CCR4 TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS FOR TREATMENT OF CCR4 POSITIVE MALIGNANCIES

Abstract

Chimeric antigen receptors for use in treating lymphoma-associated C-C chemokine receptor type 4 (CCR4) and other cancers expressing CCR4 are described.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a polypeptide, wherein the chimeric antigen receptor or polypeptide comprises: an scFv targeting CCR4, a spacer, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 ζ signaling domain, wherein the spacer comprises SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:12, or a variant thereof having 1-5 amino acid modifications.

2. The nucleic acid molecule of claim 1, wherein the transmembrane domain is selected from: a CD4 transmembrane domain or variant thereof having 1-5 amino acid modifications, a CD8 transmembrane domain or variant thereof having 1-5 amino acid modifications, a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications.

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.

4. The nucleic acid molecule of claim 1, wherein the transmembrane domain is a CD4 transmembrane domain or variant thereof having 1-5 amino acid modifications.

5. The nucleic acid molecule of claim 1, wherein the transmembrane domain is a CD4 transmembrane domain.

6. The nucleic acid molecule of claim 1, wherein the chimeric antigen receptor or polypeptide comprises a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-2 amino acid modifications, a CD8 transmembrane domain or variant thereof having 1-2 amino acid modifications, a CD28 transmembrane domain or a variant thereof having 1-2 amino acid modifications,

7. 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 having 1-5 amino acid modifications.

8. The nucleic acid molecule of claim 1, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:21.

9. The nucleic acid molecule of claim 1, wherein a linker of 3 to 15 amino acids is located between the 4-1BB costimulatory domain and the CD3 ζ signaling domain or variant thereof.

10. The nucleic acid molecule of claim 1, wherein the CAR or the polypeptide comprises the amino acid sequence of SEQ ID NO: 29, 30, 31, 38, 39, or 40 or a variant thereof having 1-5 amino acid modifications.

11. The nucleic acid molecule of claim 1, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NO:1, 40, 43, 44, or 45.

12. An expression vector comprising the nucleic acid molecule of claim 1.

13. The expression vector of claim 12, wherein the vector is a viral vector.

14. The expression vector of claim 12, wherein expression of the CCR4 CAR is under the control of an inducible promoter.

15. The expression vector of claim 14, wherein expression of the CCR4 CAR is under the control of a Tet Off system.

16. A population of human T cells transduced by a vector comprising the nucleic acid molecule of claim 1.

17. The population of human T cells of claim 14, wherein the population of human T cells comprise central memory T cells, naive memory T cells, CD4+ cells and CD8+ cells enriched from PBMC cells, T cells isolated via negative depletion, or PBMC substantially depleted for CD25+ cells and CD14+ cells.

18. A method of treating T cell lymphoma in a patient comprising administering a population of autologous or allogeneic human T cells transduced by a vector comprising the nucleic acid molecule of claim 1, wherein the T cell lymphoma comprises cells expressing CCR4.

19. The method of claim 18, wherein the population of human T cells expressing the chimeric antigen receptor or the polypeptide is administered locally or systemically.

20. The method of claim 18, wherein the CCR4-expressing cells are cancerous T cells or T-regulatory cells.

21. The method of claim 18, wherein the population of human T cells expressing the chimeric antigen receptor or the polypeptide is administered by single or repeat dosing.

22. A method of preparing CCR4 CART cells comprising: providing a population of autologous or allogeneic human T cells and transducing the T cells by a vector comprising the nucleic acid molecule of claim 1, wherein the T cells comprise PBMC cells.

23. The method of claim 22, wherein the depleted 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.

24. The method of claim 21, wherein the PBMC cells comprise CD4+ T cells or CD8+ T cells or both

25. The population of human T cells of claim 14, wherein the population of human T cells comprise 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.

26. A method of preparing T cells expressing a CCR4 CAR, comprising expanding T cells harboring the expression vector of claim 14 under conditions in which CCR4 CAR expression is not induced until a desired number of cells is produced and then inducing CCR4 CAR expression.

