CCR4 targeted chimeric antigen receptor modified t cells for treatment of CCR4 positive malignancies

Abstract

To provide chimeric antigen receptors for use in treating lymphoma-associated C-C chemokine receptor type 4 (CCR4) and other cancers expressing CCR4.SOLUTION: A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a polypeptide, where the CAR or polypeptide comprises an scFv targeting CCR4, a spacer, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain, where the spacer comprises a specific amino acid sequence or a variant thereof in which 1-5 amino acids of the specific amino acid sequence are modified.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] [Claim of priority] This application claims the benefit of U.S. Provisional Application No. 62 / 852,934, filed May 24, 2019, the entire contents of which are incorporated herein by reference.

[0002] [Technical Field] The present disclosure relates to lymphoma-associated CC chemokine receptor type 4 (CCR4)-specific chimeric antigen receptor (CAR) recombinant T cells, methods for their formulation, and methods for their use as anti-cancer agents selective for CCR4-positive cells. [Background technology]

[0003] CCR4 is expressed at high levels on malignant skin-homing T cells, and its surface expression is closely associated with enhanced skin-homing properties of cutaneous T-cell lymphoma (CTCL) cells and unfavorable disease outcomes (Non-Patent Document 1). CARs that target CCR4 using humanized variable heavy chain (Vh) and kappa light chain (Vl) portions can be derived from anti-CCR4 antibodies different from mogamulizumab (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Chang DH,Sui J,Geng S,Muvaffak A,Bai M,Fuhlbrigge RC,et al.Humanization of an anti-CCR4 antibody that kills Cutaneous T-Cell Lymphoma cells and abrogates suppression by T-regulatory cells.Mol.Cancer Ther.2012;11:2451-61 [Non-patent document 2] Perera LP,Zhang M,Nakagawa M,et al.Chimeric antigen receptor modified T cells that target chemokine receptor CCR4 as a therapeutic modality for T cell malignancies.Am.J.Hematol.2017;92(9):892-901 Summary of the Invention

[0005] Described herein are methods of treating various cancers, such as cutaneous T-cell lymphoma (CTCL), using CCR4-targeted CAR T cells (also referred to herein as CCR4 CAR T cells). Furthermore, in addition to methods of use as anti-cancer agents selective for CCR4-positive cells, the present specification describes methods for reducing the population of regulatory T cells (Tregs). Without being bound by theory, Tregs can inhibit desirable immune responses, and the methods described herein can be used to eliminate or reduce the number of CCR4-positive Tregs.

[0006] The present specification describes a nucleic acid molecule comprising a coding nucleotide sequence encoding a chimeric antigen receptor (CAR) or polypeptide, wherein the chimeric antigen receptor or polypeptide comprises an scFv targeting CCR4, a spacer, a transmembrane domain, or a 41-BB costimulatory domain, and a CD3ζ signaling domain.

[0007] In various embodiments, the transmembrane domain is selected from a CD4 transmembrane domain or variant thereof with 1 to 5 amino acid modifications, a CD8 transmembrane domain or variant thereof with 1 to 5 amino acid modifications, or a CD28 transmembrane domain or variant thereof with 1 to 5 amino acid modifications, and the spacer comprises 20 to 150 amino acids. the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-12 or a variant thereof with one to five amino acid modifications; the spacer comprises an IgG hinge region; the spacer comprises 10 to 50 amino acids, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof with one to five amino acid modifications; the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO: 21; a linker of 3 to 15 amino acids is located between the 4-1BB costimulatory domain and the CD3ζ signaling domain or a variant thereof; the CAR or polypeptide comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof with one to five amino acid modifications; and the scFv comprises the amino acid sequence of SEQ ID NO: 1.

[0008] Also disclosed herein are viral vectors comprising the nucleic acid molecules described herein; populations of human T cells (e.g., populations comprising central memory T cells) transduced with vectors comprising the nucleic acid molecules described herein. In some embodiments, the vector is an expression vector in which expression of the CCR4 CAR or CCR4 polypeptide is under the control of an inducible promoter. In some embodiments, expression of the CCR4 CAR or CCR4 polypeptide is under the control of a Tet Off system.

[0009] Also described herein are methods of treating CCR4-positive cancers (including, for example, peripheral T-cell lymphoma, adult T-cell lymphoma, anaplastic large cell lymphoma, primary cutaneous T-cell lymphoma, renal cell carcinoma, lung carcinoma, hepatocellular carcinoma, and diffuse large B-cell lymphoma) in a patient, comprising administering a population of autologous or allogeneic human T cells transduced with a vector comprising a nucleic acid molecule described herein, wherein the T-cell lymphoma comprises cells that express CCR4. In various embodiments, the chimeric antigen receptor or polypeptide is administered locally or systemically; the CCR4-expressing cells are cancerous T cells; and the chimeric antigen receptor or polypeptide is administered in a single or repeated dose.

[0010] In various embodiments, the chimeric antigen receptor or polypeptide is a huCCR4 scFv (e.g., the following: QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 1) and wherein there are no more than 10 single amino acid substitutions).

[0011] In various embodiments, the chimeric antigen receptor or polypeptide is a huCCR4 scFv (e.g., the following: QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTYYRPLDYWGQGTLVTVSSSGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 43) and wherein there are no more than 10 single amino acid substitutions).

[0012] In various embodiments, the chimeric antigen receptor or polypeptide is a huCCR4 scFv (e.g., the following: QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTYYRPLDYWGQGTLVTVSSSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 44) and wherein there are no more than 10 single amino acid substitutions).

[0013] In various embodiments, the chimeric antigen receptor or polypeptide is a huCCR4 scFv (e.g., the following: QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTYYRPLDYWGQGTLVTVSSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 45) and wherein there are no more than 10 single amino acid substitutions).

