DUAL-TARGETING CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS COMPRISING IL-13 AND CHLOROTOXIN FOR CANCER TREATMENT

Abstract

Chimeric antigen receptors having a chlorotoxin domain and an IL-13 are described. These dual targeted chimeric antigen receptors are useful for treating glioblastoma and other cancers of neuroectodermal origin.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises: a chlorotoxin domain, an IL-13 domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain, wherein a linker is located between the chlorotoxin domain and the IL-13 domain.

2. The nucleic acid molecule of claim 1, wherein the transmembrane domain is comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-20.

3. The nucleic acid molecule of claim 1, wherein the IL-13 domain comprises the amino acid sequence of SEQ ID NO: 1, or variant thereof having 1-5 single amino acid substitutions, or the amino acid sequence of SEQ ID NO: 29, or variant thereof having 1-5 single amino acid substitutions.

4. The nucleic acid molecule of claim 1, wherein the wherein the co-stimulatory domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25.

5. (canceled)

6. The nucleic acid molecule of claim 1, wherein a linker of 3 to 50 amino acids is located between the chlorotoxin domain and the IL-13 domain.

7.-10. (canceled)

11. The nucleic acid molecule of claim 1, wherein the IL-13 domain is followed by the linker that is followed by the chlorotoxin domain, or wherein the chlorotoxin domain is followed by the linker that is followed by the IL-13 domain.

12. The nucleic acid molecule of claim 1, wherein the chlorotoxin domain comprises SEQ ID NO:34.

13.-15. (canceled)

16. The nucleic acid molecule of claim 1, wherein the CAR comprises an amino acid sequence selected from SEQ ID NOs: 43-52.

17.-21. (canceled)

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

23. (canceled)

24. A population of human T cells or human NK cell harboring the nucleic acid molecule of claim 1.

25. The population of human T cells of claim 24, wherein the population of human T cells comprise central memory T cells, naive memory T cells, and/or PBMC substantially depleted for CD25+ cells and CD14+ cells.

26. A method of treating a tumor of neuroectodermal or a tumor of peripheral neuroectodermal tumor origin a glioma comprising administering a therapeutically effective amount of a population of autologous or allogeneic human T cells or NK cells harboring the nucleic acid molecule of claim 1.

27.-29. (canceled)

30. A method of preparing CAR T 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.

31. (canceled)

32. The method of claim 26, wherein the tumor is tumor is a glioblastoma.

33. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises: a toxin domain comprising an amino acid sequence selected from SEQ ID NOs: 35-38, an IL-13 domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain, wherein the chlorotoxin domain can precede or follow the IL13 domain and wherein a linker is located between the chlorotoxin domain and the IL-13 domain.

34.-49. (canceled)

50. A population of human T cells or human NK cell harboring the nucleic acid molecule of claim 33.

51. (canceled)

52. A method of treating a tumor of neuroectodermal or a tumor of peripheral neuroectodermal tumor origin or a glioma comprising administering a therapeutically effective amount of a population of autologous or allogeneic human T cells or NK cells harboring the nucleic acid molecule of claim 33.

53.-55. (canceled)

56. A method of preparing CAR T 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 33.

57.-58. (canceled)

59. A chimeric antigen receptor (CAR) comprising: a chlorotoxin domain, an IL-13 domain, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3ζ signaling domain, wherein a linker is located between the chlorotoxin domain and the IL-13 domain.

60. A population of human T cells or human NK cell expressing the CAR of claim 59.

Description

DESCRIPTION OF DRAWINGS

[0043] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0044] FIGS. 1A-1D depict representative schematics of CLTX/IL-13 CAR T-cells. FIG. 1A depicts a schematic of a representative lentiviral chimeric antigen receptor (CAR) cassette using CLTX and IL13 as the antigen-targeting domains. FIG. 1B shows a schematic depicting four representative dual-targeting CAR constructs differing in the orientations between CLTX and IL13 tumor-targeting domains, and linkers that separate CLTX from IL13, including g3 (3 amino acids) and (g4s)3 (15 amino acids). FIG. 1C depicts a representative schematic of a dual-targeting CAR, which comprises the extracellular chlorotoxin, IL-13, modifiable linker, and IgG4Fc (EQ) spacer domains, a transmembrane domain and, the cytoplasmic CD3ζ signaling domain and co-stimulatory domains. FIG. 1D shows results from experiments with representative bispecific CAR T cells where CD-19t and Fc are co-expression of the bispecific CAR and CD19t transgenes in transduced T cell subsets. Percentages of immunoreactive cells for transduced cells (“CAR”), compared with untransduced cells (“Mock”), its expression was stable through 14 day culture duration.

