 provides the following: The CAR of any preceding embodiment, wherein said intracellular domain comprises a costimulatory domain and an intracellular signaling domain. Aspect 14 provides the following:

[0023] The CAR of any of the preceding embodiments, wherein said intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 15 provides the following: the intracellular domain being The costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:38.

[0023] The CAR of any preceding embodiment, comprising: Aspect 16 provides the following:

[0023] The CAR of any of the preceding embodiments, wherein said intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 17 provides the following: the intracellular signaling domain is The intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:40.

[0023] The CAR of any preceding embodiment, comprising: Aspect 18 provides the following:

[0023] The CAR of any preceding embodiment, comprising an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an intracellular domain comprising a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. Aspect 19 provides the following: The CAR of embodiment 18, further comprising a CD8 leader sequence optionally comprising the amino acid sequence set forth in SEQ ID NO:32. Aspect 20 provides the following: The CAR of any of the preceding embodiments, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102. Aspect 21 provides the following: The CAR of any of the preceding embodiments, encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 29, 53, 61, 69, 81, 89, and 101. Aspect 22 provides the following: The CAR of any of the previous embodiments, wherein the CAR is linked to a safety switch via a self-cleavable linker, and the safety switch binds to a safety switch agent. Aspect 23 provides the following: 23. The CAR of embodiment 22, wherein the safety switch is selected from: (a) truncated EGFR (EGFRt), where the safety switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), where the safety switch agent is AP1903; and (d) herpes simplex virus type 1 thymidine kinase (HSVTK), where the safety switch agent is ganciclovir (GCV). Aspect 24 provides the following: The CAR of embodiment 22 or embodiment 23, wherein the CAR is linked to a dominant negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker. Aspect 25 provides the following: The CAR of embodiment 22 or embodiment 23, wherein said safety switch is linked to a dominant negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker. Aspect 26 provides the following: A nucleic acid comprising a polynucleotide sequence encoding an anti-CCR4 CAR, wherein the CAR comprises an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain. Aspect 27 provides the following: 27. The nucleic acid of embodiment 26, wherein said anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Aspect 28 provides the following: 28. The nucleic acid of embodiment 25 or embodiment 27, wherein said anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. Aspect 29 provides the following: The nucleic acid of any of aspects 26 to 28, wherein the anti-CCR4 antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody. Aspect 30 provides the following: 30. The nucleic acid of any of aspects 26 to 29, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single chain variable fragment (scFv). Aspect 31 provides the following: The nucleic acid of any of aspects 26 to 30, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single chain variable fragment (scFv) having a heavy chain and a light chain derived from mogamulizumab. Aspect 32 provides the following: 32. The nucleic acid of any of embodiments 26-31, wherein the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:112. Aspect 33 provides the following: 33. The nucleic acid of any of embodiments 26-32, wherein the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:114. Aspect 34 provides the following: 34. The nucleic acid of any of embodiments 26-33, wherein the anti-CCR4 antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16. Embodiment 35 provides the following: The nucleic acid of any of the preceding embodiments, wherein said CAR further comprises a CD8 alpha hinge, and optionally said CD8 alpha hinge comprises the amino acid sequence shown in SEQ ID NO:34. Aspect 36 provides the following: The transmembrane domain is Artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, the transmembrane domain of CD28, the transmembrane domain of CD3 epsilon, the transmembrane domain of CD45, the transmembrane domain of CD4, the transmembrane domain of CD5, the transmembrane domain of CD8, the transmembrane domain of CD9, the transmembrane domain of CD16, the transmembrane domain of CD22, the transmembrane domain of CD33, the transmembrane domain of CD37, the transmembrane domain of CD64, the transmembrane domain of CD80, the transmembrane domain of CD86, the transmembrane domain of OX40 (CD134), the transmembrane domain of 4-1BB (CD137), the transmembrane domain of ICOS, and the transmembrane domain of CD154. or a transmembrane domain derived from a Killer Immunoglobulin-Like Receptor (KIR). Aspect 37 provides the following:

[0023] The nucleic acid of any preceding embodiment, wherein said transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally said CD8 alpha transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:36. Embodiment 38 provides the following:

[0023] The nucleic acid of any preceding embodiment, wherein the intracellular domain comprises a costimulatory domain and an intracellular signaling domain. Aspect 39 provides the following:

