Mesothelin-targeted car t cells with increased Anti-tumor efficacy, persistence and resilience and use thereof for the treatment of mesothelin-positive cancers

Abstract

T naïve / stem cell memory (TNaïve / scm) cells expressing a chimeric antigen receptor (CAR) that targets the tumor antigen mesothelin are described. The antigen-binding domain of the CAR includes the variable domains of the highly potent anti-mesothelin antibody hYP218. Methods of using the mesothelin-targeted CAR T cells for the treatment of mesothelin-positive tumors is also described. It is demonstrated herein that the combination of the TNaïve / scm cell population and the potent mesothelin-targeted CAR enhanced the anti-tumor activity of the CAR T cells due to increased cytotoxicity, greater persistence in the tumor and a reduced exhaustion phenotype of TNaïve / scm cells.

Description

[0001] 4239-113023-02

[0002] MESOTHELIN-TARGETED CAR T CELLS WITH INCREASED ANTI TUMOR EFFICACY, PERSISTENCE AND RESILIENCE AND USE THEREOF FOR THE TREATMENT OF MESOTHELIN-POSITIVE CANCERS

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U. S. Application No. 63 / 730,757, filed December 11, 2024, which is herein incorporated by reference in its entirety.

[0005] FIELD

[0006] This disclosure concerns naive / stem cell memory T (TNaive / scm) cells expressing a chimeric antigen receptor (CAR) that targets the tumor antigen mesothelin. Use of the CAR-expressing Txaive / scm cells for the treatment of mesothelin-expressing cancers is also described.

[0007] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0008] The electronic sequence listing, submitted herewith as an XML file named 4239-113023-02.xml (38,805 bytes), created on December 1, 2025, is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] The mesothelin (MSLN) gene encodes a ~70 kDa precursor protein that is processed to a ~30 kDa N-terminal protein and a ~ 40 kDa C-terminal membrane-bound mature mesothelin (Hassan and Ho, Eur J Cancer 44:46-53, 2008). Mesothelin is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in malignant mesotheliomas (Chang et al., Cancer Res 52:181-186, 1992; Chang and Pastan, Proc Natl Acad Set USA 93:136-140, 1996) and other solid tumors, such as lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, and colorectal cancer (Hassan et al., Clin. Cancer Res. 10:3937-3942, 2004; Argani et al., Clin. Cancer Res. 7:3862-3868, 2001; Hassan et al., Appl. Immunohistochem. Mol. Morphol. 13:243-247, 2005; Ho et al., Clin Cancer Res 13(5):1571-1575, 2007; Li et al., Mol. Cancer Ther. 7:286-296, 2008; Yu et al., J Cancer 1:141-1749, 2010; Tchou et al., Breast Cancer Res Treat 133(2):799-804, 2012; Thomas et al., Lung Cancer. 101:104-110, 2016; Shiraishi et al., Oncotarget. 11(43): 3807-3817, 2020; U. S. Patent No.

[0011] 7,081,518). Due to its expression in several different types of cancer, and minimal expression in healthy tissues, mesothelin is a suitable immunotherapy target for the treatment of multiple solid tumors.

[0012] CAR-T cell therapy in patients with hematological malignancies has shown long-term complete responses (Melenhorst et al., Nature 2022;602:503-509; Porter et al., Sci Transl Med 2015;7:303ral39). However, their efficacy is highly variable and often patient-specific in solid 4239-113023-02

[0013] tumors, indicating additional factors that limit the CAR-T cell activity in targeting solid tumors (Adusumilli et al., Cancer Discov 2021;11:2748-2763; Hassan et al., Nat Med 2023;29:2099-2109; Labanieh and Mackall, Nature 2023;614:635-648; Drougkas et al., J Cancer Res Clin Oncol 2023;149:2709-2734). The immune suppressive microenvironment of solid tumors causes exhaustion of tumor-infiltrating T-cells, leading to poor persistence and significantly reduces the efficacy of CAR-T cell therapy. The lack of CAR-T cell expansion and persistence helps the tumor evade the CAR-T cell mediated killing. In addition, the ability of CAR T cells to navigate efficiently to the tumor, secondary lymphoid organs and sustain a stem phenotype population that could effectively possess memory, proliferation, differentiation, and effector potential are important factors. These functional attributes of T cells often determine the resilience of T cells thereby providing effective tumor elimination and surveillance, resulting in enduring efficacy of the treatment and the long-term tumor-free survival. However, current CAR-T cell production methods often fail to enrich the T cell population that provide effective endurance and moreover, most often include an increasingly heterogenous T cell population which may lead to a response that varies in several degrees.

[0014] SUMMARY

[0015] The present disclosure describes the use of T naive / stem cell memory (T Naive / scm ) cells to generate CAR T cells targeting mesothelin to facilitate enduring treatment efficacy against mesothelin-positive tumors. The T cell compositions disclosed herein are unique because they utilize the most desirable T cell population that possesses sternness, and this T cell population is genetically modified to express a CAR that includes an extremely potent mesothelin-targeted antibody. It is demonstrated herein that the combination of the TNaive / scm cell population and the potent mesothelin-targeted CAR enhanced the anti-tumor activity of the CAR T cells due at least to increased cytotoxicity, greater persistence in the tumor, increased differentiation potential upon antigen exposure, and a reduced exhaustion phenotype. The disclosed TNaive / scm CAR T cells also secreted lower levels of the inflammatory cytokines IFN-y and IL-6 compared to the corresponding Tsuik CAR T cells, which leads to fewer side effects in patients by preventing or decreasing the intensity of cytokine release syndrome.

[0016] Provided herein are isolated T cells expressing a CAR that specifically binds mesothelin, wherein the T cells are TNaive / scm cells. In some aspects, the CAR includes an antigen-binding domain having a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2. In some aspects, the isolated T cells express CD62L. In some examples, the CAR includes a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB intracellular co-stimulatory domain, and / or a CD3^ intracellular signaling domain. 4239-113023-02

[0017] Compositions that include a pharmaceutically acceptable carrier and the isolated T cells disclosed herein are also provided.

[0018] Further provided are methods of treating a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of the isolated T cells or compositions disclosed herein.

[0019] Also provided is a method of producing TNaive / scm cells expressing a CAR that binds mesothelin. In some aspects, the method includes culturing peripheral blood mononuclear cells (PBMCs) in the presence of a CD3 agonist, a CD28 agonist, interleukin (IL)-7, IL-15, and IL-2 to produce an activated T cell population; isolating CD62L-positive cells from the activated T cell population; and transducing the CD62L-positive cells with a nucleic acid molecule encoding the CAR. In some aspects, the CAR includes an antigen-binding domain having a VH domain and a VL domain, wherein the VH domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2.

[0020] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1C: TNaivc / scm cell isolation, transduction and expansion. (FIG. 1A) Schematic representation of enrichment of TNaive / scmcells from human PBMCs. TNaive / scmcells consist of CD3+ T cells expressing CD62L and CD45RA (top panel). Magnetic beads were used to isolate CD62L+ cells. After removal of the beads, the enriched CD62L+ cells were enriched for CD45RA by using CD45RA magnetic beads to obtain CD62L and CD45RA double positive TNaive / scmcells (bottom panel). (FIG. IB) Schematic representation indicating experimental design for generating TNhYP218 CAR T cells from PBMC. (FIG. 1C) Flow cytometry analysis of CD3+ T cells isolated from two healthy donors (ID15 and ID83) show greater than 95% enrichment of CD62L+ / CD45RA+ TNaive / scm population in comparison to about 46% in the native TBulkcells.

[0022] FIGS. 2A-2D: TNhYP218 CAR T cell generation. (FIG. 2A) TBuik and TNaive / scm were transduced with a multiplicity of infection (MOI) of 5, and analyzed for transduction efficiency using EGFR expression by flow cytometry on day 7 post- transduction. Mock indicates T cells isolated from the same donor that underwent the transduction processes except for the addition of virus and was used as a control. Representative flow cytometry from one donor is shown. (FIG. 2B) Transduction efficiency from four different healthy donors as measured by EGFR expression compared to mock show no significant transduction differences between TBuik and TNaive / scm derived CAR T cells. (FIGS.

[0023] 2C-2D) TNhYP218 CAR T cells show decreased expansion compared to TBuik and mock T cells. (FIG. 2C) Representative graph from a single donor showing decreased expansion of TNaive / scm CAR 4239-113023-02

[0024] cells in comparison to TBuik and mock T cells. (FIG. 2D) TNhYP218 CAR T cells show a statistically significant reduced expansion on day 7 of post transduction compared to mock and TBuik CAR T cells in four different healthy donors. TBUIK, TBllik CAR T cells; TNaive / scm, TN11YP218 CAR T cells.

[0025] FIGS.3A-3D: Phenotype of TN11YP218 CAR T cells. (FIGS. 3A-3B) CAR-T cell phenotype analyzed on day 7 post transduction. (FIG. 3A) Flow cytometry analysis for T aive (TN) (CD45RA+CD62L+) phenotype in TBuik and TNhYP218 CAR T cells. (FIG. 3B) Phenotype of CAR T cells from three different healthy donors. There was a statistically significant increase in the Txaive / scm cell population in TNhYP218 CAR T but a decrease in TEMRA phenotype. T central memory (TCM) (CD45RA+CD62L-), T effector memory RA+ (TEMRA) (CD45RA+CD62L+) and T effector memory (TEM) (CD45RA-CD62L-). (FIGS. 3C-3D) TBuik and TNhYP218 CAR T cells were assessed for CD69 activation marker expression. TNhYP218 CAR T cell population show reduced CD69 expression. * Indicates a p value of < 0.05. TBuik, TBUIR CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0026] FIGS.4A-4C: TNhYP218 CAR T cells are cytotoxic to mesothelin expressing cell lines but show significantly decreased IFNy secretion. (FIG. 4A) Cytotoxicity assays were performed by coculturing the mesothelin expressing cancer cell lines with either mock, TBUIR or TNhYP218 CAR T cells at increasing effector to tumor cell ratios (top). After 24 hours of co-culture, the viability of cells was measured using luciferase assay. The bar graphs indicate E:T50obtained from four different healthy donor derived CAR T cells (bottom). (FIGS. 4B-4C) TN11YP218 CAR T cells and mock control cells were co-cultured with mesothelin expressing cancer cell lines at an E:T50of 3:1 for 24 hours. The supernatant was collected and levels of IFNy and IL-2 cytokines were measured using ELISA. TBuik, TBuik CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0027] FIGS.5A-5C: TNhYP218 CAR T cells show an accelerated tumor control compared to TBulkCAR T cells against ovarian cancer tumor xenografts. (FIG. 5A) NSG mice bearing OVCAR8 tumors were infused either with 10xl06Tmock, TBuik or TNhYP218 CAR T cells or saline intravenously on day 0. Mice were monitored for tumor growth using bioluminescence imaging over time. (FIG. 5B) The radiance values of mice from different treatment groups were plotted. (FIG. 5C) Kaplan-Meier curve indicates the survival of various groups. Statistical comparison between groups is also provided. TBuik, TBuik CAR T cells; TNaive / scm, TNI1YP218 CAR T cells, D, days.