Description

DESCRIPTION OF DRAWINGS

[0042] FIG. 1 shows a schematic depicting three CCR4 CAR constructs differing in the spacer region.

[0043] FIG. 2 shows a schematic of CCR4 CAR design: Three different CCR4 CAR constructs were designed. All three of the constructs express the same codon optimized, humanized CCR4 single chain variable fragment. All of the CAR constructs also express a CD4 transmembrane (TM) domain, a 41BB costimulatory domain, a CD3 zeta (ζ) domain, and a separate protein (truncated EGFR) to mark the successful transduction of the cells with the CAR construct. The three CCR4 CAR constructs differ in their spacer domains.

[0044] FIGS. 3A-3B shows results from a 48 hr long term killing assay with CCR4 EQ CAR at 1:10 E:T (A) and rechallenge assays (B). In the rechallenge assays, after 2 days at an E:T of 1:2 tumor cells were added back so that the 1:2 E:T is re-established. Lysis was analyzed 48 hrs later.

[0045] FIG. 4 shows results from experiments with CCR4 CAR T cells where EGFRt is used as a tracking marker and its expression was stable through 7 day culture duration. Mock (untransduced) and CCR4 CAR T cells were evaluated by flow cytometry for EGFR expression to detect transduction of CARs.

[0046] FIGS. 5A-5B show results of flow cytometric analysis of CCR4 expression in different T cell subpopulations. T cell populations of depleted PBMC (CD14−CD25−; also called “dPBCM”), naïve memory T cells (Tn/mem) and central memory T cells (Tcm) were stained for CCR4 expression via flow cytometry in CD8+ (FIG. 5A) and CD4+ (FIG. 5B).

[0047] FIGS. 6A-6B shows results from a CCR4 growth curve. (A) Pan T cells from the same healthy donor with the three different CCR4 CAR constructs and tracked the live cells counts of the cells over 13 days. (C) Depleted PBMC (CD14−CD25−; also called dPBCM) cells from the same healthy donor with the three different CCR4 CAR constructs and tracked the live cells counts of the cells over 13 days.

[0048] FIGS. 7A-7D shows the ability of the CCR4 CAR2 T cells to kill CCR4 expressing T cell tumor lines. (A) A schematic representation depicts a “Killing” assay followed by a “Rechallenge” assay demonstrates the ability of a CCR4 CART cell to kill CCR4 expressing T cell tumor lines. (B) Killing and rechallenge assays using CEM cells, a CCR4+ tumor cell line. (C) Killing and rechallenge assays using MT1 cells, a CCR4+ tumor cell line. (D) Killing and rechallenge assays using LCL cells, CCR4 negative B cell tumor line.

[0049] FIGS. 8A-8C shows survival curves in three mouse models engrafted with CCR4-expressing malignant T cell lines using a variety of injection routes (IP, intraperitoneal; SC subcutaneous; IV, intravenous).

[0050] FIG. 9A-9B shows in vivo efficacy of the CCR4 CAR T cells. (A) A schematic depicts sc engraftment of CEM in NSG mice. After 4 days, mice were treated IV with the CAR T cells or mock transduced T cells and compared to a tumor only group. (B) Survival of mice treated IV with the CAR or mock transduced T cells compared to a tumor only group was monitored.

[0051] FIG. 10A-10C show the annotated amino acid sequences of CAR1 (CCR4 L CAR) (A; SEQ ID NO: 29) CAR2 (CCR4 EQ CAR) (B; SEQ ID NO: 30), and CAR3 (CCR4 ΔCH2 CAR; also called CCR4 CH3 CAR) (C; SEQ ID NO: 31).

[0052] FIG. 11 shows bioluminescent detection of tumor growth following engraftment of CEM in NSG mice treated IV with the CAR T cells or mock transduced T cells and compared to a tumor only group.

[0053] FIGS. 12A-12B show in vivo efficacy of the CCR4 CAR T cells in a HUT78 mouse model. (A) Bioluminescent detection of tumor growth following IV engraftment of HUT78 in NSG mice. After 10 days, mice were treated IV with the CART cells or mock transduced T cells. (B) Survival of mice treated IV with the CAR or mock transduced T cells was monitored.