[0045] FIG. 2 depicts T cell activation using the degranulation marker CD107a, which indicates T cell activation, of T cells transduced with different dual-targeting CAR constructs activated against co-cultured GBM cells.

[0046] FIGS. 3A-3C shows results from killing/rechallenge experiments with representative dual-targeting T cells that demonstrated the ability of CLTX/IL-13 T cells to kill tumor cell lines (e.g., GBM). FIG. 3A depicts killing and rechallenge results using PBT003-4 cells. FIG. 3B depicts killing and rechallenge results using PBT030-2 cells. FIG. 3C depicts killing and rechallenge results using PBT106 cells. Plotted are the viable tumor cell numbers, which indicate the long-term killing potential of CAR T cells, and rechallenge occurred at 48 hour intervals (arrowheads).

[0047] FIG. 4 shows the potency of representative dual-targeting CAR T cells with different intracellular signaling domains. A schematic diagram depicts representative dual-targeting CAR construct differing in intracellular co-stimulatory domains, including CD28 and 41BB. Killing assay results of dual-targeting CAR T cells with different co-stimulatory domains against GBM tumor cells show the percentages of tumor cells killed by the T cells harboring different dual-targeting CAR constructs; killing percentages were calculated by comparing with tumor cells numbers co-cultured with the same amount of mock T cells.

[0048] FIGS. 5A-5C show representative in vivo antitumor effect of dual-targeting CAR T cells against orthotopic GBM xenograft. FIG. 5A depicts a schematic showing a representative orthotopic xenograft generation followed by T cell treatment in NSG mice. FIG. 5B depicts survival of mice bearing two independent GBM models receiving different treatments (tumor only; mock-transduced T cells; Tandem-28z or Tandem-BBz CAR T cells) plotted over a 256-day monitoring period.

[0049] FIGS. 6A-6E show the annotated amino acid sequences of representative dual-targeting CAR T cells: (A) Cltx-g3-IL13(E13Y)-IgG4(PEQ)-CD28TM-CD28gg-CD3z (SEQ ID NO: 43; SEQ ID NO: 44 without the MLLLVTSLLLCELPHPAFLLIP signal peptide sequence), (B) Cltx-(g4s)3-IL13(E13Y)-IgG4(PEQ)-CD28TM-CD28gg-CD3z (SEQ ID NO: 45; SEQ ID NO: 46 without the MLLLVTSLLLCELPHPAFLLIP signal peptide sequence), (C) IL13(E13Y)-g3-Cltx-IgG4(PEQ)-CD28TM-CD28gg-CD3z (SEQ ID NO: 47; SEQ ID NO: 48 without the MLLLVTSLLLCELPHPAFLLIP signal peptide sequence), (D) IL13(E13Y)-(G45)3-Cltx-IgG4(PEQ)-CD28TM-CD28gg-CD3z (SEQ ID NO: 49; SEQ ID NO: 50 without the MLLLVTSLLLCELPHPAFLLIP signal peptide sequence), (E) Cltx-(g4s)3-IL13(E13Y)-IgG4(PEQ)-CD4tm-41BB-CD3Z (E; SEQ ID NO: 51; SEQ ID NO: 52 without the MLLLVTSLLLCELPHPAFLLIP signal peptide sequence).

DETAILED DESCRIPTION

[0050] In this disclosure the generation and anti-tumor efficacy of CAR comprising an IL-13 and a chlorotoxin are described. The bispecific CAR T cells exhibited potent cytotoxicity against multiple cancer lines. Regional intraperitoneal in vivo delivery of bispecific CAR T cells in GBM murine tumor models conferred elimination of antigen-positive disease and extension of overall survival.

[0051] The present disclosure also provides methods for treating subjects with a cancer or tumor expressing a chlorotoxin receptor and/or an IL-13Rα1 and/or IL-13Rα2 and/or IL-4R.