[0014] Also described are T cells carrying a vector expressing a CAR or polypeptide. In various embodiments, at least 20%, 30%, or 40% of the transduced human T cells are central memory T cells; at least 30% of the transduced human T cells are CD4+ and CD62L+ or CD8+ and CD62L+; the population of human T cells is autologous to the patient; and the population of human T cells is allogeneic to the patient.

[0015] CCR4-targeted CAR The CCR4-targeting CAR or CCR4-targeting polypeptide described herein comprises a CCR4-targeting scFv. In one embodiment, the scFv has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTYYRPLDYWGQGTLVTVSSSGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 43), or the following sequence linked by a flexible linker: QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTYYRPLDYWGQGTLVTVSS (SEQ ID NO: 32) and the following sequence DIVMTQSPDSLAVSLGERATMSCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSYTFGQGTKLEIK (SEQ ID NO: 33).

[0016] In one embodiment, the scFv comprises the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFASQWMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTWYRPLDYWGQGTLVTVSS (SEQ ID NO: 34) and the following sequence: DIVMTQSPDSLAVSLGERATMSCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYISSYTFGQGTKLEIK (SEQ ID NO: 35).

[0017] In one embodiment, the scFv comprises the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFASAWMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARSTYYRPLDYWGQGTLVTVSS (SEQ ID NO: 36) and the following sequence: DIVMTQSPDSLAVSLGERATMSCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYMSSYTFGQGTKLEIK (SEQ ID NO: 37).

[0018] In some embodiments, useful flexible linkers have the sequence GGGS (SEQ ID NO:46) repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, useful flexible linkers have the sequence GGGS (SEQ ID NO:47) repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0019] A useful CCR4 CAR or CCR4 polypeptide can consist of or comprise the amino acid sequence of SEQ ID NO: 38, 39, or 40 (a signal sequence-deficient mature CAR), or a CCR4 CAR or CCR4 polypeptide can consist of or comprise the amino acid sequence of SEQ ID NO: 29, 30, or 31 (a GMCSFRa signal sequence-containing immature CAR). The CAR or polypeptide can be expressed in a form that includes a signal sequence, for example, the human GM-CSF receptor alpha signal sequence (MLLLVTSLLPELPHPAFLLIP; SEQ ID NO: 43). The CAR or polypeptide can be expressed with additional sequences useful for tracking expression, for example, a T2A skip sequence and a truncated EGFRt or a truncated CD19. Thus, a CAR or polypeptide can comprise or consist of the amino acid sequence of SEQ ID NO: 1, 29, 30, 31, 38, 39, 40, 43, 44, or 45, or can comprise or consist of an amino acid sequence that is at least 95%, 96%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 29, 30, 31, 38, 39, 40, 43, 44, or 45. A CAR or polypeptide can consist of or comprise the amino acid sequence of any of SEQ ID NOs: 1, 29, 30, 31, 38, 39, 40, 43, 44, or 45 in which 1, 2, 3, 4, or up to 5 amino acid changes (preferably conservative amino acid changes) have occurred. A CAR or polypeptide can comprise SEQ ID NO: 32 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) and SEQ ID NO: 33 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) linked by a flexible linker. A CAR or polypeptide can comprise SEQ ID NO: 34 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) and SEQ ID NO: 35 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) linked by a flexible linker.The CAR or polypeptide can comprise SEQ ID NO: 36 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) and SEQ ID NO: 37 with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservative amino acid changes) linked by a flexible linker.

[0020] In one embodiment, the nucleic acids encoding the amino acid sequences of SEQ ID NOs: 1, 29-40, and 43-45 are codon-optimized.

[0021] spacer domain The CARs or polypeptides described herein can include a spacer located between the CCR4 targeting domain (i.e., the CCR4-targeting ScFv or variant thereof) and the transmembrane domain. A variety of different spacers can be used, some of which include at least a portion of a human Fc region, such as the hinge portion of a human Fc region, or a CH3 domain or variant thereof. Table 1 below provides various spacers that can be used in the CARs described herein. Table 1: Examples of spacers

[0022] [Table 1] Some spacer domains include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer domains include an immunoglobulin CH3 domain (referred to as CH3 or ΔCH2) or both a CH3 domain and a CH2 domain. The immunoglobulin-derived sequence can include one or more amino acid modifications, e.g., 1, 2, 3, 4, or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0023] The hinge / linker region can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 4) or ESKYGPPCPPCP (SEQ ID NO: 3). The hinge / linker region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO: 3), followed by the linker sequence GGGSSGGGSG (SEQ ID NO: 2), followed by the IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12). Thus, the entire linker / spacer domain can comprise the sequence ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11). In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO: 11. In some cases, the IgG4 Fc hinge / linker region is mutated at two positions (L235E; N297Q) to reduce binding by the Fc receptor (FcR).

[0024] Transmembrane domain A variety of transmembrane domains can be used. Examples of suitable transmembrane domains are listed in Table 2. When a spacer domain is present, the transmembrane domain (TM) is located carboxy-terminal to the spacer domain. Table 2: Examples of transmembrane domains

[0025] [Table 2] Costimulatory domain The costimulatory domain can be any domain suitable for use with the CD3ζ signaling domain. In some cases, the costimulatory domain is a 4-1BB costimulatory domain, including a sequence identical or at least 90%, at least 95%, or at least 98% identical to KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 24). In some cases, the 4-1BB costimulatory domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 24. The costimulatory domain is located between the transmembrane domain and the CD3ζ signaling domain. Table 3 provides examples of CD3ζ signaling domain sequences and suitable costimulatory domains. Table 3: Examples of CD3 ζ domains and costimulatory domains