[0054] FIGS. 13A-13E show inducible CAR constructs. FIG. 13A shows the shorthand of four inducible CCR4 CAR constructs. FIG. 13B shows the plasmid map of inducible CAR1 (CCR4 EQ with CD19t). FIG. 13C shows the plasmid map of inducible CAR2 (CCR4 CH3 with CD19t). FIG. 13D shows the plasmid map of inducible CAR3 (CCR4 EQ). FIG. 13E shows the plasmid map of inducible CAR4 (CCR4 CH3; also called CCR4 ΔCH2).

[0055] FIGS. 14A-14C show the annotated amino acid sequences (without the signal sequence) of CAR1 (CCR4 L CAR) (A; SEQ ID NO: 38) CAR2 (CCR4 EQ CAR) (B; SEQ ID NO: 39), and CAR3 (CCR4 ΔCH2 CAR; also called CCR4 CH3 CAR) (C; SEQ ID NO: 40).

[0056] FIGS. 15A-15B show the annotated amino acid sequences of tTA (A; SEQ ID NO: 41) and T2A-CD19t (B; SEQ ID NO: 42) as used in the plasmid maps.

DETAILED DESCRIPTION

[0057] In this disclosure the generation and anti-tumor efficacy of CAR with a humanized anti-human CCR4 scFv antigen-binding domain and a 4-1BB intracellular co-stimulatory signaling domain are described. The 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 in human leukemia/lymphoma murine tumor models conferred elimination of antigen-positive disease and extension of overall survival.

[0058] The present disclosure also provides methods for treating subjects with a T-cell cancer, including a non-Hodgkin lymphoma, a peripheral T-cell lymphoma (PTCL), an anaplastic large cell lymphoma, a lymphoblastic lymphoma, precursor T-lymphoblastic lymphoma, an angioimmunoblastic T-cell lymphoma, Cutaneous T-Cell Lymphoma (CTCL), mycosis fungoides (MF), Sézary syndrome (SS), and the like. T cell lymphomas encompass a variety of conditions including without limitation: (a) lymphoblastic lymphomas in which the malignancy occurs in primitive lymphoid progenitors from the thymus; (b) mature or peripheral T cell neoplasms, including T cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia, NK-cell leukemia, cutaneous T cell lymphoma (Mycosis fungoides and Sezary syndrome), anaplastic large cell lymphoma, T cell type, enteropathy-type T cell lymphoma, Adult T-cell leukemia/lymphoma including those associated with HTLV-1, and angioimmunoblastic T cell lymphoma, and subcutaneous panniculitic T cell lymphoma; and (c) peripheral T cell lymphomas that initially involve a lymph node paracortex and never grow into a true follicular pattern.

EXAMPLES

[0059] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0060] Materials and Methods

[0061] The following materials and methods were used in the Examples set forth herein.

[0062] Cell Lines

[0063] The CEM (adult T cell leukemia line), MT-1 (adult T cell leukemia line), and LCL (generated from PBMC; Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice. Xiuli Wang, et al. (2011) Blood) cell lines were cultured in RPMI-1640 (Lonza) containing 10% fetal bovine serum (FBS, Hyclone) (complete RPMI). The 293T cell lines were cultured in Dulbecco's Modified Eagles Medium (DMEM, Life Technologies) containing 10% FBS, 1× AA, 25 mM HEPES (Irvine Scientific), and 2 mM L-Glutamine (Fisher Scientific) (complete DMEM). All cells were cultured at 37° C. with 5% CO.sub.2. HUT78 cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium; Fisher Scientific) with 20% FBS.

[0064] DNA Constructs and Lentivirus Production

[0065] Tumor cells were engineered to express enhanced green fluorescent protein and firefly luciferase (eGFP/ffluc) by transduction with epHIV7 lentivirus carrying the eGFP/ffluc fusion under the control of the EF1α promoter as described previously (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 a monoclonal antibody clone h1567 that targets CCR4 (Chang D-H, Sui J, Geng S, Muvaffak A, Bai M, Fuhlbrigge R C, 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).