[0028] FIGS.6A-6C: TNhYP218 CAR-T cell treatment using the NCI-Meso63 mesothelioma tumor model. (FIG. 6A) NCI-Meso63 cells were implanted subcutaneously in NSG mice and tumor progression was measured. Ten million Tmock, TBuik and TNhYP218 CAR T cells were injected on day 0 and tumor volume was measured as indicated in the graph. The line labelled (a) with asterisks indicates the p value obtained after t test comparing mock with TNhYP218 CAR T cell; the line labelled (b) with asterisks denotes p value of mock vs bulk. (FIG. 6B) Five million CAR T cells were infused along with mock and saline controls in mice bearing NCI-Meso63 tumors. Tumor volume was measured and plotted. (FIG. 6C) Kaplan-Mayer curve indicates an improved survival of the 4239-113023-02

[0029] TNhYP218 CAR T treated group. Statistical comparison between various groups is also provided. TBulk, TBulk CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0030] FIGS. 7A-7C: TNhYP218 CAR-T cell treatment against KLM-1 pancreatic tumor model. (FIG. 7A) Mesothelin expressing KLM-1 pancreatic tumor cell line was injected intraperitoneally to NSG mice and tumor development was measured using bioluminescence. Ten million Tmock, TBulkand TNhYP218 cells were infused intravenously on day 0 and tumor growth was monitored over time (in days). (FIG. 7B) Bioluminescence radiance from tumor groups were plotted. (FIG. 7C) Tumor bearing mice were sacrificed on day 33, tumors were harvested and tumor weight was plotted for each group. TBuik, TBuik CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0031] FIGS.8A-8C: TNhYP218 CAR T cells are able to control KLM-1 pancreatic tumor at a lower dose compared to TBuik CAR T cells. (FIG. 8A) NSG mice with KLM-1 tumors were infused with 5x106mock, TBuik CAR T cells and TNhYP218 CAR T cells intravenously on day 0 and tumor growth was monitored using bioluminescence. (FIG. 8B) Bioluminescence radiance from tumor groups was plotted. (FIG. 8C) Kaplan-Meier curve indicates an improved survival of TNaive / scm treated KLM 1 implanted tumor. Statistical comparison between various groups is also provided. * Indicates a p value of < 0.05, ** p value of < 0.01, **** p value of < 0.0001. TBuik, Tsuik CAR T cells; TNaive / scm, TNhYP218 CAR T cells; D, days.

[0032] FIGS.9A-9D: TNhYP218 CAR T cells show increased persistence in tumor and spleen compared to TBuik CAR T cells. NSG mice implanted with KLM1 tumor were infused with 10 million mock, TBulkand TNhYP218 CAR T cells. The mice were sacrificed on day 33. Single cell suspensions were generated from tumor and spleen and analyzed for CAR T cells by flow cytometry. (FIG. 9A) Representative flow cytometry plots showing EGFR positive CAR T cells in tumor and bar graphs showing data from three mice. (FIG. 9B) EGFR positive CAR T cells identified from spleen samples. (FIG. 9C) CAR-T cells from tumor were further analyzed for their expression of PD1 and the percent of PD1+ CAR T cells were plotted. There was decreased PD1 expression in TNhYP218 CAR T cells. (FIG. 9D) CAR T cells from tumor were analyzed for naive and stem cell markers, CD62L, CD45RA and CD95, and percent of CAR T cells with expression of these markers were plotted. Statistical comparison between TBulkand TNaive / scmindicate a significant p value. * Indicates a p value of < 0.05 and ** indicates < 0.01. TBulk, TBulk CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0033] FIGS. 10A-10F: TNhYP218 CAR T cells show prolonged cytotoxicity and significant resilience upon long term antigen exposure, driven by their ability for increased proliferation. (FIG.

[0034] 10A) Schematic diagram of the experimental design. CAR T cells were co-cultured with mesothelin over-expressing ovarian cancer cell line, OVCAR8, without addition of cytokines at an initial E: T ratio of 3:1. The target cells were replenished every one to two days and the viable T cells in the media were counted using dye exclusion method. This was performed without trypsinization to avoid adherent target tumor cells from being counted. (FIG. 10B) Plot showing number of viable CAR T cells at different time points. (FIG. 10C) Percent of CAR T cells in supernatant were analyzed by flow 4239-113023-02

[0035] cytometry, which shows that TNhYP218 CAR T cells expanded better than TBuik CAR T cells and remained in expanded state for a longer duration. (FIG. 10D) Cytotoxicity of luciferase expressing adherent target cells, OVCAR8, was analyzed. The luminescence of TNhYP218 CAR-T cell treated OVCAR8 cells were compared to TBuik CAR T treated OVCAR8. The results indicate that the TN11YP218 CAR T cells show a significantly increased cytotoxicity for a longer duration than TBuik cells. (FIG. 10E) Complete loss of cytotoxicity against the target cells from the CAR T cells generated from different donors were measured. The results indicate that the TNhYP218 CAR T cells exhibited cytotoxicity for 18 days (mean) compared to a mean of 11.8 days of TBuik CAR T cells. (FIG. 10F) Cell proliferation assay. CAR T cells were pre-stained with celltrace violet, co-cultured with OVCAR8 cells for three days and analyzed using flowcytometry. TBuik, TBuik CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0036] FIGS. 11A-11D: TNhYP218 CAR T cells generated from patients exhibit increased cytotoxicity compared to TBulkCAR T cells. (FIG. 11A) Transduction efficiency using surface expression of EGFR was measured from patient derived CAR T cells by flow cytometry. Compared to mock, TBulkhad a transduction efficiency of 44% and TNhYP218 CAR T cell had an efficiency of 45%. (FIG. 11 B) Post CAR production, cell expansion was measured as fold change on days 2, 5 and 7 and plotted in the graph. (FIG. 11C) cytotoxicity assays were performed by co-culturing the mesothelin expressing cancer cell line (KLM1) with either patient derived mock, Tbuik or TNhYP218 CAR T cells at increasing effector to tumor cell ratios. After 24 hours of co-culture, the viability of cells was measured using luciferase assay. The bar graph indicates the % of cytotoxicity at E: T ratio of 1.5. (FIG. 11D) Cytotoxicity assays were performed using patient derived mock, TBuik or TN11YP218 CAR T cells by co-culturing them with mesothelin expressing OVCAR8 cells at an increasing E: T ratios. Bar graph indicates the % of cytotoxicity at an E: T ratio of 0.375. E: Tso ratios for both KLM1 and OVCAR8 were calculated using non-linear curve fitting method in Graphpad prism. TBuik, TBuik CAR T cells; TNaive / scm, TNhYP218 CAR T cells.

[0037] FIGS. 12A-12B: TNhYP218 CART cells generated from patients exhibit cytotoxicity against autologous tumor cells in vitro and accelerated tumor reduction in vivo. (FIG. 12 A) cytotoxicity assays were performed by co-culturing the mesothelin expressing patient derived cancer cell line NCI-Meso63 with autologous derived mock, TBuik or TNhYP218 CAR T cells at increasing effector to tumor cell ratios. After 24 hours of co-culture, the viability of cells was measured using CellTiter-Glo assay, and the cytotoxicity was calculated and plotted. (FIG. 12B) Patient-derived (NCI-Meso63) mock, TBuik and TNhYP218 CAR T cells were infused intravenously at a dose of 5 million CAR T cells to autologous tumor implanted NSG mice models. Tumor volume was measured and plotted. The significance of tumor volume differences between the treatment groups was measured using t test and the p value is provided. * Indicates a p value of < 0.05 and ** indicates < 0.01. 4239-113023-02

[0038] FIGS. 13A-13C: TN11YP218 CAR T cells generated from patients exhibit cytotoxicity against their autologous tumor cells at lower E: T ratios. (FIGS. 13A-13B, top panels) Cytotoxicity assays were performed by co-culturing the mesothelin expressing padent-derived cancer cell lines (NCI-Meso95, NCI-Meso29, and NCI-Meso41) widi their autologous mock T cells, TBulk or TNhYP218 CAR T cells at increasing E: T ratios. After 24 hours of co-culture, the viability of cells was measured using CellTiter-Glo assay. (FIGS. 13A-13C, bottom panels) The cytotoxicity at a lower 0.75 E: T ratio was plotted for NCI- Meso95 and NCI-Meso29, and cytotoxicity at 1.5 E: T ratio was plotted for NCI-Meso41. Statistical comparison between TBulk and TNhYP218 CAR T cells indicates a significant p- value. * Indicates a p value of < 0.05, ** indicates < 0.01, *** indicates < 0.001 and **** indicates < 0.0001.

[0039] FIGS. 14A-14D: TNhYP218 CAR T cells generated from patients secrete reduced cytokines compared to the TBuik CAR T cells upon coculture with the autologous tumor cells. Cytokine release by CAR T cells generated from patient samples (NCI-Meso63 - FIGS. 14A, 14C and NCI-Meso29 -FIGS. 14B, 14D) when cocultured with their autologous tumor cells at an E: T ratio of 1 for 72 hours. The media were sampled at the indicated time points and measured for the concentration of IFN-y (FIGS. 14A-14B) and IL-6 (FIGS. 14C-14D).

[0040] FIGS. 15A-15D: TNhYP218 cells show significantly accelerated and sustained ability for differentiation to activated CD4+ and CD8+ cells. (FIG. 15A) CAR T cells generated from healthy donors were co-cultured with a mesothelin overexpressing ovarian cancer cell line, OVCAR8, without the addition of cytokines at an initial E: T ratio of 3:1. A constant number of target tumor cells was replenished daily. Arrows indicate the time points when the T cells were analyzed by CITEseq. (FIG.

[0041] 15B) Hie proportion of cell cluster fractions generated upon CITEseq analysis of CAR-T cell products and at days 1, 3, and 7 of co-culture. (FIGS. 15C-15D) Hie percent change of TNhYP218 and TBulk CAR T cells from the product state (day 0) to days 1, 3, and 7 was calculated for (FIG. 15C) cluster 0 (activated CD4+ cells) and (FIG. 15D) cluster 1 (activated CD8+ cells).

[0042] FIGS. 16A-16B: TNhYP218 CAR T cells show significantly enhanced naive, cytotoxic and memory functions with reduced exhaustion. The TNhYP218 and TBIA CAR T cells that were continuously cocultured with OVCAR8 cells were analyzed for their single-cell transcriptomic profiles. The functional scores from the single-cell transcriptomics data were calculated based on the known functions of a set of genes. (FIG. 16A) Plot shows naive and exhaustion scores for cluster 0 (activated CD4+ T cells). (FIG. 16B) Plot shows cytotoxic and T effector memory (Tem) scores for cluster 1 (activated CD8+ T cells).

[0043] SEQUENCES

[0044] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, 4239-113023-02

[0045] as defined in 37 C. F. R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0046] SEQ ID NO: 1 is the amino acid sequence of the 11YP218 VH domain.

[0047] SEQ ID NO: 2 is the amino acid sequence of the 11YP218 VL domain.