[0026] [Table 3] In various embodiments, the costimulatory domain is selected from the group consisting of a costimulatory domain shown in Table 3 or a variant thereof with one to five (e.g., one or two) amino acid modifications, a CD28 costimulatory domain or a variant thereof with one to five (e.g., one or two) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof with one to five (e.g., one or two) amino acid modifications, and an OX40 costimulatory domain or a variant thereof with one to five (e.g., one or two) amino acid modifications. In certain embodiments, the 4-1BB costimulatory domain or a variant thereof has one to five (e.g., one or two) amino acid modifications. In certain embodiments, there are two costimulatory domains, for example, a CD28 costimulatory domain or a variant thereof with one to five (e.g., one or two) amino acid modifications (e.g., substitutions) and a 4-1BB costimulatory domain or a variant thereof with one to five (e.g., one or two) amino acid modifications (e.g., substitutions). In various embodiments, the 1 to 5 (e.g., 1 or 2) amino acid modifications are substitutions. The costimulatory domain is amino-terminal to the CD3ζ signaling domain, and a short linker of 2 to 10 amino acids, e.g., 3 amino acids (e.g., GGG), may be positioned between the costimulatory domain and the CD3ζ signaling domain.

[0027] CD3ζ Signaling Domain The CD3ζ signaling domain can be any domain suitable for use with a CD3ζ signaling domain. In some cases, the CD3ζ signaling domain comprises a sequence at least 90%, at least 95%, or at least 98% identical to RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 21). In some cases, the CD3ζ signaling domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 21.

[0028] Truncated EGFR or CD19 The CD3ζ signaling domain is followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 27) and the following sequence: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO: 28) The CD3ζ signaling domain may be followed by a truncated EGFR sequence identical to, or at least 90%, at least 95%, or at least 98% identical to, SEQ ID NO: 28. In some cases, the truncated EGFR has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 28. Alternatively, the CD3ζ signaling domain may be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 27) and the following sequence: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKR (SEQ ID NO: 26) A truncated CD19R (referred to as CD19t) may be located which is identical to, or at least 90%, at least 95%, at least 98% identical to,

[0029] "Amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. An "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. Such substitutions can change the amino acids in the resulting protein in a non-conservative manner (i.e., changing a codon from an amino acid belonging to one group of amino acids of a particular size or property to an amino acid belonging to another group) or in a conservative manner (i.e., changing a codon from an amino acid belonging to one group of amino acids of a particular size or property to an amino acid belonging to the same group). Such conservative changes generally result in less alteration of the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) amino acids with a nonpolar R group: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; 2) amino acids with an uncharged polar R group: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; 3) amino acids with a charged polar R group (negatively charged at pH 6.0): aspartic acid, glutamic acid; 4) basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (positively charged at pH 6.0). Another group is amino acids with a phenyl group: phenylalanine, tryptophan, tyrosine.

[0030] In some cases, CCR4 CAR or CCR4 polypeptides can be produced using vectors containing a T2A ribosomal skip sequence and a truncated EGFR (EGFRt) lacking the cytoplasmic signaling tail following the CAR open reading frame. This sequence provides an inert, non-immunogenic surface marker for positive selection of gene-modified cells and efficient in vivo tracking of therapeutic T cells after adoptive transfer, allowing accurate measurement of gene-modified cells upon coexpression of EGFRt. Efficient control of proliferation to avoid cytokine storms and off-target toxicity is a key hurdle for successful T cell immunotherapy. EGFRt incorporated into CCR4 CAR lentiviral vectors acts as a suicide gene, allowing elimination of CAR+ T cells in the event of treatment-related toxicity.

[0031] The CAR or polypeptide described herein is preferably produced using recombinant DNA technology, but can be produced by any means known in the art. Nucleic acids encoding several regions of the chimeric receptor can be conveniently prepared and the complete coding sequence constructed using standard techniques of molecular cloning known in the art (genomic library screening, overlap PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably T lymphocytes, most preferably autologous T lymphocytes.

[0032] Various T cell subsets isolated from patients can be transduced with vectors for CAR or polypeptide expression. Central memory T cells are one useful T cell subset. Central memory T cells can be isolated from peripheral blood mononuclear cells (PBMCs) by selecting CD45RO+ / CD62L+ cells, for example, using a CliniMACS® device, followed by immunomagnetic selection of cells expressing the desired receptor. Cells enriched for central memory T cells can be activated with anti-CD3 / CD28 and transduced with, for example, a lentiviral vector directing expression of a CCR4 CAR and a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. Activated / genetically modified CCR4 central memory T cells can be expanded in vitro using IL-2 / IL-15 and then cryopreserved. Further methods for preparing CAR T cells are described in PCT / US2016 / 043392.