[0023] The nucleic acid of any of the preceding embodiments, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 40 provides the following: the intracellular domain being The costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:38. 4. The nucleic acid of any preceding aspect, comprising: Aspect 41 provides the following:

[0023] The nucleic acid of any of the preceding embodiments, wherein said intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 42 provides the following: the intracellular signaling domain is The intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:40. 4. The nucleic acid of any preceding aspect, comprising: Aspect 43 provides the following:

[0023] The nucleic acid of any of the preceding embodiments, wherein the CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an intracellular domain comprising a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. Aspect 44 provides the following: 44. The nucleic acid of embodiment 43, wherein the CAR further comprises a CD8 leader sequence optionally comprising the amino acid sequence depicted in SEQ ID NO:32. Aspect 45 provides the following: The nucleic acid of any of the preceding embodiments, wherein the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102. Aspect 46 provides the following: The nucleic acid of any of the preceding embodiments, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 29, 53, 61, 69, 81, 89, and 101. Aspect 47 provides the following:

[0023] The nucleic acid of any of the preceding embodiments, wherein the CAR is linked to a safety switch via a self-cleavable linker, and the safety switch binds to a safety switch agent. Aspect 48 provides the following: 48. The nucleic acid of embodiment 47, wherein the safety switch is selected from: (a) truncated EGFR (EGFRt), where the safety switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), where the safety switch agent is AP1903; and (d) herpes simplex virus type 1 thymidine kinase (HSVTK), where the safety switch agent is ganciclovir (GCV). Aspect 49 provides the following: The nucleic acid of embodiment 47 or embodiment 48, wherein said CAR is linked to a dominant negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker. Embodiment 50 provides the following: The nucleic acid of embodiment 47 or embodiment 48, wherein said safety switch is linked to a dominant negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker. Aspect 51 provides the following: A vector comprising the nucleic acid according to any one of embodiments 26 to 50. Embodiment 52 provides the following: 52. The vector of embodiment 51, which is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. Embodiment 53 provides the following: 53. The vector of embodiment 52, wherein the viral vector is a lentiviral vector. Embodiment 54 provides the following: A modified immune cell or a precursor thereof comprising a CAR according to any of embodiments 1 to 25, a nucleic acid according to any of embodiments 26 to 50, and / or a vector according to any of embodiments 51 to 53. Embodiment 55 provides the following: (a) CCR4 null knockout allele; (b) suppressed CCR4 gene expression; and (c) a fusion protein comprising an anti-CCR4 scFv and a KDEL motif, and / or a nucleic acid encoding said fusion protein. The modified immune cell or progenitor thereof of embodiment 54, further comprising one or more of: Embodiment 56 provides the following: 56. The modified immune cell or progenitor thereof of embodiment 55, wherein said CCR4 null knockout allele or suppressed CCR4 gene expression is obtained via a genetic engineering technique comprising a nuclease selected from the group consisting of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease, a transcription activator-like effector nuclease (TALEN), and a zinc finger nuclease. Embodiment 57 provides the following: 57. The modified immune cell or progenitor thereof of embodiment 55 or 56, further comprising an inhibitory RNA molecule that suppresses CCR4 gene expression. Embodiment 58 provides the following: 58. The modified immune cell or a precursor thereof of embodiment 57, wherein said inhibitory RNA molecule is selected from the group consisting of RNA interference (RNAi) RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), microRNA (miRNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), single guide RNA (sgRNA), double-stranded RNA (dsRNA), ribozyme, and any combination thereof. Embodiment 59 provides the following: 59. The modified immune cell or precursor thereof of any of aspects 54 to 58, wherein the modified cell is an autologous cell. Embodiment 60 provides the following: 60. The modified immune cell or precursor thereof of any of aspects 54 to 59, wherein the modified immune cell is derived from a peripheral blood mononuclear cell (PBMC). Aspect 61 provides the following: The modified immune cell, or a precursor thereof, of embodiment 60, wherein said PBMCs comprise tumor cells. Embodiment 62 provides the following: 62. The modified immune cell or progenitor thereof of any of embodiments 54-61, wherein the cell is isolated from a human subject. Embodiment 63 provides the following: The modified immune cell or a precursor thereof of any of aspects 54 to 62, wherein the modified immune cell is a modified T cell. Embodiment 64 provides the following: A method for producing a modified immune cell or a precursor cell thereof, the method comprising introducing into said immune cell or a precursor cell thereof a nucleic acid according to any one of aspects 26 to 50, or a vector according to any one of aspects 51 to 53. Embodiment 65 provides the following: The method of embodiment 58, wherein said vector is introduced via viral transduction. Embodiment 66 provides the following: A method of treating cancer in a subject in need thereof comprising administering to the subject the modified immune cell or progenitor cell of any of aspects 54 to 63, or the modified immune cell or progenitor cell thereof produced by the method of aspect 64 or aspect 65. Aspect 67 provides the following: The method of embodiment 66, wherein said modified cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in said subject, thereby treating said cancer. Embodiment 68 provides the following: The method of embodiment 66 or 67, wherein the subject has previously been administered mogamulizumab. Aspect 69 provides the following: The method of any of aspects 66-68, wherein the cancer is refractory to mogamulizumab. Embodiment 70 provides the following: 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a modified T cell comprising an anti-CCR4 CAR, wherein the CAR comprises an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain. Aspect 71 provides the following: The method of embodiment 70, wherein said anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Aspect 72 provides the following: 72. The method of embodiment 70 or embodiment 71, wherein said anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. Aspect 73 provides the following: The method of any of aspects 70 to 72, wherein the anti-CCR4 antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody. Aspect 74 provides the following: The method of any of aspects 70 to 73, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single chain variable fragment (scFv).