[0048] SEQ ID NOs: 3-8 are the amino acid sequences of the hYP218 CDRs according to Kabat.

[0049] SEQ ID NOs: 9-14 are the amino acid sequences of the hYP218 CDRs according to IMGT.

[0050] SEQ ID NO: 15 is the amino acid sequence of the hYP218 CAR.

[0051] SEQ ID NO: 16 is the amino acid sequence encoded by the complete hYP218 CAR transgene (GMCSFR- VH-linker- VL-CD8H-CD8TM-4- 1 BB-CD3 -T2 A-hEGFRt).

[0052] SEQ ID NO: 17 is the amino acid sequence of the GMCSFR signal sequence.

[0053] SEQ ID NO: 18 is the amino acid sequence of a linker.

[0054] SEQ ID NO: 19 is the amino acid sequence of the CD8a hinge region.

[0055] SEQ ID NO: 20 is the amino acid sequence of the CD8a transmembrane domain.

[0056] SEQ ID NO: 21 is the amino acid sequence of the 4-1BB intracellular co-stimulatory domain.

[0057] SEQ ID NO: 22 is the amino acid sequence of the CD3ζ intracellular signaling domain. SEQ ID NO: 23 is the amino acid sequence of the T2A self-cleaving peptide.

[0058] SEQ ID NO: 24 is the amino acid sequence of truncated human EGFR (hEGFRt).

[0059] SEQ ID NO: 25 is a nucleotide sequence encoding the hYP218 CAR.

[0060] SEQ ID NO: 26 is a nucleotide sequence of the complete hYP218 CAR transgene (EF- 10 / 11YP218-C AR / EGFRt).

[0061] SEQ ID NO: 27 is a nucleotide sequence encoding the GMCSFR signal sequence.

[0062] SEQ ID NO: 28 is a nucleotide sequence encoding the hYP218 scFv ( VH-linker- VL). SEQ ID NO: 29 is a nucleotide sequence encoding the CD8a hinge region.

[0063] SEQ ID NO: 30 is a nucleotide sequence encoding the CD8a transmembrane domain. SEQ ID NO: 31 is a nucleotide sequence encoding the 4-1BB intracellular co-stimulatory domain.

[0064] SEQ ID NO: 32 is a nucleotide sequence encoding the CD3ζ intracellular signaling domain.

[0065] SEQ ID NO: 33 is a nucleotide sequence encoding the T2A self-cleaving peptide.

[0066] SEQ ID NO: 34 is a nucleotide sequence encoding hEGFRt.

[0067] DETAILED DESCRIPTION

[0068] I. Abbreviations

[0069] CAR chimeric antigen receptor

[0070] CDR complementarity determining region 4239-113023-02

[0071] E: T effector to target ratio

[0072] EGFR epidermal growth factor receptor

[0073] EGFRt truncated epidermal growth factor receptor

[0074] ELISA enzyme-linked immunosorbent assay

[0075] ET50 ratio of effector to target cells required to achieve 50% maximum killing of target cells

[0076] IFN interferon

[0077] IL interleukin

[0078] MOI multiplicity of infection

[0079] MSLN mesothelin

[0080] NK natural killer

[0081] PBMC peripheral blood mononuclear cell

[0082] PD1 programmed cell death protein 1

[0083] TCM T central memory

[0084] TEM T effector memory

[0085] TEMRA T effector memory RA+

[0086] TIL tumor infiltrating lymphocyte

[0087] TNaive / scm T naive / stem cell memory

[0088] TScmT stem cell memory

[0089] TN T naive

[0090] VH variable heavy

[0091] VL variable light

[0092] II. Summary of Terms

[0093] Unless otherwise noted, technical terms are used according to conventional usage.

[0094] Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, die term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not 4239-113023-02

[0095] intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0096] 4-1BB: A co-stimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4-1BB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response. 4-1BB is also known as TNF receptor superfamily member 9 (TNFRSF9) and CD137. 4-1BB sequences are publicly available, such as under NCBI Gene ID 3604. An exemplary amino acid sequence of 4-1BB is set forth herein as SEQ ID NO: 21.

[0097] Acute myeloid leukemia (AML): A cancer of the blood that originates from granulocytes or monocytes in the bone marrow. AML is the most common type of acute leukemia in adults. This type of leukemia is typically treated with chemotherapy, radiation therapy, stem cell transplant and / or other targeted therapies. AML is also known as acute myeloid leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia.

[0098] Administration: To provide or give a subject an agent, such as T cells expressing a mesothelin-targeted CAR, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0099] Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen (such as mesothelin). Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) domain and the variable light (VL) domain, respectively. Together, the VH domain and the VL domain are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0100] Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U. S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia etal., Nature 342:877, 1989; and Al-Lazikani et al., 4239-113023-02

[0101] JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue): W521-524, 2012; “Paratome CDRs’’) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn / aligncdr / abrsa.php).

[0102] A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VLdomain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.

[0103] A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0104] A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species (such as mouse).

[0105] A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

[0106] Binding affinity: Affinity of an antibody (or CAR) for an antigen (such as mesothelia). In one aspect, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another aspect, binding affinity is measured by an antigen / antibody dissociation rate. In another aspect, a binding affinity is measured by a competition radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In other aspects, antibody affinity is measured by flow cytometry, surface plasmon reference, or biolayer 4239-113023-02

[0107] interferometry (BLI). An antibody that “specifically binds” an antigen (such as mesothelin) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

[0108] In some examples, a CAR (such as a mesothelin-targeted CAR provided herein) specifically binds to a target (such as mesothelin) with a binding constant that is at least 103M-1greater, 104M-1greater or 105M-1greater than a binding constant for other molecules in a sample or subject. In some examples, a CAR has an equilibrium constant (KD) of 5 |1M or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a CAR binds to a target, such as mesothelin, with a binding affinity of at least about 1 x 10‘6M, at least about 0.5 x 10‘6M, at least about 1 x 10‘7M, at least about 0.5 x 10'7M, at least about 1 x 10'8M, at least about 0.5 x 10'8M, at least about 1 x 10'9M, at least about 0.5 x 10'9M, or at least about 0.1 x IO-9. In certain aspects, a specific binding agent that binds to its target has a dissociation constant (Kd) of <1000 nM, <750 nM, 500 nM, <250 nM, <100 nM, <50 nM, <25 nM, <10 nM, <5 nM, <2.5 nM, <1 nM, <0.5 nM, <0.25 nM, <0.01 nM, or <0.001 nM (e.g., 10-6M or less, e.g., from 10-6M to 10-10M, e.g., from 10-10M to 10-12M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs), which is based on BLI technology. In some examples, Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N. J.).

[0109] Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make milk). Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2 / neu protein. Triple negative breast cancer is also called ERnegative PR-negative HER2 / neu-negative breast cancer.

[0110] CD3: A component of the T cell receptor (TCR) complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. Low expression of the antigen results in impaired immune response. CD3ζ is also known as CD247. Sequences for CD3 are publicly available, such as under NCBI Gene ID 919. An exemplary amino acid sequence of the CD3ij intracellular signaling domain is set forth herein as SEQ ID NO: 32.

[0111] CD8 subunit alpha (CD8a): A transmembrane glycoprotein predominantly expressed by cytotoxic T lymphocytes, but can also be expressed by NK cells, cortical thymocytes and dendritic cells. CD8α mediates efficient cell-cell interactions within the immune system. The CD8 protein acts as a coreceptor with the TCR on T cells to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains. CD8a sequences are publicly available, such as under NCBI Gene ID 925. Exemplary amino acid sequences 4239-113023-02

[0112] of CD8α hinge and CD8α transmembrane domains are set forth herein as SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

[0113] CD62L: A cell surface adhesion protein that belongs to a family of adhesion / homing receptors. CD62L, also known as L-selectin, is a marker of certain T cell subsets, including central memory T cells and naive T cells. TNaïve / scmcells express CD62L. This protein plays a role in trafficking of T cells to lymph nodes. See NCBI Gene ID 6402 for human CD62L.

[0114] Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a mesothelin-expressing tumor. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison’s Principles of Internal Medicine, 14th edition; Perry etal., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nded., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.) Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of a CAR that binds mesothelin, used in combination with a radioactive or chemical compound. In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as anti-PDl or anti-PDLl (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA-4).

[0115] Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a scFv) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3Q. Typically, CARs are comprised of an antigen-binding moiety, a hinge / spacer element, a transmembrane domain, and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3^ or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and / or DAP10. In some examples, the CAR is multispecific (such as bispecific) or bicistronic. A multispecific CAR is a single CAR molecule comprised of at least two antigen-binding domains (such as scFvs) that each bind a different antigen or a different epitope on the same antigen (see, for example, US 2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule having two antigen-binding domains that each bind a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules that are linked by 4239-113023-02

[0116] a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both of the antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337).

[0117] Cholangiocarcinoma: A type of cancer that develops in cells that line the bile ducts in the liver.

[0118] Colorectal cancer: Colorectal cancer starts in the colon or the rectum. These cancers can also be called colon cancer or rectal cancer, depending on where they start. Colon cancer and rectal cancer are often grouped together because they have many features in common.

[0119] Complementarity determining region (CDR): Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site. The light and heavy chains of an Ig each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2 and HCDR3, respectively.

[0120] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the activity or affinity of a protein, such as the affinity of an antibody or CAR to mesothelin. For example, a monoclonal antibody or CAR that specifically binds mesothelin can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind mesothelin. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody or CAR specifically binds mesothelin. Non-conservative substitutions are those that reduce an activity or binding to mesothelin.

[0121] Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0122] 1) Alanine (A), Serine (S), Threonine (T);

[0123] 2) Aspartic acid (D), Glutamic acid (E);

[0124] 3) Asparagine (N), Glutamine (Q);

[0125] 4) Arginine (R), Lysine (K);

[0126] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0127] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0128] Degenerate variant: A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

[0129] Framework region: Amino acid sequences interposed between CDRs. Framework regions include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding. 4239-113023-02

[0130] Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins.

[0131] Heterologous: Originating from a separate genetic source or species.

[0132] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular' antigen (an “antigenspecific response”). In one aspect, an immune response is a T cell response, such as a CD4+response or a CD8+response. In another aspect, the response is a B cell response, and results in the production of specific antibodies.

[0133] Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antibodies or CARs) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component occurs, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0134] Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a drug or a detectable label. In some aspects herein, the linker connects a VH domain to a VL domain of an scFv (such as an scFv targeting mesothelin).

[0135] Lung cancer: Cancer that forms in tissues of the lung, usually in the cells lining air passages. The two main types are small cell lung cancer and non-small cell lung cancer (NSCLC). These types can be diagnosed using microscopy.