[0052] T cells expressing a CAR comprising chlorotoxin (or a variant thereof) and IL-13 (or a variant thereof) can be useful in treatment of cancers such as glioblastoma, as well as other cancers expressing a receptor for chlorotoxin (or a variant thereof) or a receptor for IL-13 (or a variant thereof), which include, but are not limited to: primary brain tumors and gliomas (glioblastoma multiforme WHO Grade IV, anaplastic astrocytoma WHO Grade III, low-grade astrocytoma WHO Grade II, pilocytic astrocytoma WHO Grade I, other ungraded gliomas, oligodendroglioma, gliosarcoma, ganglioglioma, meningioma, ependymona), neuroectodermal tumors (medulloblastoma, neuroblastoma, ganglioneuroma, melanoma (metastatic), melanoma (primary), pheochromocytoma, Ewing's sarcoma, primitive neuroectodermal tumors, small cell lung carcinoma, Schwannoma), other brain tumors (epidermoid cysts, brain tumors of unknown pathology, pituitary gland of glioblastoma multiforme pt., metastatic tumors to brain of unknown tissue origin), other tumors (breast cancer, breast cancer metastases, kidney cancer, kidney cancer metastases, liver cancer, liver cancer metastases, lung cancer, lung cancer metastases, lymphoma, lymphoma metastases, ovarian cancer, ovarian cancer metastases, pancreatic cancer, pancreatic cancer metastases, prostate cancer, prostate cancer metastases, colorectal cancer, colorectal cancer metastases, and combinations thereof), combinations thereof, and the like.

EXAMPLES

[0053] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

[0054] The following materials and methods were used in the Examples set forth herein. Dual targeting CART cells of this disclosure are generally referred to as CLTX/IL-13 CAR and IL-13/CLTX CAR interchangeably throughout the disclosure and does not specifically indicate the orientation of the two domains.

Cell Lines

[0055] The GBM cell lines (e.g., PBT-106, PBT-030-2, and PBT-003-4) were cultured in RPMI-1640 (Lonza) containing 20% fetal bovine serum (FBS, Hyclone) and 1× antibiotic-antimycotic (1× AA, Gibco) (complete RPMI). The cancer 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% CO2.

DNA Constructs and Lentivirus Production

[0056] 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 (Brown et al, Cancer Res, 2009).

[0057] Lentivirus was generated as previously described (Brown et al, Mol Ther, 2018). Briefly, 293T cells were transfected with packaging plasmid and CAR lentiviral backbone plasmid using a modified calcium phosphate method. Viral supernatants were collected after 3 to 4 days and treated with 2 mM magnesium and 25 U/mL Benzonase® endonuclease (EMD Millipore). 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 HT1080 cells based on CD19t expression or EGFRt expression.

T Cell Isolation, Lentiviral Transduction, and Ex Vivo Expansion

[0058] 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.

[0059] T cell activation and transduction was performed as described previously (Wang et al, Sci Transl Med, 2020). Briefly, freshly thawed dPBMC were washed once and cultured in complete X-VIVO 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® (Life Technologies), protamine sulfate (APP Pharmaceuticals), cytokine mixture (as stated above) and desired lentivirus at a multiplicity or infection (MOI) of 1 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. CART cells were positively selected for CD19t or EGFRt using the EasySep™ CD19 Positive Enrichment Kit I or II (StemCell Technologies) according to the manufacturer's protocol. 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.

Flow Cytometry

[0060] 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), MUC16 (Abcam, Clone X75 or EPSISR23), biotinylated Protein-L (GenScript USA) (25), Fc ( ), Donkey Anti-Rabbit Ig (Invitrogen), Goat Anti-Mouse Ig (BD Biosciences), and/or 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).

In Vitro Tumor Killing and Rechallenge Assays

[0061] 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 CD45-negative cell counts relative to that observed when targets were co-cultured with Mock (untransduced) T cells. In some embodiments, rechallenge assays, 24-168 hours after completion of the killing assay, CAR T 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. In some embodiments, multiple rechallenge assays follow a killing assay. In a representative initial rechallenge assay, 24-168 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. Afterwards, one or more subsequent rechallenge assays were conducted 24-168 hours after completion of the initial rechallenge 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.

In Vivo Tumor Studies

[0062] All animal experiments were performed under protocols approved by the City of Hope Institutional Animal Care and Use Committee. For in vivo tumor studies, GBM cells (5.0×10.sup.6) were prepared in a final volume of 500 μl HBSS−/− and engrafted in 6 to 8 week old female NSG mice by injection. In some embodiments, engraftment comprises intraperitoneal (i.p.) injection, subcutantous (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 8 for OV90 and day 14 for OVCAR3, T cells were prepared in 1× PBS, and mice were treated with 500 μL i.p. or 200 μL intravenous (i.v.) injection of 5.0×10.sup.6 Mock or Cltx/IL-13 CAR T cells. In the GBM tumor model, we tested the impact of repeat treatment with i.v. Cltx/IL-13 CAR T cells starting at day 4. In some embodiments, this was followed by treatments at additional indicated days post tumor engraftment. Humane endpoints were used in determining survival. 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. At pre-determined time points or at moribund status, mice were euthanized and tissues and/or ascites fluid were harvested and processed for flow cytometry and/or immunohistochemistry as described below.