[0023] Aspect 75 provides the following: The method of any of aspects 70 to 74, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single chain variable fragment (scFv) having a heavy chain and a light chain derived from mogamulizumab.

[0023] Aspect 76 provides the following: 76. The method of any of embodiments 70-75, wherein the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:112. Aspect 77 provides the following: 77. The method of any of embodiments 70-76, wherein the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:114. Aspect 78 provides the following: 78. The method of any of embodiments 70-77, wherein the anti-CCR4 antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16. Aspect 79 provides the following: The method of any of the preceding embodiments, wherein said CAR further comprises a CD8 alpha hinge, optionally wherein said CD8 alpha hinge comprises the amino acid sequence set forth in SEQ ID NO:34. Embodiment 80 provides the following: The transmembrane domain is Artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, the transmembrane domain of CD28, the transmembrane domain of CD3 epsilon, the transmembrane domain of CD45, the transmembrane domain of CD4, the transmembrane domain of CD5, the transmembrane domain of CD8, the transmembrane domain of CD9, the transmembrane domain of CD16, the transmembrane domain of CD22, the transmembrane domain of CD33, the transmembrane domain of CD37, the transmembrane domain of CD64, the transmembrane domain of CD80, the transmembrane domain of CD86, the transmembrane domain of OX40 (CD134), the transmembrane domain of 4-1BB (CD137), the transmembrane domain of ICOS, and the transmembrane domain of CD154. or a transmembrane domain derived from a Killer Immunoglobulin-like Receptor (KIR). Aspect 81 provides the following:

[0023] The method of any of the preceding aspects, wherein said transmembrane domain comprises a CD8 alpha transmembrane domain, optionally wherein said CD8 alpha transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:36. Embodiment 82 provides the following:

[0023] The method of any preceding aspect, wherein the intracellular domain comprises a costimulatory domain and an intracellular signaling domain. Embodiment 83 provides the following:

[0023] The method of any of the preceding embodiments, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins within the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Embodiment 84 provides the following: the intracellular domain being The costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:38. 4. The method of any preceding aspect, comprising: Embodiment 85 provides the following:

[0023] The method of any of the preceding embodiments, wherein said intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Embodiment 86 provides the following: the intracellular signaling domain is The intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:40. 4. The method of any preceding aspect, comprising: Aspect 87 provides the following:

[0023] The method of any of the preceding embodiments, wherein said CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an intracellular domain comprising a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. Embodiment 88 provides the following: The method of embodiment 87, wherein the CAR further comprises a CD8 leader sequence optionally comprising the amino acid sequence set forth in SEQ ID NO:32. Aspect 89 provides the following: The method of any of the preceding embodiments, wherein the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:30, 54, 62, 70, 82, 90, and 102. Aspect 90 provides the following: The method of any of the preceding embodiments, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:29, 53, 61, 69, 81, 89, and 101. Aspect 91 provides the following:

[0023] The method of any of the preceding embodiments, wherein the CAR is linked to a safety switch via a self-cleavable linker, and the safety switch binds to a safety switch material. Embodiment 92 provides the following: 92. The method of embodiment 91, wherein the safety switch is selected from: (a) truncated EGFR (EGFRt), where the safety switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), where the safety switch agent is AP1903; and (d) herpes simplex virus type 1 thymidine kinase (HSVTK), where the safety switch agent is ganciclovir (GCV). Aspect 93 provides the following:

[0046] The method of embodiment 91 or embodiment 92, wherein said CAR is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker. Aspect 94 provides the following: 93. The method of embodiment 91 or embodiment 92, wherein said safety switch is linked to the dominant negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker. Embodiment 95 provides the following: The method of any of aspects 91-94, further comprising administering the safety-switch agent to the subject. Aspect 96 provides the following: The method of any of aspects 70-95, wherein said modified T cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in said subject, thereby treating said cancer. Aspect 97 provides the following: The method of any of aspects 70 to 96, wherein the modified T cells are derived from PBMCs. Aspect 98 provides the following: 98. The method of embodiment 97, wherein said PBMCs comprise tumor cells. Aspect 99 provides the following: The method of any of aspects 70 to 98, wherein the modified T cell is a human T cell. The embodiment 100 provides: 99. The method of any of aspects 70 to 99, wherein the modified T cells are autologous. Aspect 101 provides the following: The method of any one of aspects 70 to 100, wherein the subject is a human. Aspect 102 provides: The method of any of aspects 70-101, wherein the subject has previously been administered mogamulizumab. Aspect 103 provides the following: The method of any of aspects 70 to 102, wherein the cancer is refractory to mogamulizumab.

[0371] Other Aspects The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the spirit and scope of the present invention. The appended claims are intended to be construed to cover all such embodiments and equivalent variations.

Claims

1. An anti-CC chemokine receptor 4 (CCR4) chimeric antigen receptor (CAR), comprising an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain.

2. The CAR of claim 1, wherein the anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:

6.

3. The CAR of claim 1 or claim 2, wherein the anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO:

12.

4. The CAR of any one of claims 1 to 3, wherein the anti-CCR4 antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

5. The CAR of any one of claims 1 to 4, wherein the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv).

6. The CAR of any one of claims 1 to 5, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single-chain variable fragment (scFv) having heavy and light chains derived from mogamulizumab.

7. The CAR of any one of claims 1 to 6, wherein the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

112.

8. The CAR of any one of claims 1 to 7, wherein the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

114.

9. The CAR of any one of claims 1 to 8, wherein the anti-CCR4 antigen binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

16.

10. The CAR of any one of claims 1 to 9, wherein the CAR further comprises a CD8 alpha hinge, and optionally the CD8 alpha hinge comprises the amino acid sequence shown in SEQ ID NO:

34.

11. The transmembrane domain is Artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, the transmembrane domain of CD28, the transmembrane domain of CD3 epsilon, the transmembrane domain of CD45, the transmembrane domain of CD4, the transmembrane domain of CD5, the transmembrane domain of CD8, the transmembrane domain of CD9, the transmembrane domain of CD16, the transmembrane domain of CD22, the transmembrane domain of CD33, the transmembrane domain of CD37, the transmembrane domain of CD64, the transmembrane domain of CD80, the transmembrane domain of CD86, the transmembrane domain of OX40 (CD134), the transmembrane domain of 4-1BB (CD137), the transmembrane domain of ICOS, and the transmembrane domain of CD154. The CAR of any one of claims 1 to 10, comprising a transmembrane domain selected from the group consisting of: or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

12. The CAR of any one of claims 1 to 11, wherein the transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally the CD8 alpha transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

36.

13. The CAR of any one of claims 1 to 12, wherein the intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

14. 14. The CAR of any one of claims 1-13, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

15. the intracellular domain being The costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:

38. The CAR of any one of claims 1 to 14, comprising:

16. The CAR of any one of claims 1-15, wherein the intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

17. the intracellular signaling domain is The intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:

40. The CAR of any one of claims 1 to 16, comprising:

18. 18. The CAR of any one of claims 1 to 17, comprising an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain.

19. The CAR of claim 18, further comprising a CD8 leader sequence optionally comprising the amino acid sequence set forth in SEQ ID NO:

32.

20. The CAR of any one of claims 1 to 19, comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 30, 54, 62, 70, 82, 90, and 102.

21. The CAR of any one of claims 1 to 20, encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 29, 53, 61, 69, 81, 89, and 101.