[0136] Mesothelin: A 40 kDa cell-surface glycosylphosphatidylinositol (GPI)-linked glycoprotein. The human mesothelin protein is synthesized as a 70 kD precursor which is then proteolytically processed. The 30 kD amino terminus of mesothelin is secreted and is referred to as megakaryocyte potentiating factor (Yamaguchi et al., J. Biol. Chem. 269:805 808, 1994). The 40 kD carboxyl terminus remains bound to the membrane as mature mesothelin (Chang et al., Natl. Acad. Sci. USA 93:136 140, 1996). Exemplary nucleic acid and amino acid sequences of mesothelin are as described in PCT Publication No. WO 97 / 25,068; U. S. Patent No. 6,083,502; Chang and Pastan, Int. J. Cancer 57:90, 1994; Chang and Pastan, Proc. Natl. Acad. Sci USA 93:136, 1996; Brinkmann et al., Int. J. Cancer 71:638, 1997; and Chowdhury et al., Mol. Immunol. 34:9, 1997. Amino acid sequences of human mesothelin are publicly available, such as under GENBANK Accession No. AAH09272. Mesothelin also refers to mesothelin proteins or polypeptides which remain intracellular as well as secreted and / or isolated extracellular mesothelin protein.

[0137] Mesothelin-positive cancer: A cancer that expresses or overexpresses mesothelin.

[0138] Examples of mesothelin-positive cancers include, but are not limited to, mesothelioma, lung cancer, 4239-113023-02

[0139] stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer (such as triple negative breast cancer), thymic carcinoma, colorectal cancer, ovarian cancer, acute myeloid leukemia (or any other tumor that expresses or overexpresses mesothelin).

[0140] Mesothelioma: A type of neoplasm derived from the lining cells of the pleura and peritoneum which grows as a thick sheet covering the viscera, and is composed of spindle cells or fibrous tissue which may enclose gland-like spaces lined by cuboidal cells. Mesotheliomas often originate in the tissue lining the lung, heart or abdomen. In some cases, mesotheliomas are caused by exposure to asbestos.

[0141] Naive / stem cell memory T (TNaive / scm) cells: A population of naive T cells with some stem cell properties, as well as memory and proliferation capacity (Arcangeli et al., J Clin Invest 2022;132(12):el50807; Gattinoni et al., Nat Med 2011;17:1290-1297). TNaive / scm cells express at least CD3 and CD62L and may also express CD45RA.

[0142] Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden’’ which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and / or can metastasize is referred to as “malignant.”

[0143] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0144] Ovarian cancer: Cancer that forms in tissues of the ovary (one of a pair of female reproductive glands in which the ova, or eggs, are formed). Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells).

[0145] Pancreatic cancer: A disease in which malignant (cancer) cells are found in the tissues of the pancreas. Also called exocrine cancer.

[0146] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the CAR-expressing cells and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. 4239-113023-02

[0147] For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0148] Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0149] Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0150] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.

[0151] Methods of alignment of sequences for comparison are well-known. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A.

[0152] 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0153] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0154] Homologs and variants of a VLor a VH of an antibody that specifically binds mesothelin, or a fragment thereof are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length 4239-113023-02

[0155] alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).

[0156] Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. A skilled person will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0157] Stomach cancer: Cancer that forms in tissues lining the stomach. Also called gastric cancer.

[0158] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. In some aspects, a subject is a human with a mesothelin-positive cancer.

[0159] Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount of a CAR-expressing cell necessary to inhibit or suppress growth of a tumor. In one aspect, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor (such as a mesothelin-positive cancer), such as reduce a tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and / or reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size / volume / number prior to treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as a mesothelin-positive cancer), such as increase survival time by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, for example as compared to a survival time compared to a subject with no treatment or a different treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as a mesothelin-positive cancer), such as increase survival time by at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months, for example as compared a survival time compared to a subject with no treatment or a different treatment. 4239-113023-02

[0160] In some aspects, combinations of these affects are achieved. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

[0161] Thymic carcinoma: Cancer that forms in the cells that cover the outside surface of the thymus. The thymus is a small organ that lies in the upper chest above the heart and under the breastbone.

[0162] Transduced: A virus or vector “transduces” a cell when it transfers nucleic acid into the cell.

[0163] Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.

[0164] III. Introduction

[0165] The naive / stem cell memory T (TNaive / scm) cell population is a part of the T cell repertoire that has shown quasi-stemness, memory, and proliferation capacity (Arcangeli et al.,. J Clin Invest 2022;132; Gattinoni et al., Nat Med 2011;17: 1290- 1297). Naive T cells in humans have an extended lifespan and increased ability to home and survive in secondary lymphoid organs, which also provides an enduring therapeutic benefit (Gattinoni et al., Nat Med 2017;23:18-27). The present disclosure uses the TNaive / scm population to generate CAR T cells targeting mesothelin to facilitate enduring treatment efficacy against mesothelin-positive tumors. The T cell compositions disclosed herein utilize a superior T cell population that possesses sternness, and this T cell population is genetically modified to express a chimeric antigen receptor (CAR) that includes an extremely potent mesothelin-targeted antibody (hYP218). It is demonstrated herein that the combination of the TNaive / scm cell population and the potent hYP218-containing mesothelin-targeted CAR (referred to herein as “TNhYP218 CAR T cells”) enhanced the anti-tumor activity of the CAR T cells due at least to greater persistence in the tumor.

[0166] CAR T cell therapies typically use unselected CD3+ T cells (TBUII) to manufacture CAR T cells for treatment of patients. To make the TNhYP218 CAR T cells disclosed herein, TNaive / scm cells were enriched using CD62L (and optionally CD45RA) selection (FIG. 1A). CD62L is an L-selectin, which is a marker for naive T cells. CD62L is also a lymph node homing molecule, highly expressed in central memory T cells. TNaive / scm cells are often selected based on CD62L expression along with CD45RA marker positivity. These cells show decreased activation markers such as CD69. CD62L has previously been used as a marker for clinical and preclinical studies (Arcangeli et al., J Clin Invest 2022;132; Larson et al., Cancer Discov 2023;13:580-597). As demonstrated herein, CD62L selection (with or without CD45RA selection) significantly enriched the TNaive / scm population compared to unselected T cells (TBulk). 4239-113023-02

[0167] The data provided herein demonstrate that (1) TNhYP218 CAR T cells exhibit transduction efficiency, expansion, and in vitro cytotoxicity against mesothelin expressing cell lines that is comparable to TBulkCAR T cells; (2) TNhYP218 CAR T cells have decreased IFNγ expression and increased IL-2 secretion when co-cultured with target tumor cells, indicating lower toxicity than TBulkCAR T cells, as well as increased potential for expansion and persistence; (3) TNhYP218 CAR T cells show anti-tumor efficacy in three tumor models, with faster tumor clearance in the OVCAR-8 model and increased anti-tumor activity in the NCI-Meso63 and mesothelioma PDX models, at a lower dose as compared to TBulkCAR T cells; (4) TNhYP218 CAR T cells have increased persistence in the tumor compared to TBulkCAR T cells; (5) TNhYP218 CAR T cells have increased resilience upon repeated exposure to mesothelin-expressing tumor cells; (6) TNhYP218 CAR T cells generated from patient’s autologous T cells have increased cytotoxicity compared to TBulkCAR T cells, demonstrating their increased efficiency; (7) TNhYP218 CAR T cells generated from the T cells of patients with cancer resulted in decreased IFN-y and IL-6 secretion when cocultured with their own tumor cells and could therefore lead to decreased side effects in patients by preventing or decreasing intensity of cytokine release syndrome; (8) TNhYP218 CAR T cells showed accelerated and sustained ability for differentiation to activated CD4+ and CD8+ T cells upon coculturing with mesothelin expressing tumor cells, demonstrating their enhanced sternness compared to TBulk CAR T cells; and (9) TNhYP218 CAR T cells have increased cytotoxicity and showed a naive, effector memory and reduced exhaustion phenotype compared to TBulk CAR T cells, leading to improved antitumor activity.

[0168] IV. Mesothelin-Targeted Chimeric Antigen Receptor-Expressing T Cells

[0169] Provided herein are isolated T cells expressing a CAR that specifically binds mesothelin, wherein the T cells are TNaive / scm cells. In some aspects, the CAR includes an antigen-binding domain having a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of the VH domain of antibody hYP218 (SEQ ID NO: 1) and the VL domain includes the CDR1, CDR2 and CDR3 sequences of the VL domain of antibody hYP218 (SEQ ID NO: 2). In some aspects, the CAR further includes a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0170] In some aspects, the isolated T cells express CD62L. In some examples, the isolated T cells further express CD45RA.

[0171] In some aspects, the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. 4239-113023-02

[0172] In some aspects, the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0173] In some aspects, the CDR sequences of the antigen-binding domain include SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. In other aspects, the CDR sequences of the antigen-binding domain include SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0174] In some aspects, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 2. In some examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.

[0175] In some aspects of the CAR, the hinge region is a CD8α hinge region; the transmembrane domain is a CD8α transmembrane domain; the intracellular co-stimulatory domain is a 4- IBB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3^ intracellular signaling domain. In some examples, the amino acid sequence of the CD8α hinge region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19, or includes or consists of SEQ ID NO: 19. In some examples, the amino acid sequence of the CD8α transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20, or includes or consists of SEQ ID NO: 20. In some examples, the amino acid sequence of the 4-1BB intracellular co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21, or includes or consists of SEQ ID NO: 21. In some examples, the amino acid sequence of the CD3ζ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22, or includes or consists of SEQ ID NO: 22.

[0176] In some examples, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15, or includes or consists of SEQ ID NO: 15.

[0177] In some aspects, the isolated T cells include a heterologous nucleic acid molecule encoding the CAR. In some examples, the nucleotide sequence of the heterologous nucleic acid molecule is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 25 or SEQ ID NO: 26, or includes or consists of SEQ ID NO: 25 or SEQ ID NO: 26. 4239-113023-02

[0178] Also provided are compositions that include a pharmaceutically acceptable carrier and an isolated T cell disclosed herein. Compositions are further described in section V.

[0179] Further provided are methods of treating a mesothelin-positive cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of an isolated T cell or composition disclosed herein. In some examples, the mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, colorectal cancer, or acute myeloid leukemia, but can be any other tumor that expresses or overexpresses mesothelin. In some examples, the subject is a human subject. Methods of treatment are further described in section VI.

[0180] A. Antibody and CAR Amino Acid Sequences

[0181] Provided below are the amino acid sequences of the variable heavy (VH) and variable light (VL) domain of humanized antibody YP218 (hYP218). The CDR residues according to Kabat are underlined and the CDR residues according to IMGT are shown in bold font. The sequence of each CDR is also listed in Table 1.