[0033] Methods for preparing useful T cell populations are described, for example, in WO 2017 / 015490 and WO 2018 / 102761. In some cases, it may be useful to use natural killer (NK) cells, such as allogeneic NK cells derived from peripheral blood or umbilical cord blood. NK cells may also be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database listings, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In the case of conflict, the present specification will control where there is a definition. Other features and advantages of the invention will be apparent from the following description and drawings, and from the claims. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram showing three CCR4 CAR constructs with different spacer domains. [Figure 2] A schematic diagram of the CCR4 CAR design is shown. Three different CCR4 CAR constructs were designed. All three constructs express the same codon-optimized humanized CCR4 single-chain variable fragment. All CAR constructs express the CD4 transmembrane (TM) domain, the 4-1BB costimulatory domain, the CD3 zeta (ζ) domain, and another protein (truncated EGFR), indicating successful transduction of cells with the CAR constructs. Each of the three CCR4 CAR constructs has a different spacer domain. [Figure 3] (A and B) Graphs showing the results of a 48-hour long-term bactericidal assay using CCR4 EQ CAR at 1:10 E:T (A) and a re-dosing assay (B). In the re-dosing assay, tumor cells were returned to 1:2 E:T after 2 days at 1:2 E:T to re-establish a 1:2 E:T. Lysates were analyzed after 48 hours. [Figure 4] Figure 1 shows the results of an experiment using CCR4 CAR T cells with stable expression of EGFRt as a tracking marker over a 7-day culture period. Mock (untransduced) and CCR4 CAR T cells were evaluated by flow cytometry to detect CAR transduction. [Figure 5] Figure 5A shows the results of flow cytometry analysis of CCR4 expression in different T cell subpopulations. Figure 5B shows the results of flow cytometry analysis of CCR4 expression in different T cell subpopulations. Depleted PBMCs (CD14-CD25-; also referred to as "dPBCM"), naive memory T cells (Tn / mem), and central memory T cells (Tcm) were stained for CCR4 expression via flow cytometry in CD8+ (Figure 5A) and CD4+ (Figure 5B). [Figure 6] A and B are graphs showing results from CCR4 growth curves. (A) Viable cell counts of Pan T cells from the same healthy donor with three different CCR4 CAR constructs were followed over 13 days. (B) Viable cell counts of depleted PBMC (CD14-CD25-; also known as dPBCM) cells from the same healthy donor with three different CCR4 CAR constructs were followed over 13 days. [Figure 7A] Figure 1 shows the killing ability of CCR4 CAR2 T cells against T cell tumor lines expressing CCR4. Schematic of a "re-administration" assay following a "killing" assay shows the killing ability of CCR4 CAR T cells against T cell tumor lines expressing CCR4. [Figure 7B] Graph showing the results of a killing and rechallenge study using CCR4+ tumor cell line, CEM cells, demonstrating the killing ability of CCR4 CAR2 T cells against T cell tumor lines expressing CCR4. [Figure 7C] Graph showing the results of a killing and re-administration assay using MT1 cells, a CCR4+ tumor cell line, demonstrating the killing ability of CCR4 CAR2 T cells against T cell tumor lines expressing CCR4. [Figure 7D] Graph showing the results of a killing and rechallenge study using LCL cells, a CCR4-negative B-cell tumor line, demonstrating the killing ability of CCR4 CAR2 T cells against T-cell tumor lines expressing CCR4. [Figure 8] A to C are graphs showing survival curves in three mouse models transplanted with CCR4-expressing malignant T cell lines using various injection routes (IP, intraperitoneal; SC, subcutaneous; IV, intravenous). [Figure 9] A and B show the in vivo efficacy of CCR4 CAR T cells. (A) Schematic of sc engraftment of CEM in NSG mice. Four days later, mice were intravenously administered CAR T cells or mock-transduced T cells and compared to the tumor-only group. (B) Graph showing the survival follow-up of mice intravenously administered CAR or mock-transduced T cells compared to the tumor-only group. [Figure 10A]FIG. 1 shows the annotated amino acid sequence of CAR1 (CCR4 L CAR) (A; SEQ ID NO: 29). [Figure 10B] FIG. 1 shows the annotated amino acid sequence of CAR2 (CCR4 EQ CAR) (B; SEQ ID NO: 30). [Figure 10C] FIG. 1 shows the annotated amino acid sequence of CAR3 (CCR4 ΔCH2 CAR; also referred to as CCR4 CH3 CAR) (C; SEQ ID NO: 31). [Figure 11] Graph showing bioluminescence detection of tumor growth after engraftment of CEM in NSG mice intravenously treated with CAR T cells or Mock-transduced T cells compared to tumor-only groups. [Figure 12] (A and B) Graphs showing in vivo efficacy of CCR4 CAR T cells in the HUT78 mouse model. (A) Bioluminescent detection of tumor growth after IV engraftment of HUT78 in NSG mice 10 days after intravenous administration of CAR T cells or mock-transduced T cells. (B) Survival follow-up of mice intravenously treated with CAR or mock-transduced T cells. [Figure 13A] A to E are diagrams showing inducible CAR constructs. A is a schematic diagram of four inducible CCR4 CAR constructs. [Figure 13B] A to E show the inducible CAR constructs. B shows the plasmid map of inducible CAR1 (CD19t-containing CCR4 EQ). [Figure 13C] A to E are diagrams showing inducible CAR constructs. C shows the plasmid map of inducible CAR2 (CD19t-containing CCR4 CH3). [Figure 13D] A to E show the inducible CAR constructs, and D shows the plasmid map of inducible CAR3 (CCR4 EQ). [Figure 13E] A to E show the inducible CAR constructs, respectively. E shows the plasmid map of inducible CAR4 (CCR4 CH3; also called CCR4 ΔCH2). [Figure 14A]FIG. 1 shows the annotated amino acid sequence (without signal sequence) of CAR1 (CCR4 L CAR) (SEQ ID NO: 38). [Figure 14B] FIG. 1 shows the annotated amino acid sequence (without signal sequence) of CAR2 (CCR4 EQ CAR) (SEQ ID NO: 39). [Figure 14C] FIG. 1 shows the annotated amino acid sequence (without the signal sequence) of CAR3 (CCR4 ΔCH2 CAR; also referred to as CCR4 CH3 CAR) (SEQ ID NO: 40). [Figure 15] A and B show the annotated amino acid sequences of tTA (A; SEQ ID NO: 41) and T2A-CD19t (B; SEQ ID NO: 42) used in the plasmid map. DETAILED DESCRIPTION OF THE INVENTION

[0036] This disclosure describes the generation and antitumor efficacy of CARs containing a humanized anti-human CCR4 scFv antigen-binding domain and a 4-1BB intracellular costimulatory signaling domain. CCR4 CAR T cells exhibited potent antigen-dependent cytotoxicity against multiple CCR4-expressing human T-cell cancer lines. Intravenous in vivo delivery of CCR4 CAR T cells eliminated antigen-positive disease and prolonged survival in a human leukemia / lymphoma mouse tumor model.