22. 22. The CAR of any one of claims 1 to 21, wherein the CAR is linked to a safety switch via a self-cleavable linker, and the safety switch binds to a safety switch substance.

23. The safety switch, (a) truncated EGFR (EGFRt), where the safety-switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety-switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), wherein the safety-switch agent is AP1903; and (d) Herpes simplex virus type 1 thymidine kinase (HSVTK) in which the safety switch agent is ganciclovir (GCV). The CAR of claim 22, wherein the CAR is selected from the group consisting of:

24. The CAR of claim 22 or claim 23, which is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

25. The CAR of claim 22 or claim 23, wherein the safety switch is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

26. A nucleic acid comprising a polynucleotide sequence encoding an anti-CCR4 CAR, wherein the CAR comprises an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain.

27. The nucleic acid of claim 26, wherein the anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:

6.

28. The nucleic acid of claim 26 or claim 27, wherein the anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO:

12.

29. The nucleic acid of any one of claims 26 to 28, wherein the anti-CCR4 antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

30. The nucleic acid of any one of claims 26 to 29, wherein the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv).

31. The nucleic acid of any one of claims 26 to 30, wherein the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv) having heavy and light chains derived from mogamulizumab.

32. 32. The nucleic acid of any one of claims 26 to 31, wherein the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

112.

33. 33. The nucleic acid of any one of claims 26 to 32, wherein the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

114.

34. The nucleic acid of any one of claims 26 to 33, wherein the anti-CCR4 antigen binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

16.

35. The nucleic acid of any one of claims 26 to 34, wherein the CAR further comprises a CD8 alpha hinge, and optionally, the CD8 alpha hinge comprises the amino acid sequence shown in SEQ ID NO:

34.

36. The transmembrane domain is Artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, the transmembrane domain of CD28, the transmembrane domain of CD3 epsilon, the transmembrane domain of CD45, the transmembrane domain of CD4, the transmembrane domain of CD5, the transmembrane domain of CD8, the transmembrane domain of CD9, the transmembrane domain of CD16, the transmembrane domain of CD22, the transmembrane domain of CD33, the transmembrane domain of CD37, the transmembrane domain of CD64, the transmembrane domain of CD80, the transmembrane domain of CD86, the transmembrane domain of OX40 (CD134), the transmembrane domain of 4-1BB (CD137), the transmembrane domain of ICOS, and the transmembrane domain of CD154.

36. The nucleic acid of any one of claims 26 to 35, comprising a transmembrane domain selected from the group consisting of: or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

37. 37. The nucleic acid of any one of claims 26 to 36, wherein said transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally said CD8 alpha transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

36.

38. The nucleic acid of any one of claims 26 to 37, wherein the intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

39. 39. The nucleic acid of any one of claims 26-38, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

40. the intracellular domain being The costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:

38. The nucleic acid of any one of claims 26 to 39, comprising:

41. 41. The nucleic acid of any one of claims 26-40, wherein the intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

42. the intracellular signaling domain is The intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:

40. The nucleic acid of any one of claims 26 to 41, comprising:

43. 43. The nucleic acid of any one of claims 26 to 42, wherein the CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain.

44. The nucleic acid of claim 43, wherein the CAR further comprises a CD8 leader sequence optionally comprising the amino acid sequence set forth in SEQ ID NO:

32.

45. The nucleic acid of any one of claims 26 to 44, wherein the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 30, 54, 62, 70, 82, 90, and 102.

46. The nucleic acid of any one of claims 26 to 45, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 29, 53, 61, 69, 81, 89, and 101.

47. 47. The nucleic acid of any one of claims 26 to 46, wherein the CAR is linked to a safety switch via a self-cleavable linker, and the safety switch binds to a safety switch substance.

48. The safety switch, (a) truncated EGFR (EGFRt), where the safety-switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety-switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), wherein the safety-switch agent is AP1903; and (d) Herpes simplex virus type 1 thymidine kinase (HSVTK) in which the safety switch agent is ganciclovir (GCV).

48. The nucleic acid of claim 47, selected from:

49. The nucleic acid of claim 47 or claim 48, wherein the CAR is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

50. 49. The nucleic acid of claim 47 or claim 48, wherein the safety switch is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

51. A vector comprising the nucleic acid of any one of claims 26 to 50.

52. 52. The vector of claim 51, which is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

53. The vector of claim 52, wherein the viral vector is a lentiviral vector.

54. 52. A modified immune cell or a precursor thereof comprising a CAR according to any one of claims 1 to 25, a nucleic acid according to any one of claims 26 to 50, and / or a vector according to any one of claims 51 to 53.