[0182] hYP218 VH domain (SEQ ID NO: 1) EVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWVRQAPGKGLEWVSCIYTAGSGST YYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSTANTRSTYYLNLWGQGTLV TVSS

[0183] hYP218 VL domain (SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFS GSGSGTDFTLTISSLQPEDVATYYCQSYAYFDSNNWHAFGGGTKVEIKA

[0184] Table 1. CDR sequences of hYP218

[0185] CDR Kabat SEQ ID IMGT SEQ ID NO: NO: HCDR1 FYFYAC 3 FDLGFYFY 9 HCDR2 CIYTAGSGSTYYASWAKG 4 IYTAGSGS 10 HCDR3 STANTRSTYYLNL 5 ARSTANTRSTYYLNL 11 LCDR1 QASQRISSYLS 6 QRISSY 12 LCDR2 GASTLAS 7 GAS 13 LCDR3 QSYAYFDSNNWHA 8 QSYAYFDSNNWHA 14

[0186]

[0187] hYP218 CAR amino acid sequence (SEQ ID NO: 15): 4239-113023-02

[0188] MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWV RQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT CQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQSYAYFDSNNWHAFGGGTKVEIKATSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R

[0189] Residues of

[0190] CAR Component

[0191] SEQ ID NO: 15

[0192] Signal sequence 1-22

[0193] VH domain 25-148

[0194] Linker 149-163

[0195] VL domain 164-275

[0196] CD8α hinge region 278-322

[0197] CD8α TM domain 323-343

[0198] 4- IBB intracellular co-stimulatory domain 344-385

[0199] CD3ζ intracellular signaling domain 386-497

[0200]

[0201] GMCSFR-VH-linker-VL-CD8H-CD8TM-4-1BB-CD3ζ-T2A-hEGFRt (SEQ ID NO: 16) MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACWV RQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT CQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQSYAYFDSNNWHAFGGGTKVEIKATSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP REGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIK HFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFEN LEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQ KTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPRE FVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKY ADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM GMCSFR signal sequence amino acid sequence (SEQ ID NO: 17) MLLLVTSLLLCELPHPAFLLIP

[0202] Linker sequence (SEQ ID NO: 18)

[0203] GGGGSGGGGSGGGGS 4239-113023-02

[0204] CD8α hinge region (SEQ ID NO: 19) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0205] CD8α transmembrane domain (SEQ ID NO: 20)

[0206] IYIWAPLAGTCGVLLLSLVIT

[0207] 4- IBB intracellular co-stimulatory domain (SEQ ID NO: 21) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0208] CD3ζ intracellular signaling domain (SEQ ID NO: 22) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0209] T2A self-cleaving peptide amino acid sequence (SEQ ID NO: 23) EGRGSLLTCGDVEENPGP

[0210] hEGFRt amino acid sequence (SEQ ID NO: 24) RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTV KEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIIS GNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSC RNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGP HCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATG MVGALLLLLVVALGIGLFM

[0211] B. Antibody and CAR Nucleic Acid Sequences

[0212] Provided below are the nucleic acid sequences of the hYP218-based CAR constructs and components thereof.

[0213] hYP218 CAR nucleotide sequence (SEQ ID NO: 25):

[0214] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGA TCCCACATATGGAAGTACAACTGGTAGAGTCAGGAGGCGGCCTCGTACAACCAGGTGGG TCCCTTCGGCTTTCATGTGCCGCTTCTGGTTTCGATCTCGGTTTCTACTTCTACGCTTGTTG GGTACGCCAAGCCCCTGGAAAAGGGTTGGAGTGGGTGTCATGTATTTACACCGCCGGAT CTGGGTCTACGTATTATGCCTCCTGGGCCAAAGGAAGGTTCACTATTAGCCGCGATAACT CCAAGAACACGCTCTACTTGCAAATGAATAGCCTTAGGGCAGAGGACACAGCAGTATAC TACTGCGCGCGGAGTACGGCGAACACCAGGTCCACATACTATCTCAACCTCTGGGGGCA AGGAACCCTCGTGACCGTTAGCTCTGGTGGAGGCGGTTCAGGAGGTGGTGGAAGCGGTG GAGGGGGTAGTGATATTCAAATGACGCAGTCACCGTCAAGCCTCTCAGCCTCAGTTGGA GACCGAGTGACCATTACCTGTCAAGCGTCCCAGAGAATCAGTAGCTACTTGTCCTGGTAT CAGCAGAAGCCCGGAAAAGTTCCTAAGTTGCTGATTTATGGGGCATCTACATTGGCTTCT GGGGTGCCATCCAGATTTTCAGGTAGCGGATCCGGGACCGATTTCACCTTGACCATAAGT TCATTGCAGCCCGAGGATGTGGCAACCTATTATTGCCAGTCCTACGCCTATTTTGACTCA AATAATTGGCACGCTTTTGGAGGTGGGACGAAAGTCGAGATTAAGGCGACTAGTACCAC 4239-113023-02

[0215] GACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGA CTTCGCCTGTGACATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTG TCACTGGTTATCACCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTT ATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCG TACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTA CGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC EF-la / hYP218-CAR / EGFRt (SEQ ID NO: 26):

[0216] GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGA ACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCT CCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGT TCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCCCTGATCATTGTCGATCCTACCATCC ACTCGACACACCCGCCAGGGCCCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGT TACCACACCCAGCATTCCTCCTGATCCCACATATGGAAGTACAACTGGTAGAGTCAGGAG GCGGCCTCGTACAACCAGGTGGGTCCCTTCGGCTTTCATGTGCCGCTTCTGGTTTCGATCT CGGTTTCTACTTCTACGCTTGTTGGGTACGCCAAGCCCCTGGAAAAGGGTTGGAGTGGGT GTCATGTATTTACACCGCCGGATCTGGGTCTACGTATTATGCCTCCTGGGCCAAAGGAAG GTTCACTATTAGCCGCGATAACTCCAAGAACACGCTCTACTTGCAAATGAATAGCCTTAG GGCAGAGGACACAGCAGTATACTACTGCGCGCGGAGTACGGCGAACACCAGGTCCACAT ACTATCTCAACCTCTGGGGGCAAGGAACCCTCGTGACCGTTAGCTCTGGTGGAGGCGGTT CAGGAGGTGGTGGAAGCGGTGGAGGGGGTAGTGATATTCAAATGACGCAGTCACCGTCA AGCCTCTCAGCCTCAGTTGGAGACCGAGTGACCATTACCTGTCAAGCGTCCCAGAGAATC AGTAGCTACTTGTCCTGGTATCAGCAGAAGCCCGGAAAAGTTCCTAAGTTGCTGATTTAT GGGGCATCTACATTGGCTTCTGGGGTGCCATCCAGATTTTCAGGTAGCGGATCCGGGACC GATTTCACCTTGACCATAAGTTCATTGCAGCCCGAGGATGTGGCAACCTATTATTGCCAG TCCTACGCCTATTTTGACTCAAATAATTGGCACGCTTTTGGAGGTGGGACGAAAGTCGAG ATTAAGGCGACTAGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCAT CGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAG TGCACACGAGGGGGCTGGACTTCGCCTGTGACATCTACATCTGGGCGCCCTTGGCCGGGA CTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCAAACGGGGCAGAAAGAAACTCCTGT ATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGT AGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCA GGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGCGAGGGCAGAGGAAGTCTTCTAACATGCGGTGAC GTGGAGGAGAATCCCGGCCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTA CCACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAA TTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCC ATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACT CCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTT TTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAA ATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAAC ATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCA GGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTC 4239-113023-02

[0217] CGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGC CAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGC GTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGA GGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCC TGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGT GCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGA AAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATC CAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCT AAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCC CTGGGGATCGGCCTCTTCATG GMCSFR signal sequence (SEQ ID NO: 27) ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGA TCCCA VH-linker-VL (SEQ ID NO: 28) GAAGTACAACTGGTAGAGTCAGGAGGCGGCCTCGTACAACCAGGTGGGTCCCTTCGGCT TTCATGTGCCGCTTCTGGTTTCGATCTCGGTTTCTACTTCTACGCTTGTTGGGTACGCCAA GCCCCTGGAAAAGGGTTGGAGTGGGTGTCATGTATTTACACCGCCGGATCTGGGTCTACG TATTATGCCTCCTGGGCCAAAGGAAGGTTCACTATTAGCCGCGATAACTCCAAGAACACG CTCTACTTGCAAATGAATAGCCTTAGGGCAGAGGACACAGCAGTATACTACTGCGCGCG GAGTACGGCGAACACCAGGTCCACATACTATCTCAACCTCTGGGGGCAAGGAACCCTCG TGACCGTTAGCTCTGGTGGAGGCGGTTCAGGAGGTGGTGGAAGCGGTGGAGGGGGTAGT GATATTCAAATGACGCAGTCACCGTCAAGCCTCTCAGCCTCAGTTGGAGACCGAGTGACC ATTACCTGTCAAGCGTCCCAGAGAATCAGTAGCTACTTGTCCTGGTATCAGCAGAAGCCC GGAAAAGTTCCTAAGTTGCTGATTTATGGGGCATCTACATTGGCTTCTGGGGTGCCATCC AGATTTTCAGGTAGCGGATCCGGGACCGATTTCACCTTGACCATAAGTTCATTGCAGCCC GAGGATGTGGCAACCTATTATTGCCAGTCCTACGCCTATTTTGACTCAAATAATTGGCAC GCTTTTGGAGGTGGGACGAAAGTCGAGATTAAGGCGACTAGT CD8a hinge region (SEQ ID NO: 29) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCT GTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGG CTGGACTTCGCCTGTGAC CD8a transmembrane domain (SEQ ID NO: 30) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCA CC

[0218] 4-1BB intracellular co-stimulatory domain (SEQ ID NO: 31) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGAT GTGAACTG CD3 intracellular signaling domain (SEQ ID NO: 32) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCT CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG 4239-113023-02

[0219] GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0220] T2A sequence (SEQ ID NO: 33) GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0221] hEGFRt sequence (SEQ ID NO: 34) CGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCT ACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCG GTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGAT ATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAAC AGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACA TGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTC AAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAA TACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCA ACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCC GAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGG CAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGA ACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCA CAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGC GTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGC AGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCC AGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGT GGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG

[0222] V. Compositions

[0223] Provided herein are compositions that include mesothelin-targeted CAR-expressing Tnaive / scm cells as disclosed herein and a pharmaceutically acceptable carrier.

[0224] The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The CAR T cell composition can be formulated for systemic or local (such as intratumor) administration. In some aspects, the CAR T cell composition is formulated for parenteral administration, such as intravenous administration.

[0225] The compositions for administration can include a solution of CAR T cells in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration or quantity of CAR T cells in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body 4239-113023-02

[0226] weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

[0227] The compositions that include CAR T cells can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the compositions can be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary cour se of treatment may be with more than one separate dose, for instance 1-10 doses, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner.

[0228] Typical dosages of the CAR T cell compositions or additional agents can range from about IxlO5to about 1x10’ CAR T cells / kg, such as from about 5xl05to about 5xl08CAR T cells / kg. In some examples, the dosage is at least about 5xl05CAR T cells / kg, at least about 6xl05CAR T cells / kg, at least about 7xl05CAR T cells / kg, at least about 8xl05CAR T cells / kg, at least about 9xl05CAR T cells / kg, at least about 1×106CAR T cells / kg, at least about 2xl06CAR T cells / kg, at least about 3x106CAR T cells / kg, at least about 4x106CAR T cells / kg, at least about 5x106CAR T cells / kg, at least about 6xl06CAR T cells / kg, at least about 7xl06CAR T cells / kg is at least about 8xl06CAR T cells / kg, at least about 9xl06CAR T cells / kg, at least about 1×107CAR T cells / kg, at least about 2xl07CAR T cells / kg, at least about 3xl07CAR T cells / kg, at least about 4xl07CAR T cells / kg, at least about 5xl07CAR T cells / kg, at least about 6xl07CAR T cells / kg, at least about 7xl07CAR T cells / kg, at least about 8xl07CAR T cells / kg, at least about 1×108CAR T cells / kg, at least about 2xl08CAR T cells / kg, at least about 3xl08CAR T cells / kg, at least about 4xl08CAR T cells / kg, or at least about 5xl08CAR T cells / kg.