[0037] The present disclosure also provides methods for treating a subject with a T-cell cancer, such as non-Hodgkin's lymphoma, peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma, lymphoblastic lymphoma, precursor T-cell lymphoma, angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma (CTCL), mycosis fungoides (MF), or Sézary syndrome (SS). T-cell lymphomas include (a) lymphoblastic lymphomas, which are malignant tumors arising from primitive lymphoid precursor cells derived from the thymus; (b) T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, NK-cell leukemia, cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), anaplastic large cell lymphoma, T-cell type, enteropathic T-cell lymphoma, adult T-cell leukemia / lymphoma, including those associated with HTLV-1, and angioimmunoblastic T-cell lymphoma, and subcutaneous panniculus T-cell lymphoma; and (c) Peripheral T-cell lymphomas that initially involve the paracortical lymph node but do not grow into a true follicular pattern; including, but not limited to: [Example] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Materials and Methods The following materials and methods were used in the examples described herein.

[0038] [Cell line] CEM (adult T-cell leukemia), MT-1 (adult T-cell leukemia), and LCL (derived from PBMC; engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice) cell lines (Xiuli Wang et al. (2011) Blood) were cultured in RPMI-1640 (Lonza) (complete RPMI) containing 10% fetal bovine serum (FBS, Hyclone). 293T cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) (complete DMEM) containing 10% FBS, 1X AA, 25 mM HEPES (Irvine Scientific), and 2 mM L-glutamine (Fisher Scientific). All cells were cultured at 37°C with 5% CO2. HUT78 cells were cultured in IMDM (Iscove's modified Dulbecco's medium; Fisher Scientific) with 20% FBS.

[0039] [DNA constructs and lentivirus production] Tumor cells were engineered to express enhanced green fluorescent protein and firefly luciferase (eGFP / fluc) by transduction with eHIV7 lentivirus carrying an eGFP / ffluc fusion under the control of the EF1α promoter, as described above (Lenalidomide Enhances the Function of CS1 Chimeric Antigen Receptor-Redirected T Cells Against Multiple Myeloma (Wang et al.) Clinical Cancer Research 2018). The humanized scFv sequence used in the CAR construct was obtained from the monoclonal antibody clone h1567, which targets CCR4 (Non-Patent Document 1).

[0040] Lentivirus was produced using a modified polyethylenimine (PEI)-mediated transfection method (Optimization of lentiviral vector production using polyethylenimine-mediated transfection, Yong Tang, et al., Oncology Letters, 2015). Briefly, 293T cells were transfected with the packaging plasmid and CAR lentiviral backbone plasmid using the modified PEI method. The viral supernatant was harvested 3–4 days later. The supernatant was concentrated by high-speed centrifugation, and the lentiviral pellet was resuspended in phosphate-buffered saline (PBS)-lactose solution (4 g lactose per 100 mL of PBS) and stored in aliquots at -80°C. Lentiviral titers were quantified using Jurkat cells based on EGFRt expression.

[0041] [T cell isolation, lentiviral transduction, and ex vivo expansion] Leukapheresis transfusion products were obtained from consenting study participants (healthy donors) under a protocol approved by the City of Hope Internal Review Board (IRB). On the day of leukapheresis, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Ficoll-Paque (GE Healthcare) followed by multiple washes with PBS / EDTA (Miltenyi Biotec). Cells were placed overnight on a rotator at room temperature (RT), then washed and resuspended in X-VIVO T cell medium (Lonza) containing 10% FBS (complete X-VIVO). A maximum of 5.0 × 10 9 PBMCs were incubated with anti-CD14 and anti-CD25 microbeads (Miltenyi Biotec) for 30 minutes at room temperature and magnetically removed using a CliniMACS® system (Miltenyi Biotec) according to the manufacturer's protocol. These depleted PBMCs (dPBMCs) were frozen in a CryoStor® CS5 (StemCell Technologies) until further processing.

[0042] T cell activation and transduction were performed as previously described (Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer. Priceman Saul J, et al. 2018. Oncoimmunology). Briefly, freshly thawed dPBMCs were washed once and cultured in complete X-vivo medium containing 100 U / mL recombinant human IL-2 (rhIL-2, Novartis Oncology) and 0.5 ng / mL recombinant human IL-15 (rhIL-15, CellGenix). For CAR lentiviral transduction, T cells were cultured with CD3 / CD28 Dynabeads® (LifeTechnologies), protamine sulfate (APP Pharmaceuticals), a cytokine mixture (as described above), and the desired lentivirus at a multiplicity of infection (MOI) of 1–3 the day after bead stimulation. Cells were then cultured and replenished with fresh complete X-vivo medium containing cytokines every 2–3 days. After 7 days, the beads were magnetically removed and the cells were further expanded in complete X-vivo medium containing cytokines to achieve the desired cell yield. After further expansion, the cells were frozen in CryoStor® CS5 prior to in vitro functional assays and in vivo tumor models.