55. (a) CCR4 null knockout allele; (b) suppressed CCR4 gene expression; and (c) a fusion protein comprising an anti-CCR4 scFv and a KDEL motif, and / or a nucleic acid encoding said fusion protein.

55. The modified immune cell or progenitor thereof of claim 54, further comprising one or more of:

56. 56. The modified immune cell or progenitor thereof of claim 55, wherein the CCR4 null knockout allele or suppressed CCR4 gene expression is obtained through a genetic engineering technique involving a nuclease selected from the group consisting of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease, transcription activator-like effector nuclease (TALEN), and zinc finger nuclease.

57. 57. The modified immune cell or progenitor thereof of claim 55 or 56, further comprising an inhibitory RNA molecule that suppresses CCR4 gene expression.

58. 58. The modified immune cell or precursor thereof of claim 57, wherein the inhibitory RNA molecule is selected from the group consisting of RNA interference (RNAi) RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), microRNA (miRNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), single guide RNA (sgRNA), double-stranded RNA (dsRNA), ribozyme, and any combination thereof.

59. 59. The modified immune cell or a precursor thereof of any one of claims 54 to 58, wherein the modified cell is an autologous cell.

60. 60. The modified immune cell or a precursor thereof of any one of claims 54 to 59, wherein said modified immune cell is derived from a peripheral blood mononuclear cell (PBMC).

61. 61. The modified immune cell, or a precursor thereof, of claim 60, wherein the PBMC comprises a tumor cell.

62. 62. The modified immune cell, or a precursor thereof, of any one of claims 54 to 61, which is a cell isolated from a human subject.

63. 63. The modified immune cell or a precursor thereof of any one of claims 54 to 62, wherein the modified immune cell is a modified T cell.

64. 52. A method for producing an engineered immune cell or a precursor thereof, comprising introducing into said immune cell or a precursor thereof a nucleic acid according to any one of claims 26 to 50 or a vector according to any one of claims 51 to 53.

65. 65. The method of claim 64, wherein the vector is introduced via viral transduction.

66. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a modified immune cell or progenitor cell described in any one of claims 54 to 63, or a modified immune cell or a progenitor cell thereof produced by the method of claim 64 or claim 65.

67. The pharmaceutical composition of claim 66, wherein the modified cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in the subject, thereby treating the cancer.

68. 68. The pharmaceutical composition of claim 66 or 67, wherein the subject has previously been administered mogamulizumab.

69. The pharmaceutical composition of any one of claims 66-68, wherein the cancer is refractory to mogamulizumab.

70. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a modified T cell comprising an anti-CCR4 CAR, wherein the CAR comprises an anti-CCR4 antigen-binding domain, a transmembrane domain, and an intracellular domain.

71. The pharmaceutical composition of claim 70, wherein the anti-CCR4 antigen binding domain comprises at least one heavy chain variable region (HCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:

6.

72. The pharmaceutical composition of claim 70 or claim 71, wherein the anti-CCR4 antigen binding domain comprises at least one light chain variable region (LCDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO:

12.

73. The pharmaceutical composition of any one of claims 70 to 72, wherein the anti-CCR4 antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

74. The pharmaceutical composition of any one of claims 70 to 73, wherein the anti-CCR4 antigen binding domain is an anti-CCR4 single chain variable fragment (scFv).

75. The pharmaceutical composition of any one of claims 70 to 74, wherein the anti-CCR4 antigen-binding domain is an anti-CCR4 single-chain variable fragment (scFv) having heavy and light chains derived from mogamulizumab.

76. 76. The pharmaceutical composition of any one of claims 70-75, wherein the anti-CCR4 antigen binding domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

112.

77. The pharmaceutical composition of any one of claims 70-76, wherein the anti-CCR4 antigen binding domain comprises a light chain variable region comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

114.

78. The pharmaceutical composition of any one of claims 70-77, wherein the anti-CCR4 antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

16.

79. The pharmaceutical composition of any one of claims 70-78, wherein the CAR further comprises a CD8 alpha hinge, optionally wherein the CD8 alpha hinge comprises the amino acid sequence shown in SEQ ID NO:

34.