[0229] In particular examples, the subject is administered a CAR T cell or composition thereof, or additional agent(s), on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the CAR T cell composition and / or additional agent(s) for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0230] In some aspects, the CAR T cell composition is administered intravenously, intraperitoneally, intratumorally, intrapleurally, subcutaneously or by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some aspects, the initial period of treatment (e.g., administration of the therapeutic agent daily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In a related aspect, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain aspects, the dosing regimen of the therapeutic 4239-113023-02

[0231] agent is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off.

[0232] The compositions disclosed herein can also be administered by other routes, including via inhalation, oral, or topical. In some examples, the composition is administered via fine-needle.

[0233] The CAR T cell compositions may be provided in sterile solutions of known concentration. The CAR T cell solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 5xl05to about 1×108CAR T cells / kg of body weight. CAR T cell compositions can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.

[0234] Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic composition as a central core. In microspheres, the therapeutic composition is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.

[0235] Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0236] Polymers can be used for ion-controlled release of the CAR T cell compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (e.g., see Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic compositions are known (see U. S. Patent Nos. 5,055,303; 5,188,837; 4239-113023-02

[0237] 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0238] VI. Methods of Treating Mesothelin-Positive Cancers

[0239] Further provided are methods of treating a mesothelin-positive cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of the isolated T cells or compositions disclosed herein. In some examples, the mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, colorectal cancer, AML, or any other cancer / tumor that expresses or overexpresses mesothelin.

[0240] The CAR T cell compositions disclosed herein can be administered to slow or inhibit the growth of tumor cells, inhibit the metastasis of tumor cells, and / or increase the survival of a subject having a tumor, such as a mesothelin-positive tumor, such as a mesothelin-positive solid tumor. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, increase the survival of a subject having a tumor, and / or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a cancer that expresses mesothelin, such as, but not limited mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, colorectal cancer, or AML. In one aspect, the subject is a human subject diagnosed with a cancer that expresses mesothelin. In one aspect, the subject is a veterinary subject diagnosed with a cancer that expresses mesothelin.

[0241] In some examples, the disclosed method reduces tire size and / or volume of a mesothelin-positive cancer by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even 100% reduction the cancer (such as a reduction of 10-95%, 10-80%, or 20-50%), for example as compared to the size and / or volume of a mesothelin-positive cancer prior to treatment with a therapeutically effective amount of a CAR T cell composition provided herein, or as compared to a treatment that does not include administration of the CAR T cell composition provided herein.

[0242] Also provided herein is a method of reducing tumor growth or metastasis of a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of the CAR T cells or compositions disclosed herein. In some examples, such a method reduces the size, volume, and / or number of a mesothelin-positive cancer metastases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even 100% reduction the metastases (such as a reduction of 10-95%, 10-80%, or 20-50%), for example as compared to the size, volume, and / or number of a mesothelin-positive cancer metastases prior to treatment with a therapeutically effective amount of the CAR T cell composition provided herein, or as compared to a treatment that does not include administration of the CAR T cell composition provided herein. 4239-113023-02

[0243] A therapeutically effective amount of a CAR T cell composition disclosed herein can depend upon the severity of the disease, the type of disease, and the general state of tire patient’s health. A therapeutically effective amount of the CAR T cell composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

[0244] Administration of the CAR T cell compositions disclosed herein can also be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical resection of a tumor). Any suitable anti-cancer agent can be administered in combination with the CAR T cell compositions disclosed herein, such as administered prior to, concurrently with, or following administration of the CAR T cell composition. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. antiandrogens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

[0245] Non-limiting examples of alkylating agents for use with the CAR T cell compositions include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0246] Non-limiting examples of antimetabolites for use with the CAR T cell compositions include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0247] Non-limiting examples of natural products for use with the CAR T cell compositions include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0248] Non-limiting examples of miscellaneous agents for use with the CAR T cell compositions include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

[0249] Non-limiting examples of hormones and antagonists for use with the CAR T cell compositions include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, 4239-113023-02

[0250] Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

[0251] Non-limiting examples of immunomodulators that can be used with the CAR T cell compositions include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Irnreg of New Orleans, La.), SK& F 106528, and TNF (tumor necrosis factor; Genentech).

[0252] Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.

[0253] VII. Methods of Producing CAR-Expressing Naive / Stem Cell Memory T cells

[0254] Also provided are methods of producing isolated naive / stem cell memory T (TNaive / scm) cells expressing a chimeric antigen receptor (CAR) that specifically binds mesothelin. In some aspects, the method includes culturing peripheral blood mononuclear cells (PBMCs) in the presence of one or more agents (such as cytokines) that induce the production of an activated T cell population; isolating CD62L-positive cells from the activated T cell population; and transducing the CD62L-positive cells with a nucleic acid molecule encoding the CAR. In some aspects, the one or more agents that induce production of the activated T cell population includes one or more of a CD3 agonist, a CD28 agonist, interleukin (IL)-7, IL-15, and IL-2. In some examples, the one or more agents includes each of a CD3 agonist, a CD28 agonist, IL-7, IL-15, and IL-2. In some examples, the PBMCs are cultured in the presence of a CD3 agonist, a CD28 agonist, IL-7, IL-15, and / or IL-2 for about 2 days, such as about 36 hours, about 48 hours or about 60 hours.

[0255] In some examples, the PBMCs are cultured with IL-2 at a concentration of about 20 to about 600 IU, such as about 40 to about 500 IU, about 60 to about 400 IU, about 80 to about 300 IU, or about 100 to about 200 IU. In specific examples, the PBMCs are cultured with IL-2 at a concentration of about 100 IU.

[0256] In some examples, the PBMCs are cultured with IL- 15 at a concentration of about 1 to about 20 ng / ml, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 ng / ml. In specific examples, the PBMCs are cultured with IL- 15 at a concentration of about 5 ng / ml.

[0257] In some examples, the PBMCs are cultured with IL-7 at a concentration of about 1 to about 20 ng / ml, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, 4239-113023-02

[0258] about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 ng / ml. In specific examples, the PBMCs are cultured with IL-7 at a concentration of about 5 ng / ml.

[0259] In some examples, the PBMCs are cultured with a CD3 agonist: CD28 agonist ratio of about 1:10 to about 1:100, such as about 1:20 to about 1:80, or about 1:40 to about 1:60. In specific examples, the PBMCs are cultured widi a CD3 agonist: CD28 agonist ratio of about 1: 10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90 or about 1:100. In specific examples, the CD3 agonist is an anti-CD3 antibody and the CD28 agonist is an anti-CD28 antibody. Kits for activating T cells using CD3 and CD28 agonists are commercially available (such as from Miltenyi Biotec).

[0260] In some aspects, the CD62L-positive cells are selected using CD62L magnetic beads.

[0261] In some aspects, the method further includes enriching the isolated CD62L-positive cells for cells expressing both CD62L and CD45RA prior to transduction.

[0262] In some aspects, the transduced T cells express CD62L and CD45RA.

[0263] In some aspects, the PBMCs are obtained from one or more healthy donors (e.g., donors that do not have cancer).

[0264] In some aspects, the CAR includes an antigen-binding domain having a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2. In some examples, the CAR further includes a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0265] In some aspects, the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

[0266] In some aspects, the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively includes SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0267] In some aspects, the CDR sequences of the antigen-binding domain include SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. In other aspects, the CDR sequences of the antigen-binding domain include SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

[0268] In some aspects, the amino acid sequence of the VH domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ 4239-113023-02

[0269] ID NO: 2. In some examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.

[0270] In some aspects of the CAR, the hinge region is a CD8α hinge region; the transmembrane domain is a CD8α transmembrane domain; the intracellular co-stimulatory domain is a 4- IBB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3ζ intracellular signaling domain. In some examples, the amino acid sequence of the CD8α hinge region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19, or includes or consists of SEQ ID NO: 19. In some examples, the amino acid sequence of the CD8α transmembrane domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 20, or includes or consists of SEQ ID NO: 20. In some examples, the amino acid sequence of the 4-1BB intracellular co-stimulatory domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21, or includes or consists of SEQ ID NO: 21. In some examples, the amino acid sequence of the CD3ζ intracellular signaling domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22, or includes or consists of SEQ ID NO: 22.

[0271] In some examples, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15, or includes or consists of SEQ ID NO: 15.

[0272] In some aspects, the nucleotide sequence of the nucleic acid molecule encoding the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 25 or SEQ ID NO: 26, or includes or consists of SEQ ID NO: 25 or SEQ ID NO: 26.

[0273] In some aspects, the nucleic acid molecule encoding the CAR includes a vector. In some examples, the vector is a lentivirus vector, an adeno-associated virus vector or an adenovirus vector.

[0274] EXAMPLES

[0275] The following examples are provided to illustrate particular features of certain aspects of tire disclosure, but the scope of the claims should not be limited to those features exemplified.

[0276] Example 1: Enrichment of TNaive / scm cells

[0277] PBMCs from healthy donors were used for isolation and enrichment of TNaive / scm cells (FIG.

[0278] 1A). PBMCs were thawed and grown in the presence of humanized CD3 and CD28 agonists and cytokines IL-7, IL-15 and IL-2 for two days. CD62L+ cells were isolated using CD62L magnetic beads from the activated T cell population. After removal of the magnetic beads, the cells were further enriched for CD45RA+ cells using magnetic beads (FIG. 1B). This process produced greater than 95% of the pure population of CD62L+ / CD45RA+ TNaive / scm cells as compared to bulk T cells from 4239-113023-02

[0279] several healthy donors (FIG. 1C). Similar results were obtained using only CD62L+ selection. Thus, autologous TNhYP218 CAR T cells generated using only CD62L+ selection can be used to obtain Txaive / scm Cells.

[0280] Example 2: TNatvescm cells show comparable CAR transduction efficiency

[0281] TNaive / scmand TBulkcells from several donors were transduced with the hYP218 CAR construct EF-la / hYP218-CAR / EGFRt (Tomar et al., Mol Cancer Ther 2022;21: 1195-1206) with an MOI of 5 and allowed to expand in the presence of IL-2, IL-7, and IL-15. Transduction efficiency was determined using EGFR expression by flow cytometry on day 7 post-transduction. Transduction efficiency of TNaive / scm was comparable to that of TBulkcells (FIG. 2A). The median transduction efficiency of the TNaive / scmCAR T cells was 63% and TBulkCAR T cells was 45% (FIG. 2B). However, the fold expansion of TNhYP218 CAR T cells was less than that of TBulkCAR T cells (FIG. 2C) with median fold increase of TBulkbeing 49.5 (38-62) and that of TNaive / scm being 24 (18-28) (FIG. 2D). Thus, it is possible to generate TNhYP218 CAR T cells with comparable efficiency.