[0043] [Flow cytometry] For flow cytometry analysis, cells were soaked in FACS buffer (containing 2% FBS and 1x AA, Ca 2+ , Mg 2+ or Hank's balanced salt solution without phenol red (HBSS - / -Cells were resuspended in PBS (Life Technologies). Cells were incubated with primary antibodies for 30 minutes at 4°C in the dark. For secondary staining, cells were washed twice before incubation with Brilliant Violet 510 (BV510), fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein complex (PerCP), PerCP-Cy5.5, PE-Cy7, allophycocyanin (APC), or APC-Cy7 (or APC-eFluor780)-conjugated antibodies for 30 minutes at 4°C in the dark. Antibodies used were CD3 (BD ​​Biosciences, clone SK7), CD4 (BD Biosciences, clone SK3), CD8 (BD Biosciences, clone SK1), CD14 (BD Biosciences, clone MΦP9), CD19 (BD Biosciences, clone SJ25C1), CD25 (BD Biosciences, clone 2A3), mouse CD45 (BioLegend, clone 30-F11), CD45 (BD Biosciences, clone 2D1), CD69 (BD Biosciences, clone L78), CD137 (BD Biosciences, clone 4B4-1), MUC1 (Biolegend, clone 16A), biotinylated Protein-L (GenScript USA), CCR4 (clone L291H4), and streptavidin (BD Biosciences). Cell viability was measured using 4',6-diamidino-2-phenylindole (DAPI, Sigma). Flow cytometry was performed on a MACSQuant Analyzer 10 (Miltenyi Biotec), and data were analyzed with FlowJo software (v10, TreeStar).

[0044] In vitro tumor killing assay and rechallenge assay For tumor killing assays, CAR T cells and tumor targets were co-cultured in 96-well plates at the indicated effector:tumor (E:T) ratios in complete x-vivo in the absence of exogenous cytokines for 24–72 hours and analyzed by flow cytometry as described above. Tumor killing by CAR T cells was calculated from the number of GFP-positive tumor cells compared to that observed when targets were co-cultured with mock (untransduced) T cells. For re-administration assays, 24–72 hours after completion of the killing assay, CAR T cells and tumor targets were co-cultured again at the indicated effector:tumor (E:T) ratios in complete x-vivo in the absence of exogenous cytokines for 24–72 hours and analyzed by flow cytometry as described above.

[0045] [In vivo tumor testing] All animal experiments were performed under protocols approved by the City of Hope Institutional Animal Care and Use Committee. For in vivo tumor studies, CEM cells (3.0 × 10 6 ) to a final volume of 150 μl HBSS - / - T cells were prepared in PBS and injected into 6-8 week-old female or male NSG mice for engraftment. In some embodiments, engraftment involves subcutaneous (sc) or intravenous (iv) injection. Tumor growth was tracked at least weekly by biophotonic imaging (Xenogen, LagoX), and flux signals were analyzed using Living Image software (Xenogen). For imaging, mice were intraperitoneally injected with 150 μL of D-luciferin potassium salt (Perkin Elmer) suspended in PBS at 4.29 mg / mouse. Once the flux signal reached the desired level, day 4 or 5 T cells were prepared in 1X PBS and injected into mice at 3.0 × 10 6Mock or CCR4 CAR2 T cells were injected intravenously (iv) in a volume of 150 μL. The effects of intravenous CCR4 EQ CAR T cell treatment were investigated in the CEM tumor model starting on day 4. Survival was determined using humane endpoints. Mice were euthanized when they showed signs of distress, such as respiratory distress, obvious weight loss, decreased mobility, or morbidity. At designated time points or when moribund, mice were euthanized, and tissues were collected and processed for flow cytometry and / or immunohistochemistry, as described below. Peripheral blood was collected from isoflurane-anesthetized mice by retro-orbital bleeding through a heparinized capillary tube (Chase Scientific) into a polystyrene tube containing heparin / PBS solution (1000 units / mL, Sagent Pharmaceuticals). The volume of each RO blood draw (approximately 120 μL / mouse) was recorded for cell quantification per μL of blood. Red blood cells (RBCs) were lysed with 1× red blood cell lysis buffer (Sigma) according to the manufacturer's protocol, then washed, stained, and analyzed by flow cytometry as described above. [Example]

[0046] Construction of CCR4 CAR T cells containing different linkers The following studies show that CCR4 CARs can be stably expressed on primary T cells. Three CCR4 targeting CAR constructs were designed (Figures 1 and 2). All three constructs expressed the same codon-optimized humanized CCR4 single-chain variable fragment. The CAR constructs also contained the CD4 transmembrane domain (TM), 4-1BB costimulatory domain, and CD3 zeta domain. The CAR was co-expressed with a truncated EGFR, which served as a marker for successful cell transduction with the CAR construct. Each of the three CCR4 CAR constructs had a different linker (Figure 2). Without being bound by theory, the different lengths of the extracellular portions of the constructs may affect the CAR's ability to bind antigen and transmit activation signals after antigen binding. These differences may result in different killing potencies against CCR4-expressing tumor cells (Figure 1). As previously reported (Priceman SJ, Gerdts EA, Tilakawardane D, Kennewick KT, Murad JP, Park AK, Jeang B, Yamaguchi Y, Yang X, Urak R, Weng L, Chang WC, Wright S, Pal S, Reiter RE, Wu AM, Brown CE, Forman SJ. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer. Oncoimmunology. 2018;7(2):e1380764) and other cell types, such as enriched T cells (EasySep Human T cell isolation Kit. StemCell Technologies), we transduced peripheral blood mononuclear cells (PBMCs) from healthy human donors that had been depleted of CD14+ and CD25+ cells (dPBMCs) with the CCR4 CAR lentivirus. CAR expression was demonstrated using flow cytometry as described above, using EGFRt as a tracking marker. All three CCR4 CAR constructs were stably expressed in T cells (Figure 4). Seven days after transduction of dPBMC cells, cells were stained with anti-CD3 to label T cells and with anti-EGFR to indicate successful uptake of the CCR4 CAR constructs. The CCR4 L CAR, CCR4 EQ CAR, and CCR4±CH2 CAR were all stably expressed 7 days after transduction (Figure 4), and EGFR expression was shown to be stable up to 28 days after the start of transduction (data not shown). [Example]