[0066] Lentivirus was generated 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 packaging plasmid and CAR lentiviral backbone plasmid using a modified PEI method. Viral supernatants were collected after 3 to 4 days. Supernatants were concentrated via high-speed centrifugation and lentiviral pellets were resuspended in phosphate-buffered saline (PBS)-lactose solution (4 g lactose per 100 mL PBS), aliquoted and stored at −80° C. Lentiviral titers were quantified using jurkat cells based on EGFRt expression.

[0067] T Cell Isolation, Lentiviral Transduction, and Ex Vivo Expansion

[0068] Leukapheresis products were obtained from consented research participants (healthy donors) under protocols approved by the City of Hope Internal Review Board (IRB). On the day of leukapheresis, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) followed by multiple washes in PBS/EDTA (Miltenyi Biotec). Cells were rested overnight at room temperature (RT) on a rotator, and subsequently washed and resuspended in X-VIVO T cell medium (Lonza) containing 10% FBS (complete X-VIVO). Up to 5.0×10.sup.9 PBMC were incubated with anti-CD14 and anti-CD25 microbeads (Miltenyi Biotec) for 30 min at RT and magnetically depleted using the CliniMACS® system (Miltenyi Biotec) according to the manufacturer's protocol and these were termed depleted PBMCs (dPBMC). dPBMC were frozen in CryoStor® CS5 (StemCell Technologies) until further processing.

[0069] T cell activation and transduction was performed as described previously (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 dPBMC were washed once and cultured in complete X-VIVO containing 100 U/mL recombinant human IL-2 (rhlL-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® (Life Technologies), protamine sulfate (APP Pharmaceuticals), cytokine mixture (as stated above) and desired lentivirus at a multiplicity or infection (MOI) of 1-3 the day following bead stimulation. Cells were then cultured in and replenished with fresh complete X-VIVO containing cytokines every 2-3 days. After 7 days, beads were magnetically removed, and cells were further expanded in complete X-VIVO containing cytokines to achieve desired cell yield. Following further expansion, cells were frozen in CryoStor® CS5 prior to in vitro functional assays and in vivo tumor models. Purity and phenotype of CAR T cells were verified by flow cytometry.

[0070] Flow Cytometry

[0071] For flow cytometric analysis, cells were resuspended in FACS buffer (Hank's balanced salt solution without Ca2+, Mg2+, or phenol red (HBSS−/−, Life Technologies) containing 2% FBS and 1× AA). Cells were incubated with primary antibodies for 30 minutes at 4° C. in the dark. For secondary staining, cells were washed twice prior to 30 min incubation at 4° C. in the dark with either 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. Antibodies against 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) were used. Cell viability was determined using 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Flow cytometry was performed on a MACSQuant Analyzer 10 (Miltenyi Biotec), and the data was analyzed with FlowJo software (v10, TreeStar).

[0072] In Vitro Tumor Killing and Rechallenge Assays

[0073] For tumor killing assays, CAR T cells and tumor targets were co-cultured at indicated effector:tumor (E:T) ratios in complete X-VIVO in the absence of exogenous cytokines in 96-well plates for 24 to 72 h and analyzed by flow cytometry as described above. Tumor killing by CAR T cells was calculated by comparing GFP positive tumor cell counts relative to that observed when targets were co-cultured with Mock (untransduced) T cells. For rechallenge assays, 24-72 hours after completion of the killing assay, CART cells and tumor targets were again co-cultured at indicated effector:tumor (E:T) ratios in complete X-VIVO in the absence of exogenous cytokines in 96-well plates for 24 to 72 h and analyzed by flow cytometry as described above.