[0282] Example 3: TNhYP218 CAR T cells exhibit significantly low differentiation and activation phenotype upon expansion

[0283] Phenotypic alterations post transduction and expansion process were evaluated in TNhYP218 and TBuik CAR T cells. A significantly large proportion of hYP218 CAR T cells retained their naive phenotype (CD45RA+CD62L+) 7 days post-transduction (FIGS. 3A-3B). Moreover, compared to TBulk, TNhYP218 CAR T cells exhibited a significantly decreased TEMRA (CD45RA+CD62L-) phenotype. There were no significant differences in T central memory (TCM) (CD45RA-CD62L+) and T effector memory (TEM) (CD45RA-CD62L-) phenotypes between the two groups (FIG. 3B).

[0284] The activation and differentiation status were further evaluated by assessing CD69 expression, which is reported to be downregulated in responders of tumor-infiltrating lymphocytes (TIL) therapy (Krishna el al., Science 2020;370:1328-1334). A large population of TNhYP218 CAR T cells were negative for CD69, suggesting a decreased activation profile (FIGS. 3C-3D). These findings indicate that TNhYP218 CAR T cells show less differentiation, increased sternness and low activation phenotypes compared to TBulkCAR T cells. This indicates the uniqueness of the methodology to develop the potent CAR T cells. This process selects less activated T cells which were shown to be associated with therapeutic efficiency in adoptive T-cell therapy. Therefore, TNhYP218 CAR T cells may be significantly more clinically effective than the TBulkCAR-T cell therapy.

[0285] Example 4: TNhYP218 CAR T cells exhibit in vitro cytotoxicity

[0286] The cytotoxicity of TBulkcells and TNhYP218 CAR T cells was compared by co-culturing them with mesothelin-expressing tumor cell lines at increasing effector to target (E: T) ratios. The 4239-113023-02

[0287] ovarian cancer cell line OVCAR8, the pancreatic cancer cell line KLM1, and a patient derived malignant mesothelioma cell line NCI-Meso63, with variable MSLN expression, were used. TBuik cells and TNhYP218 CAR T cells demonstrated comparable cytotoxicity in the OVCAR8, KLM1 and NCI-Meso63 cell lines with ET50ratios being 1.3, 2.1 and 2.4 for TBulkand 1.3, 2.4 and 2.3 for TNhYP218 CAR T cells, respectively (FIG. 4A). These E: T ratios were derived from the experiments which were repeated with CAR T cells produced from different healthy donors. Additionally, the untransduced mock T cells that are devoid of CAR, did not show any non-specific killing of mesothelin overexpressing tumor cells (FIG. 4A). Thus, the TNhYP218 CAR T cells are as effective as TBuik CAR T cells.

[0288] Example 5: TN11YP218 CAR T cells exhibit significantly decreased IFNy and increased IL-2 secretion

[0289] To further assess the differences in functional capabilities of TBuik cells and TNhYP218 CAR T cells, cytokine release was determined upon short term (24 hours) co-culturing with MSLN expressing cell lines. IFNγ release was significantly less in TNaive / scmCAR T cells than in TBulkCAR T cells (FIG. 4B). However, IL-2 expression on the other hand was increased in the TNhYP218 CAR T cells (FIG. 4C). Studies have shown that an increase in IFNγ is associated with increase in T cell exhaustion, and IL-2 mediates T cell expansion and lineage stability ( Abiko et al., Br J Cancer 2015;112:1501-1509; Spolski et al., Nat Rev Immunol 2018;18:648-659). Hence, the functional modulation by differential secretion of cytokines by TNhYP218 CAR T cells would have increased persistence as well as expansion potential in tumors.

[0290] Example 6: TNhYP218 CAR T cells show tumor regression in multiple in vivo tumor models Two intra-peritoneal mesothelin-overexpressing tumor models were used to evaluate the efficacy of TNhYP218 CAR T cells. In an ovarian cancer model, NSG mice with established OVCAR8 tumors were treated with either 10 million hYP218 TBulk, or TNhYP218 CAR T cells via intravenous injection. The control groups were treated either with saline or untransduced T cells (mock). Tumor progression was monitored over several weeks using luminescence. Both TNhYP218 and hYP218 TBuik CAR T groups started to show tumor regression from day 17 post treatment and were completely tumor free by day 30, with TNhYP218 CAR T cells showing a significantly accelerated tumor regression at early time points (Day 17 and 24) compared to TBuik CAR T cells (FIG. 5A and B). These mice had better survival than the saline or mock group (FIG. 5C).

[0291] The efficacy of the CAR T cells was also tested in a patient-derived NCI-meso63 mesothelioma mouse model (FIG. 6). A significant decrease in tumor volume was evident when animals in the TBulkand TNhYP218 groups were treated with 10 million CAR T cells (FIG. 6A). However, early tumor elimination was observed in the TNhYP218 CAR T cell treated group compared to the TBulkCAR T cell treated group at a lower treatment dose of 5 million CAR T cells 4239-113023-02

[0292] (FIG. 6B). Additionally, the TNhYP218 CAR T cell-treated animals also survived longer compared to the TBuik CAR- treated animals (FIG. 6C).

[0293] NSG mice with established KLM1 tumors were treated with 10 million CAR T cells, (mock, TBulk, TNhYP218) via intravenous injection. The control group was treated with saline. Tumors were measured by bioluminescence. FIGS. 7A-7C show significant reduction in tumor size in the TBulkand TNhYP218 groups, compared to the mock and saline groups. There was also a significant reduction in tumor weight in the treated groups. Reduced tumor growth was observed in mice treated with 5 million TNhYP218 CAR T cells compared to mock and TBulkCAR T cell treated tumors (FIGS. 8A-8B). Moreover, the TNhYP218CAR T cell-treated mice had significantly longer survival compared to the mock and TBulkCAR T cell-treated groups (FIG. 8C). The long-term treatment benefits associated with the TNhYP218 CAR T cell therapy demonstrated the exclusive functional qualities of TNaive / scmsuch as quasi-stemness.

[0294] Thus, TNhYP218 CAR T cells are significantly more efficient at lower doses in reducing tumor size and growth as well as improving survival. Additionally, the effective long term tumor control by TNhYP218 CAR T cells indicates that they can proliferate, differentiate, and perform cytotoxic functions in vivo, which is further indicative of their sternness and persistence.

[0295] Example 7: TNhYP218 CAR T cells show increased persistence in pancreatic tumor models It was hypothesized that TNhYP218 CAR T cells persist in solid tumors not only by localizing and surviving in the tumor, but also in secondary lymphoid organs such as the spleen. To study the persistence of TNhYP218 and TBulkCAR T cells, NSG mice were implanted with KLM1 tumors and treated with 10 million CAR T cells. Mice were sacrificed on day 33, and tumor and spleen were harvested and analyzed for the presence of CAR T cells by flow cytometry. FIG. 9 shows a significantly increased persistence of TNhYP218 CAR T cells in tumor (FIG. 9 A) and spleen (FIG. 9B). This data demonstrates that TNhYP218 CAR T cells were able to survive in the tumor as well as home to and survive in secondary lymphoid organs, such as the spleen.

[0296] Additionally, it was demonstrated that in tumors, the TNhYP218 CAR T cells displayed significantly decreased PD1 expression compared to TBulkCAR T cells (FIG. 9C). This decreased PD1 expression in tumors suggests that TNhYP218 CAR T cells have decreased exhaustion. Analysis of blood by flow cytometry at the same time (day 33) failed to identify CAR T cells in the TNaive / scmand TBulkCAR T cell treated groups. An increased population of CAR T cells with naive and sternness markers, such as CD62L+ (median 30.5 compared to 22 for TBulk), CD45RA+ (median 12.5 compared to 7.5 for TBulk), CD95+ (median 12.4 compared to 5.1 for TBulk), in the TNhYP218 CAR T cell-treated tumor group was observed (FIG. 9D). These markers indicate a less differentiated population in TNhYP218 CAR T cells. Thus, TNhYP218 CAR T cells continue to persist in tumor and spleen, with reduced exhaustion and increased naive and stem phenotypes in the tumor. 4239-113023-02

[0297] Example 8: TNhYP218 CAR T cells show prolonged cytotoxicity and significant resilience upon long term antigen exposure, driven by their ability for proliferation

[0298] The TNhYP218 CAR T cells were further assessed for their ability for long-term expansion, survival, and cytotoxicity upon continued antigen exposure, which was defined as “resilience” of the CAR T cells. To test this, TBulkand TNhYP218 CAR T cells were exposed to mesothelin expressing OVCAR8 cells, which were replenished every day with fresh cancer cells. Cytotoxicity, cell number and percentage of CAR T population were evaluated (FIG. 10A). These assays were performed by growing cells in media lacking cytokines, including IL-2. Upon co-culture, the viable cell number of the TNhYP218 CAR T cells were significantly increased compared to TBulkCAR T cells (FIG. 10B). Mock T cells were significantly reduced within a few days after exposure to tumor cells. TNhYP218 CAR T cells also remained in culture for a longer duration. Importantly, TNhYP218 CAR T cells remained significantly elevated for an extended period (FIGS. 10B-10C). Often, the number of cells failed to represent the cytotoxic potential of the CAR T cells. The measurement of cytotoxicity indicated that TNhYP218 CAR T cells have significantly increased cytotoxicity (50-100% relative to mock) for a prolonged period compared to TBulkCAR T cells (FIG. 10D). TNhYP218 CAR T cells had a median of 13.5 days with a cytotoxicity of more than 50%, however the median cytotoxicity of TBulkCAR T cells was 5.5 days (FIG. 10E). Thus, the TNaive / scm CAR T cells display increased resilience upon repeated exposure to mesothelin-expressing tumor cells.

[0299] Example 9: TNhYP218 CART cells show significantly increased proliferation upon repetitive antigen exposure

[0300] Since increased TNaive / scm CAR T cells were observed in the resilience assays as well as in mouse models, it was hypothesized that the TNaive / scm CAR T cells would also exhibit increased proliferation leading to their rapid expansion upon antigen encountering. The CFSE dye dilution approach indicated a rapid proliferation of TNhYP218 CAR T cells upon encountering the antigen (FIG. 10F). This indicates the unique ability of TNhYP218 CAR T cells to expand faster and generate enough cells needed to contain the tumor cells.