[0063] 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 4 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. Cells from i.p. ascites fluid was collected from euthanized mice by injecting 5 mL cold 1× PBS into the i.p. cavity, which was drawn up via syringe and stored on ice until further processing. RBC-depleted ascites was washed, stained, and analyzed by flow cytometry for tumor-associated glycoprotein expression and CAR T cells using antibodies and methods described above.

Example 1: Construction of Bispecific CLTX/IL-13 CAR T Cells Containing Differing Linkers and Differing in the Orientations Between CLTX and IL-13 Tumor-Targeting Domains

[0064] The studies described below show that dual-targeting CAR can be stably expressed on primary T cells.

[0065] A number of bispecific CAR constructs were designed (FIGS. 1A-1C). A representative schematic of a lentiviral chimeric antigen receptor (CAR) cassette used depicts CLTX and IL-13 as the antigen-targeting domains, where transcription of the CLTX/IL-13 CAR, as well as the associated T2A ribosomal skip and truncated CD19 (CD19t) sequences were driven by the EF1 promoter (EF1p). The CAR constructs also included a transmembrane domain (TM), a costimulatory domain (e.g. CD28 or 41BB), a CD3 zeta domain. The CARs were co-expressed with truncated CD19t, which served as a marker for the successful transduction of the cells with the CAR construct.

[0066] Four representative CLTX/IL-13 CAR constructs differ in the orientations between CLTX and IL-13 tumor-targeting domains and/or the linker between these two domains (FIG. 1B). Without being bound by theory, differing lengths in the linker of the construct may provide differences in the CARs ability to bind an antigen and/or receptor and transmit activation signals after binding. Without being bound by theory, differing the orientation of the two tumor-targeting domains of the construct may provide differences in the CARs ability to bind an antigen and/or receptor and transmit activation signals after binding. These differences could also result differential killing of the targeted tumor cells.

[0067] In some embodiments, CLTX/IL-13 CAR lentivirus was used to transduce human healthy donor-derived peripheral blood mononuclear cells depleted of CD14+ and CD25+ cells (dPBMC) and/or T.sub.CM/SCM/N cells, 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.

[0068] Flow cytometric analysis of healthy donor T cells (HD417.1 T.sub.CM/SCM/N) engineered to express the dual-targeting CAR show successful CAR expression (FIG. 1D). Anti-CD19 and anti-Fc staining showed sucessful co-expression of a representative CLTX/IL-13 CAR and CD19t transgenes in transduced T cell subsets. Percentages of immunoreactive cells for transduced cells (CLTX/IL-13 CAR), compared with untransduced cells (Mock), 14 days after CD3/CD28 bead stimulation are shown to prove the capability to transduce human T cells with a dual-targeting CAR (FIG. 1D).

Example 2: Dual-Targeting CAR T Cells Activated Against Tumor Cells

[0069] The studies described below examined activation of dual-targeting CAR T cells with different targeting domain orientations and linker designs.

[0070] Schematic diagrams of representative dual-targeting CAR constructs differing in the orientations between CLTX and IL13 tumor-targeting domains, and linkers that separate CLTX with IL13, including g3 (3 amino acids) and (g4s)3 (15 amino acids) are shown in FIG. 1B.

[0071] T cells transduced with different dual-targeting CAR constructs were activated against co-cultured GBM cells (FIG. 2). CAR T cells were cocultured with indicated GBM cells (PBT003-4, PBT030-2, and PBT106), at an effector:target (E:T) ratio=1:1 (20,000 T cells, 20,000 target cells) for 5 hours. Results show the percentage of CAR T cells that express the degranulation marker CD107a, which indicates T cell activation (FIG. 2). Results for untransduced (mock) T cells and T cells transduced with only CLTX or IL-13 are shown for comparison. Bispecific CAR T-cells were activated against all three GBM cell lines.

Example 3: Validation that Dual-Targeting CAR T Cells Kill Tumor Cells

[0072] The studies described below examined effector potency of dual-targeting CAR T cells with different tumor-targeting domain orientations, linker designs, and costimulatory regions.