[0074] In Vivo Tumor Studies

[0075] 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.sup.6) were prepared in a final volume of 150 μl HBSS−/− and engrafted in 6 to 8 week old female or male NSG mice by injection. In some embodiments, engraftment comprises subcutaneous (s.c.) injection or intravenous (i.v.) injection. Tumor growth was monitored at least once a week via biophotonic imaging (Xenogen, LagoX) and flux signals were analyzed with Living Image software (Xenogen). For imaging, mice were i.p. injected with 150 μL D-luciferin potassium salt (Perkin Elmer) suspended in PBS at 4.29 mg/mouse. Once flux signals reached desired levels, day 4 or 5, T cells were prepared in 1×PBS, and mice were treated with 150 μL intravenous (i.v.) injection of 3.0×10.sup.6 Mock or CCR4 CAR2 T cells. In the CEM tumor model, the impact of treatment with i.v. CCR4 EQ CAR T cells was examined starting at day 4. Humane endpoints were used in determining survival. Mice were euthanized upon signs of distress such as labored or difficulty breathing, apparent weight loss, impaired mobility, or evidence of being moribund. At pre-determined time points or at moribund status, mice were euthanized and tissues were harvested and processed for flow cytometry and/or immunohistochemistry as described below.

[0076] Peripheral blood was collected from isoflurane-anesthetized mice by retro-orbital (RO) bleed through heparinized capillary tubes (Chase Scientific) into polystyrene tubes containing a heparin/PBS solution (1000 units/mL, Sagent Pharmaceuticals). Volume of each RO blood draw (approximately 120 μL/mouse) was recorded for cell quantification per μL blood. Red blood cells (RBCs) were lysed with 1× Red Cell Lysis Buffer (Sigma) according to the manufacturer's protocol and then washed, stained, and analyzed by flow cytometry as described above.

Example 1: Construction of CCR4 CAR T Cells Containing Differing Linkers

[0077] The studies described below show that CCR4 CAR can be stably expressed on primary T cells.

[0078] Three CCR4 targeting CAR constructs were designed (FIGS. 1 and 2). All three of the constructs expressed the same codon optimized, humanized CCR4 single chain variable fragment. The CAR constructs also included a CD4 transmembrane domain (TM), a 41BB costimulatory domain, a CD3 zeta domain. The CARs were co-expressed with truncated EGFR, which serve as a marker for the successful transduction of the cells with the CAR construct. The three CCR4 CAR constructs differ in their linker (FIG. 2). Without being bound by theory, differing lengths in the extracellular portion of the construct may provide differences in the CARs ability to bind the antigen and transmit activation signals after antigen binding. These differences could also result differential killing of CCR4 expressing tumor cells (FIG. 1).

[0079] CCR4 CAR lentivirus was used to transduce human healthy donor-derived peripheral blood mononuclear cells depleted of CD14+ and CD25+ cells (dPBMC), as previously described (Priceman S J, Gerdts E A, Tilakawardane D, Kennewick K T, Murad J P, Park A K, Jeang B, Yamaguchi Y, Yang X, Urak R, Weng L, Chang W C, Wright S, Pal S, Reiter R E, Wu A M, Brown C E, Forman S J. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer. Oncoimmunology. 2018; 7(2):e1380764) as well as other cells types such as enriched T-cells (EasySep Human T cell isolation Kit. StemCell Technologies). Using EGFRt as a tracking marker, flow cytometry was used to show CAR expression as described above. All three CCR4 CAR constructs were stably expressed in T cells (FIG. 4). Seven days after dPBMC cells were transduced, cells were stained with anti-CD3 to mark T cells and anti-EGFR to mark successful incorporation of the CCR4 CAR construct. CCR4 L CAR, CCR4 EQ CAR, and CCR4 ΔCH2 CAR were all stably expressed at 7 days post-transduction (FIG. 4) and expression of EGFR was shown to be stable up to 28 days after the start of transduction (data not shown).

Example 2: CCR4 Expression in T Cell Populations

[0080] The studies described below examined CCR4 CAR T cell expansion and activity in different T cell subpopulations, such as PBMC (CD14−, CD25−), and pan-T cells.

[0081] The starting T cell population used to generate CAR T cells may influence the potency with which the CAR T cells can eliminate its target cells. There may also be differences in the proliferation of the cells during the 14 day manufacturing process.