[0047] CCR4 expression in T cell populations In the following study, we investigated the proliferation and activity of CCR4 CAR T cells in different T cell subpopulations, including PBMCs (CD14-, CD25-) and pan-T cells. The starting T-cell population used to generate CAR T cells may affect their ability to eliminate target cells, and there may be differences in cell expansion during the 14-day manufacturing process. The inventors transduced three different CCR4 CAR constructs into different T cell populations from the same healthy donor (Figure 1) and tracked the viable cell counts over 13 days. The CCR4 EQ CAR and CCR4 #CH2 CAR showed the best overall growth (Figures 6A-6B). The inventors found that the CCR4 L CAR grew poorly in multiple independent studies using CCR4 L CAR cells generated from different healthy donors in T cell populations at different starting times (data not shown). [Example]

[0048] Validation that CCR4 CAR T cells selectively target CCR4-positive cells in vitro To determine whether CCR4 CAR T cells exhibit selective activity against CCR4-positive cancer cells, CCR4 CAR T cells were grown in the presence of either CCR4-positive or CCR4-negative cancer cells, and the proportion of cancer cells killed was quantified. To evaluate the antigen-dependent activity of our CCR4 CAR T cells, we performed coculture assays using CCR4-positive and CCR4-negative tumor targets at E:T ratios of 1:2 to 1:10 and measured their killing potential. The CCR4-positive T cell tumor lines used were MT-1 and CEM. The CCR4-negative cell line used was LCL, a B cell tumor cell line. CCR4 EQ CAR T cells and tumor cells were cocultured at a ratio of 1 T cell per 10 tumor cells (1:10 E:T). Tumor cell killing (% specific lysis) was tested after 48 hours using flow cytometry. After 48 hours, antigen-specific T cell-mediated killing activity of CCR4 EQ CAR T cells against mock T cells was evident in the CEM tumor line (Figure 7B, top) and the MT-1 tumor cell line (Figure 7C, top). CCR4 EQ CAR2 T cells demonstrated sustained killing activity, with 100% or near lysis, in both the CEM tumor line (Figure 7B, top) and the MT-1 tumor cell line (Figure 7C, top). These results, with an E:T ratio of 1:10, demonstrate the potent killing activity of CCR4 EQ CAR T cells. CCR4 EQ CAR T cells minimally killed CCR4-negative LCL cells (Figure 7D, top). CCR4 L CAR T cells and CCR4±CH2 CAR T cells also killed CCR4+ tumor cells (data not shown). To further test whether tumor cell killing persisted when CCR4 EQ CAR T cells were re-administered, CAR T cells and tumor cells were cultured at a 1:2 ratio (1 T cell:2 tumor cells) for 48 hours. After 48 hours, additional tumor cells were added to the co-culture wells at a 1:2 E:T ratio, and after another 48 hours, the % specific lysis of tumor cells was measured using flow cytometry. Surprisingly, we found that CCR4 EQ CAR T cells retained their killing ability against CCR4+ tumor lines even when re-administered (Figures 7B-7C, bottom). Both CCR4 L CAR T cells and CCR4 #CH2 CAR T cells killed CCR4+ tumor cells when re-administered (data not shown). [Example]

[0049] Establishment of an in vivo malignant T cell mouse model To evaluate the therapeutic potential of CCCR4 CAR T cells in vivo, three mouse models using different injection routes were established. Human endpoints were used to determine survival curves for NSG mice engrafted with CCR4-expressing malignant T cell lines. Mice were euthanized upon signs of distress, such as respiratory distress, obvious weight loss, decreased mobility, or morbidity. Mice were engrafted systemically or locally with HUT78-, CEM-, and MT-1-delivered cells via intraperitoneal, subcutaneous, and intravenous injection (Figure 8). [Example]

[0050] In vivo validation of delivered CCR4 CAR T cells in a mouse model demonstrates potent antitumor activity and extends mouse survival. To evaluate the in vivo efficacy of CCR4 CAR T cells in selectively targeting CCR4-positive cells in the CEM model, CCR4 CAR T cells were delivered and tumor size and survival time were assessed over time. CEM cells were transduced with lentivirus to express firefly luciferase (fluc) and tumor growth was tracked by noninvasive optical imaging. Four days after subcutaneous tumor injection, mice were treated with systemic intravenous (i.v.) delivery of either Mock or CCR4 EQ CAR2 T cells (3.0 × 10 6 ) (Figure 9A). In mice that received intravenous injection of CCR4 EQ CAR T cells, a rapid antitumor effect was observed, reaching a maximum antitumor response 1–2 weeks after administration. The antitumor response in the mice persisted for 3–4 weeks, but tumor recurrence was eventually observed in the mice. Delivery of CCR4 EQ CAR T cells significantly extended the survival time of the mice (Figure 9B and Figure 11). [Example]

[0051] Verification that delivered CCR4 CAR T cells exhibit potent antitumor activity and prolonged mouse survival in vivo in a mouse model To evaluate the in vivo efficacy of CCR4 CAR T cells in a disseminated model, CCR4 CAR T cells were delivered into a HUT78 mouse model and tumor size and survival were assessed over time. HUT78 cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium; Fisher Scientific) containing 20% ​​FBS. For the HUT78 in vivo model, CD4 and CD8 cells were enriched from PBMCs by incubation with anti-CD4 and anti-CD8 microbeads (Miltenyi Biotech). Other T cell populations used were T cells generated by negative selection of PBMCs. A human T cell isolation kit from stem cells was also used. For in vivo tumor studies, HUT78 cells (1.0 × 10 6 ) was prepared in a final volume of 150 μl of HBSS- / - and injected into 6-8 week-old female or male NSG mice for engraftment. The HUT78 animal model is a widely used model. In one embodiment, engraftment involves subcutaneous (sc) or intravenous (iv) injection. HUT78 cells were transduced with lentivirus to express firefly luciferase (ffluc), and tumor growth was tracked by noninvasive optical imaging. Ten days after intravenous tumor injection, mice were treated with either Mock or CCR4 EQ CAR2 T cells (3.0 × 10 6 ) via systemic intravenous (iv) administration (Figure 12A). Antitumor effects were observed in mice treated with CCR4 EQ CAR T cells after treatment. Delivery of CCR4 EQ CAR T cells significantly prolonged mouse survival (Figure 12B). [Example]