[0073] Schematic diagrams of representative dual-targeting CAR constructs differing in the orientations between CLTX and IL13 tumor-targeting domains, and linkers that separate CLTX with IL13, including g3 (3 amino acids) and (g4s)3 (15 amino acids) are shown in FIG. 1B

[0074] These dual-targeting T cells exhibit differential long-term effector function across different constructs (FIGS. 3A-3C). T cells engineered with different dual-targeted CAR constructs were co-cultured with GBM cells at an effector:target ratio=1:4 (4,000 T cells, 16,000 target cells), and rechallenged with 32,000 GBM cells every 48 hours (arrowheads). Plotted are the remaining viable tumor cell numbers, which indicate the long-term killing potential of the bispecific CAR T cells (FIGS. 3A-3C).

[0075] After 24 hours, T cell-mediated killing activity was evident with all four of the bispecific CAR T cells in the GBM tumor cell lines (PBT003-4 in FIG. 3A, PBT030-2 in FIG. 3B, and PBT106 in FIG. 3C) demonstrating the potent killing ability of these dual-targeting T CAR constructs. These dual-targeting CAR T cells also demonstrated potent killing activity in each rechallenge assay.

[0076] Experiments also tested effector potency of dual-targeting CAR T cells with different intracellular signaling domains. A schematic diagram of representative dual-targeting CAR constructs differing in intracellular co-stimulatory domains, including CD28 and 41BB, is shown in FIG. 4.

[0077] Using flow cytometry, we then tested for the killing of tumor cells (% specific lysis) of GBM tumor cells (including PBT030-2 and PBT106 cells) after 48 hours. Tumor cells were co-cultured with the indicated bispecific CAR T cells at an effector:target ratio=1:4 (4,000 T cells, 16,000 target cells) for 48 hours, and killing percentages were calculated by comparing with tumor cells numbers co-cultured with the same amount of mock T cells. The results show percentages of tumor cells killed by the dual-targeting CAR T cells. Both the CAR T cells with the CD28 domain and the CAR T cells with 41BB domain effectively killed GBM tumor cells (including PBT030-2 and PBT106 cells) (FIG. 4). Dual-targeting CAR T cells with different co-stimulatory domains are able to eliminate GBM cells.

[0078] All bispecific CAR T cells used herein led to effective, potent, and/or sustained killing of one or more of the GBM tumor cells lines.

Example 4: Validation that Bispecific CLTX/IL-13 CAR T Cells Delivered In Vivo in a Mouse Model Exhibit Potent Anti-Tumor Activity and Confer Extended Lifespan to the Mice

[0079] To evaluate therapeutic potential and in vivo efficacy of representative dual-targeting CAR T cells to selectively target and eliminate tumor cells an in vivo model, CLTX/IL-13 CAR T cells were delivered to a huGBM mouse model, and tumor size and survival was evaluated over time.

[0080] Experiments were conducted to show the antitumor effect of dual-targeting CAR T cells against orthotopic GBM xenograft. A schematic shows a representative model used herein: PBT106 orthotopic xenograft generation and CAR T cell treatment in NSG mice (FIG. 5A). Intracranial engraftment of PBT106 or PBT103B-IL13Ra2 GBM cells (100,000/mouse) were allowed to grow for 7 days before treating with Mock T cells or dual-targeting CAR T cells (500,000/mouse). Humane endpoints were used in determining survival curves of NSG mice engrafted with GBM cells and treated with T cells.

[0081] Survival of GBM-bearing mice receiving different treatments was plotted over a 256-day monitoring period (FIG. 5B). Mice with tumor growth were euthanized within 24 hours after discovery of neurological symptoms. Results show that the mice treated with dual-targeting CAR T cells (either Tandem-28z or Tandem-BBz) had significantly improved survival of human GBM bearing mice. Delivery of a composition comprising bispecific CAR T cells significantly extended survival of mice (FIG. 5B).

[0082] Additionally, tumor growth in each group of mice was monitored through bioluminescent imaging (FIG. 5C). GBM cells were lentivirally transduced to express ffluc to allow for tracking of tumor growth via non-invasive optical imaging. The bioluminescent intensity of each mouse (dotted lines) and the geometric means (solid lines) within each group report on the tumor size associated with each group (untreated, “tumor only”; untransduced, “mock”; bispecific CAR T cell treated, “CAR”). Rapid anti-tumor effects were observed in mice treated with the dual-targeting CAR2 T cells, reaching a maximal anti-tumor response 1-3 weeks following treatment (FIG. 5C).

OTHER EMBODIMENTS

[0083] 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.

[0084] All references are herein incorporated in their entirety for any and all purposes.