[0082] We transduced different populations of T cells from the same healthy donor with the 3 different CCR4 CAR constructs (FIG. 1) and tracked the live cells counts of the cells over 13 days. We found that CCR4 EQ CAR and CCR4 ΔCH2 CAR proliferated the best overall (FIGS. 6A-6B). We have observed that CCR4 L CAR has poorer proliferation in several independent studies with CCR4 L CAR cells generated from different healthy donors in a variety of starting T cell populations (data not shown).

Example 3: Validation that CCR4 CAR T Cells Selectively Target CCR4-Positive Cells in Vitro

[0083] To determine if CCR4 CAR T cells demonstrate selective activity against CCR4-positive cancer cells, the CCR4 CAR T cells were grown in presence of either CCR4-positive or CCR4-negative cancer cells and the percentage of cancerous cells killed was quantified.

[0084] To assess antigen-dependent activity of our CCR4 CAR T cells, co-cultured assays with CCR4-positive and -negative tumor targets were conducted at an E:T ratio between 1:2 and 1:10 to determine 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. We co-cultured CCR4 EQ CART cells and tumor cells at a ratio of 1 T cell for every 10 tumor cells (1:10 E:T). Using flow cytometry we then tested for the killing of tumor cells (% specific lysis) after 48 hours.

[0085] After 48 hours, antigen-specific T cell-mediated killing activity was evident with CCR4 EQ CART cells relative to Mock T cells in the CEM tumor line (FIG. 7B, top) and the MT-1 tumor cell line (FIG. 7C, top). CCR4 EQ CAR2 T cells had sustained killing at or near 100% lysis in both the CEM tumor line (FIG. 7B, top) and the MT-1 tumor cell line (FIG. 7C, top). These results at an E:T of 1:10 demonstrate the potent killing ability of CCR4 EQ CAR T cells. CCR4 EQ CAR T cells showed minimal killing of CCR4-negative LCL cells (FIG. 7D, top). CCR4 L CAR T cells and CCR4 ΔCH2 CAR T cells also killed CCR4+ tumor cells (data not shown).

[0086] Additionally, to test if CCR4 EQ CAR T cells can continue to kill tumor cells if rechallenged, we co-cultured CART cells and tumor cells at a ratio of 1:2 (1 T cell:2 tumor cells) for 48 hrs. After the 48 hours, we added additional tumor cells to the co-culture wells to an E:T of 1:2, and after an additional 48 hours, used flow cytometry to determine the % specific lysis of the tumor cells. Surprisingly, we found that the CCR4 EQ CAR T cells were able to kill CCR4+ tumor lines even when rechallenged (FIGS. 7B-7C, bottom). Both CCR4 L CAR T cells and CCR4 ΔCH2 CAR T cells also killed CCR4+ tumor cells when rechallenged (data not shown).

Example 4: Establishment of In Vivo Malignant T Cell Mouse Models

[0087] To evaluate the therapeutic potential of the CCCR4 CART cells in vivo, three mouse models were established using a variety of injection routes.

[0088] Humane endpoints were used in determining survival curves of NSG mice engrafted with CCR4 expressing malignant T cell lines. Mice were euthanized upon signs of distress such as a distended belly due to ascites, labored or difficulty breathing, apparent weight loss, impaired mobility, or evidence of being moribund. Mice were engrafted cells delivered systemically or locally with HUT78, CEM, and MT-1 via i.p., s.c., and i.v. injection (FIG. 8).

Example 5: Validation that CCR4 CAR T Cells Delivered In Vivo in a Mouse Model Exhibit Potent Anti-Tumor Activity and Confer Extended Lifespan to the Mice

[0089] To evaluate in vivo efficacy of CCR4 CAR T cells to selectively target CCR4-positive cells in the CEM model, CCR4 CAR T cells were delivered and tumor size and survival was evaluated over time.