[0052] Inducible CCR4 CAR T cells In some situations, it is desirable to control the expression of the CCR4 CAR. For example, after introducing a vector into a population of T cells, the cells are expanded to prepare a sufficient number of cells for therapeutic use. During this expansion phase, it is desirable to reduce or nearly eliminate CCR4 CAR expression, e.g., to reduce any fratricide. A system using Tet-Off regulation can be useful (Das et al. 2016 Current Gene Therapy 16:156). Thus, an expression vector for CCR4 CAR expression can express the CCR4 CAR under the control of an inducible promoter containing several copies of the tet operator and a minimal promoter. The vector can also encode a fusion protein containing a transcriptional activation domain of VP16 fused to TetR. In the presence of tetracycline or doxycycline, CCR4 CAR expression is suppressed. In the absence of tetracycline or doxycycline, CCR4 is expressed. Thus, T cells carrying a nucleic acid encoding a CCR4 CAR can be expanded under conditions in which CCR4 CAR expression is suppressed. Once the desired number of cells is obtained, expression is induced by removing the tetracycline or doxycycline. Figure 13A shows a schematic diagram of the four inducible CCR4 CAR constructs. Figure 13B shows the plasmid map of inducible CAR1 (CCR4 EQ with CD19t). Figure 13C shows the plasmid map of inducible CAR2 (CCR4 CH3 with CD19t). Figure 13D shows the plasmid map of inducible CAR3 (CCR4 EQ). Figure 13E shows the plasmid map of inducible CAR4 (CCR4 CH3).

[0053] Other embodiments While the present invention has been described in conjunction with its detailed description, it will be understood that the above description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the claims. All references are incorporated herein in their entirety for all purposes.

Claims

1. 1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or polypeptide, wherein the CAR or polypeptide is one of the following: A nucleic acid molecule comprising an scFv that targets CCR4, a spacer, a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain, wherein the spacer comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof in which 1 to 5 amino acids of the amino acid sequence of SEQ ID NO: 9 are modified.

2. 2. The nucleic acid molecule of claim 1, wherein the transmembrane domain is selected from a CD4 transmembrane domain or a variant thereof modified by 1 to 5 amino acids, a CD8 transmembrane domain or a variant thereof modified by 1 to 5 amino acids, or a CD28 transmembrane domain or a variant thereof modified by 1 to 5 amino acids, and optionally wherein the transmembrane domain is a CD4 transmembrane domain.

3. The nucleic acid molecule of claim 1, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 32 and the amino acid sequence of SEQ ID NO: 33, or the amino acid sequence of SEQ ID NO: 34 and the amino acid sequence of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 36 and the amino acid sequence of SEQ ID NO: 37, or wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 1, 40, 43, 44 or 45.

4. The nucleic acid molecule of claim 1, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof modified by 1 to 5 amino acids, and / or wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof modified by 1 to 5 amino acids, and / or wherein the linker is 3 to 15 amino acids and is located between the 4-1BB costimulatory domain and the CD3ζ signaling domain or a variant thereof.

5. The nucleic acid molecule of claim 1, wherein the CAR or polypeptide comprises the amino acid sequence of SEQ ID NO: 31 or 40 or a variant thereof in which 1 to 5 amino acids are modified.

6. An expression vector comprising the nucleic acid molecule of any one of claims 1 to 5.

7. The expression vector according to claim 6 , wherein the vector is a viral vector, and / or the expression of the CCR4 CAR is controlled by an inducible promoter, and optionally the expression of the CCR4 CAR is controlled by a Tet Off system.

8. A human T cell population transduced with a vector comprising the nucleic acid molecule of any one of claims 1 to 5.

9. 9. The human T cell population of claim 8, comprising central memory T cells, naive memory T cells, CD4+ cells and CD8+ cells enriched from PBMC cells, T cells isolated by negative depletion, or PBMCs substantially depleted of CD25+ cells and CD14+ cells, or optionally comprising PBMC cells that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% CD14-negative and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% CD25-negative.

10. 10. An autologous or allogeneic human T cell population transduced with a vector comprising the nucleic acid molecule of any one of claims 1 to 5 for use in a method for treating a T cell lymphoma that expresses CCR4 in a patient, wherein the method comprises administering the human T cell population to the patient, and the T cell lymphoma comprises cells that express CCR4.

11. 11. The autologous or allogeneic human T cell population of claim 10, wherein the human T cell population expressing the chimeric antigen receptor or polypeptide is administered locally or systemically and / or in a single dose or multiple doses.

12. The autologous or allogeneic human T cell population of claim 10, wherein the CCR4-expressing cells are cancerous T cells or regulatory T cells.

13. 1. A method for preparing isolated CCR4 CAR T cells, comprising: providing an isolated population of autologous or allogeneic human T cells; and transducing T cells of the isolated autologous or allogeneic human T cell population with a vector comprising the nucleic acid molecule of any one of claims 1 to 5, wherein: The method, wherein the T cells comprise PBMC cells.

14. 14. The method of claim 13, wherein the PBMC cells are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% CD14 negative and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% CD25 negative, and / or the PBMC cells comprise CD4+ T cells or CD8+ T cells or both.

15. 8. A method for preparing isolated T cells that express a CCR4 CAR, the method comprising: growing T cells comprising the expression vector of claim 6 or 7 under conditions where CCR4 CAR expression is not induced until a predetermined number of cells are produced, and then inducing expression of the CCR4 CAR.

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