80. The transmembrane domain is Artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the transmembrane domains of the alpha, beta, or zeta chains of the T cell receptor, the transmembrane domain of CD28, the transmembrane domain of CD3 epsilon, the transmembrane domain of CD45, the transmembrane domain of CD4, the transmembrane domain of CD5, the transmembrane domain of CD8, the transmembrane domain of CD9, the transmembrane domain of CD16, the transmembrane domain of CD22, the transmembrane domain of CD33, the transmembrane domain of CD37, the transmembrane domain of CD64, the transmembrane domain of CD80, the transmembrane domain of CD86, the transmembrane domain of OX40 (CD134), the transmembrane domain of 4-1BB (CD137), the transmembrane domain of ICOS, and the transmembrane domain of CD154. or a transmembrane domain derived from a Killer Immunoglobulin-Like Receptor (KIR).

81. 81. The pharmaceutical composition of any one of claims 70 to 80, wherein said transmembrane domain comprises a CD8 alpha transmembrane domain, and optionally said CD8 alpha transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

36.

82. The pharmaceutical composition of any one of claims 70-81, wherein the intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

83. 83. The pharmaceutical composition of any one of claims 70-82, wherein the intracellular domain comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or variants thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

84. the intracellular domain being The costimulatory domain of 4-1BB, optionally comprising the amino acid sequence set forth in SEQ ID NO:

38.

84. The pharmaceutical composition of any one of claims 70 to 83, comprising:

85. 85. The pharmaceutical composition of any one of claims 70-84, wherein said intracellular domain comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

86. the intracellular signaling domain is The intracellular signaling domain of CD3ζ or a variant thereof, optionally comprising the amino acid sequence set forth in SEQ ID NO:

40.

86. The pharmaceutical composition of any one of claims 70 to 85, comprising:

87. 87. The pharmaceutical composition of any one of claims 70-86, wherein the CAR comprises an anti-CCR4 antigen binding domain comprising an anti-CCR4 scFv, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain.

88. The pharmaceutical composition of claim 87, wherein the CAR further comprises a CD8 leader sequence optionally comprising the amino acid sequence set forth in SEQ ID NO:

32.

89. The pharmaceutical composition of any one of claims 70-88, wherein the CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 30, 54, 62, 70, 82, 90, and 102.

90. The pharmaceutical composition of any one of claims 70-89, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 29, 53, 61, 69, 81, 89, and 101.

91. 91. The pharmaceutical composition of any one of claims 70-90, wherein the CAR is linked to a safety switch via a self-cleavable linker, and the safety switch binds to a safety switch substance.

92. The safety switch, (a) truncated EGFR (EGFRt), where the safety-switch agent is an anti-EFGR antibody, optionally cetuximab; (b) CD20, where the safety-switch agent is an anti-CD20 antibody, optionally rituximab; (c) inducible caspase 9 (iCasp9), wherein the safety-switch agent is AP1903; and (d) Herpes simplex virus type 1 thymidine kinase (HSVTK) in which the safety switch agent is ganciclovir (GCV).

92. The pharmaceutical composition of claim 91, wherein said pharmaceutical composition is selected from the group consisting of

93. The pharmaceutical composition of claim 91 or claim 92, wherein the CAR is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

94. 93. The pharmaceutical composition of claim 91 or claim 92, wherein the safety switch is linked to a dominant-negative TGFb receptor type II (dnTGFbRII) via a self-cleavable linker.

95. The pharmaceutical composition of any one of claims 91 to 94, for use in combination with said safety switch material.

96. The pharmaceutical composition of any one of claims 70-95, wherein said modified T cells deplete CCR4 positive T cells and do not deplete CCR4 negative T cells in said subject, thereby treating said cancer.

97. The pharmaceutical composition of any one of claims 70-96, wherein the modified T cells are derived from PBMCs.

98. The pharmaceutical composition of claim 97, wherein the PBMCs comprise tumor cells.

99. The pharmaceutical composition of any one of claims 70-98, wherein the modified T cell is a human T cell.

100. The pharmaceutical composition of any one of claims 70-99, wherein the modified T cells are autologous.

101. The pharmaceutical composition of any one of claims 70 to 100, wherein the subject is a human.

102. The pharmaceutical composition of any one of claims 70-101, wherein the subject has previously been administered mogamulizumab.

103. The pharmaceutical composition of any one of claims 70-102, wherein the cancer is refractory to mogamulizumab.

Images