[0090] CEM cells were lentivirally transduced to express firefly luciferase (ffluc) to allow for tracking of tumor growth via non-invasive optical imaging. At 4 days post tumor s.c. injection, mice were treated with Mock or CCR4 EQ CAR2 T cells (3.0×10.sup.6) by systemic intravenous (i.v.) delivery (FIG. 9A). Rapid anti-tumor effects were observed in mice treated with CCR4 EQ CAR T cells via i.v. delivery, reaching a maximal anti-tumor response 1-2 weeks following treatment. Anti-tumor responses in mice were durable for 3-4 weeks, but ultimately tumor recurrences were observed in mice. Delivery of CCR4 EQ CAR T cells significantly extended survival of mice (FIG. 9B and FIG. 11).

Example 6: Validation that CCR4 CAR T Cells Delivered In Vivo in a Mouse Model Exhibit Potent Anti-Tumor Activity and Confer Extended Lifespan to the Mice

[0091] To evaluate in vivo efficacy of CCR4 CAR T cells in a disseminated model, CCR4 CAR T cells were delivered to the HUT78 mouse model, and tumor size and survival was evaluated over time.

[0092] HUT78 were cultured in IMDM (Iscove's Modified Dulbecco's Medium; Fisher Scientific) with 20% FBS. For the HUT78 in vivo model, CD4 and CD8 enriched cells from PBMC via incubation of PBMC with anti-CD4 and anti-CD8 microbeads (Miltenyi Biotech). Another T cell population used were T cells generated by negative selection of PBMC. Human T cell isolation kit from StemCell was also used.

[0093] For in vivo tumor studies, HUT78 cells (1.0×10.sup.6) were prepared in a final volume of 150 μl

[0094] HBSS−/− and engrafted in 6 to 8 week old female or male NSG mice by injection. HUT78 Animal model is a disseminated model. In some embodiments, engraftment comprises subcutaneous (s.c.) injection or intravenous (i.v.) injection. HUT78 cells were lentivirally transduced to express firefly luciferase (ffluc) to allow for tracking of tumor growth via non-invasive optical imaging. At 10 days post tumor i.v. injection, mice were treated with Mock or CCR4 EQ CAR2 T cells (3.0×10.sup.6) by systemic intravenous (i.v.) (FIG. 12A). Anti-tumor effects were observed in mice treated with CCR4 EQ CAR T cells following treatment. Delivery of CCR4 EQ CART cells significantly extended survival of mice (FIG. 12B).

Example 7: Inducible CCR4 CAR T Cells

[0095] In some circumstances it is desirable to control expression of a CCR4 CAR. For example, after a vector is introduced into population of T cells, the cells are expanded to prepare a sufficient number of cells to use therapeutically. During this expansion stage, it can be desirable to reduce or nearly eliminate expression of the CCR4 CAR, for example, to reduce any fratricide. A system that uses Tet-Off control can be useful (Das et al. 2016 Current Gene Therapy 16:156). Thus, an expression vector for expression of a CCR4 CAR can express the CCR4 CAR under the control of an inducible promoter that includes several copies of the tet operator and minimal promoter. The vector can also encode a fusion protein comprising the transcription activation of domain of VP16 fused to TetR. In the presence of tetracycline or doxycycline, expression of the CCR4 CAR is repressed. When tetracycline or doxycycline is not present, the CCR4 is expressed. Thus, T cells harboring a nucleic acid encoding CCR4 CAR can be expanded under conditions in which CCR4 CAR expression is repressed. When a desired number of cells is obtained, tetracycline or doxycycline is withdrawn to induce expression.

[0096] FIG. 13A depicts schematic diagrams of four inducible CCR4 CAR constructs. FIG. 13B shows the plasmid map of inducible CAR1 (CCR4 EQ with CD19t). FIG. 13C shows the plasmid map of inducible CAR2 (CCR4 CH3 with CD19t). FIG. 13D shows the plasmid map of inducible CAR3 (CCR4 EQ). FIG. 13E shows the plasmid map of inducible CAR4 (CCR4 CH3).

OTHER EMBODIMENTS

[0097] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0098] All references are herein incorporated in their entirety for any and all purposes.