[0301] Next, PBMCs from patients with mesothelin expressing tumors were used to generate TBulkand TNaive / scmCAR T cells. Patient PBMCs were activated with CD3 and CD28 agonists and cultured for two days with cytokines IL-7, IL- 15 and IL-2. The TNaive / scm cells were isolated using CD62L+ and CD45RA markers as described above. The cells were then transduced and expanded for another 7 days in culture. The TNhYP218 CAR T cells showed 44% transduction efficiency, comparable to TBuik CAR T cells with 45% efficiency. Moreover, both types of CAR T cells expanded well with a fold expansion (on day 7) of 60 for TNhYP218 cells, 87.5 for TBulkCAR T cells and 157.5 for mock (FIGS. 11A-1 IB). The cytotoxicity of these patient derived CAR T cells, which showed increased cell killing by TNhYP218 CAR T cells compared to TBulkCAR T cells, was further investigated (FIGS. 11C-11D). TNhYP218 CAR T cells showed significantly lower ET50 as well as increased 4239-113023-02

[0302] cytotoxicity in lower E: T ratios (1.5 and 0.375) compared to TBulkCAR. The cytotoxicity of the patient derived CAR T cells was further tested in autologous cells. TNhYP218 CAR T cells showed a significantly lower ET50 compared to the TBulk(0.54 for TNhYP218 and 5.69 for TBulk) indicating increased efficacy against the autologous tumor cells (FIG. 12A). To validate this observation of superior efficiency by TN11YP218 CAR T cells, the autologous tumors generated in NSG mice model were treated with saline, mock, TBuik and TNhYP218 CAR T cells. The in vivo treatment results indicated that treatment with the TNhYP218 CAR T cells decreased tumors faster than treatment with the TBulkCAR T cells, indicating significant improvement in treatment efficacy by TNhYP218 CAR T cells (FIG. 12B).

[0303] Thus, TNhYP218 CAR T cells are superior in their ability to efficiently treat tumors even at lower numbers, which could be attributed to their sternness and proliferation capabilities.

[0304] Example 10: TNhYP218 CAR T cells generated from patients with cancer exhibit increased cytotoxicity against autologous tumor cells compared to TBulkCAR T cells

[0305] CAR T cells were generated from three additional patients with mesothelioma (NCI-Meso95, NCI-Meso29 and NCI-Meso41). Their cytotoxicity was tested against autologous tumor cells NCI-Meso95, NCI-Meso29 and NCI-Meso41, respectively (FIGS. 13A-13C, top panels). Both the TNhYP218 CAR T and TBulkCAR T cells were cytotoxic to their autologous tumor cells. However, in comparison to TBuik CAR T cells, the TNhYP218 CAR T cells showed enhanced cytotoxicity even at very low E: T ratios of 0.75 or 1.5 (FIGS. 13A-13C, bottom panels). These results demonstrate that TNhYP218 CAR T cells generated from patients are much more active than TBulkCAR T cells generated from the same patients. These results confirm and expand on the results obtained from a single patient with mesothelioma (NCI-Meso63) that showed patient-derived TNhYP218 CAR T cells were more potent than TBuik CAR T cells against the patient’s own cancer cells and autologous tumors in mice (FIGS. 11C-1D and FIGS. 12A-12B).

[0306] Example 11: TNhYP218 CAR T cells generated from patients secrete reduced cytokines compared to TBuik CAR T cells upon coculture with their autologous tumor cells

[0307] Cytokine secretion by patient derived TNhYP218 and TBuikCAR T cells following coculture with their autologous tumor cells was evaluated. These CAR T cells were co-cultured with autologous tumor cells at an effector-to-target (E: T) ratio of 1:1, and cytokine levels of IFN-γ and IL-6 were measured at multiple time points. Upon coculture, TNhYP218 CAR T cells secreted lower levels of IFN-y compared to TBulkCAR T cells in two independent patient samples (NCI-Meso63 and NCI-Meso29) (FIGS. 14A-14B). Similarly, upon coculture with autologous tumor cells, IL-6 secretion was reduced in TNhYP218 CAR T cells across several time points (FIGS. 14C-14D). Collectively, these findings demonstrate that TNhYP218 CAR T cells result in decreased release of the inflammatory 4239-113023-02

[0308] cytokines compared to TBuik CAR T cells. This indicates TNhYP218 CAR T cells may be more effective by preventing or decreasing the intensity of cytokine release syndrome in patients and therefore limiting toxicity.

[0309] Example 12: TNhYP218 CAR T cells show accelerated and sustained ability for differentiation to activated CD4+ and CD8+ T cells

[0310] CAR T cells were assessed for their ability to undergo long-term expansion, differentiation, and cytotoxicity upon continued coculture with tumor cells. To determine this, mock T cells, TBulkand TNhYP218 CAR T cells were cocultured with mesothelin-expressing OVCAR8 cells and CAR T cells were replenished with fresh OVCAR8 cells at a constant number that was used at the beginning of the assay. In the first three days of coculture, significant cytotoxicity by TNhYP218 and TBuik CAR T cells was observed. However, TBulkCAR T cells displayed a rapid reduction of cytotoxicity on day 4 compared to TNhYP218 CAR T cells, which continued to kill the target cells above the E:T50ratio until day 7 (FIG. 15A). To understand the cellular and transcriptomic dynamics that drive the efficiency of TNhYP218 CAR T cells, single-cell sequencing was performed using CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) that targets T cell-associated genes. The CD3+ T cell data was further analyzed and 16 clusters were identified, which were annotated with a unique pattern of gene sets associated with the cluster (FIG. 15B).

[0311] Two new clusters were identified that were formed in TNhYP218 and TBulkCAR T cells upon coculturing with the cancer cells. These two clusters included activated CD4 (cluster 0) and activated CD8 T cells (cluster 1). Importantly, when compared to day 0, the newly formed cluster 0 (activated CD4+; FIG. 15C) and cluster 1 (activated CD8+; FIG. 15D) showed an accelerated and sustained increase in TNhYP218 CAR T cells compared to TBulkCAR T cells. These results demonstrate the increased differentiation potential of TNhYP218 CAR T cells upon antigen exposure (FIGS. 15C-15D), leading to an enduring efficacy that was observed in the resilience assay (FIG. 15A).

[0312] Example 13: TNhYP218 CAR T cells have increased cytotoxicity and show a naive, effector memory and reduced exhaustion phenotype

[0313] The single-cell transcriptomics of CD3+ T cell data generated from the resilience assay (FIG.

[0314] 15A) was further analyzed. The two new clusters formed upon coculture with the target tumor cells, activated CD4 (cluster 0) and activated CD8 (cluster 1) were further quantitated for their functions. The functional scores associated with naive, effector memory, cytotoxicity and exhaustion were calculated based on the marker gene expression associated with each function. These functional scores showed that in cluster 0 (activated CD4+), there was significantly reduced exhaustion in the TNhYP218 CAR T cells, which was accompanied by an increased and sustained naive phenotype at different timepoints (FIG. 16A). These functional differences may explain the improved and sustained 4239-113023-02

[0315] activity of TNhYP218 CAR T cells compared to TBuik CAR T cells. Additionally, the functional scores also showed increased cytotoxic T cell and effector memory function in cluster 1 (activated CD8+) in TNhYP218 CAR T cells compared to TBulkCAR T cells (FIG. 16B). Thus, TNhYP218 CAR T cells are a superior product compared to TBuik CAR T cells, and this is driven by their increased cytotoxicity potential and having a naive, effective memory and reduced exhaustion phenotype.

[0316] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239-113023-02CLAIMS1. An isolated T cell expressing a chimeric antigen receptor (CAR) that specifically binds mesothelin, wherein the T cell is a naive / stem cell memory T (TNatve / scm) cell that expresses CD62L, and wherein the CAR comprises:an antigen-binding domain comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2;a hinge region;a transmembrane domain;an intracellular co-stimulatory domain; andan intracellular signaling domain.

2. The isolated T cell of claim 1, wherein the T cell further expresses CD45RA.

3. The isolated T cell of claim 1 or claim 2, wherein the amino acid sequences of the VH domain CDR1, CDR2 and CDR3 respectively comprise:SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; orSEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

4. The isolated T cell of any one of claims 1-3, wherein the amino acid sequences of the VL domain CDR1, CDR2 and CDR3 respectively comprise:SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; orSEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

5. The isolated T cell of any one of claims 1-4, wherein:the amino acid sequence of the VH domain is at least 95% identical to SEQ ID NO: 1 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 1; and / orthe amino acid sequence of the VL domain is at least 95% identical to SEQ ID NO: 2 and comprises the CDR1, CDR2 and CDR3 sequence of SEQ ID NO: 2.

6. The isolated T cell of any one of claims 1-5, wherein:the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 2.4239-113023-027. The isolated T cell of any one of claims 1-6, wherein:the hinge region is a CD8a hinge region;the transmembrane domain is a CD8a transmembrane domain;the intracellular co-stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and / or the intracellular signaling domain is a CD3^ intracellular signaling domain.

8. The isolated T cell of claim 7, wherein:the amino acid sequence of the CD8a hinge region comprises SEQ ID NO: 19;the amino acid sequence of the CD8a transmembrane domain comprises SEQ ID NO: 20; the amino acid sequence of the 4-1BB intracellular co-stimulatory domain comprises SEQ ID NO: 21; and / orthe amino acid sequence of the CD3ζ intracellular signaling domain comprises SEQ ID NO: 22.

9. The isolated T cell of any one of claims 1-8, wherein the amino acid sequence of the CAR comprises SEQ ID NO: 15.

10. The isolated T cell of any one of claims 1-9, comprising a heterologous nucleic acid molecule encoding the CAR.

11. The isolated T cell of claim 10, wherein the nucleotide sequence of the heterologous nucleic acid molecule comprises SEQ ID NO: 25 or SEQ ID NO: 26.

12. A composition comprising the isolated T cell of any one of claims 1-11 and a pharmaceutically acceptable carrier.

13. A method of treating a mesothelin-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of the isolated T cell of any one of claims 1-11 or the composition of claim 12.

14. The method of claim 13, wherein the mesothelin-positive cancer is mesothelioma, lung cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, thymic carcinoma, colorectal cancer, or acute myeloid leukemia.

15. The method of claim 13 or claim 14, wherein the subject is a human subject.4239-113023-0216. A method of producing isolated naive / stem cell memory T (TNaive / scm) cells expressing a chimeric antigen receptor (CAR) that specifically binds mesothelin. comprising:culturing peripheral blood mononuclear cells (PBMCs) in the presence of a CD3 agonist, a CD28 agonist, interleukin (IL)-7, IL-15, and IL-2 to produce an activated T cell population;isolating CD62L-positive cells from the activated T cell population; andtransducing the CD62L-positive cells with a nucleic acid molecule encoding the CAR.

17. The method of claim 16, wherein the CAR comprises:an antigen-binding domain comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2;a hinge region;a transmembrane domain;an intracellular co-stimulatory domain; andan intracellular signaling domain.

18. The method of claim 17, wherein:the hinge region is a CD8α hinge region;the transmembrane domain is a CD8a transmembrane domain;the intracellular co-stimulatory domain is a 4-1BB intracellular co-stimulatory domain; and / orthe intracellular signaling domain is a CD3ζ intracellular signaling domain.

19. The method of any one of claims 16-18, wherein the amino acid sequence of the CAR comprises SEQ ID NO: 15.

20. The method of any one of claims 16-19, wherein the nucleic acid molecule encoding the CAR comprises the nucleotide sequence of SEQ ID NO: 25 or SEQ ID NO: 26.

21. The method of any one of claims 16-20, wherein the nucleic acid molecule encoding the CAR comprises a vector.

22. The method of claim 21, wherein the vector is a lentivirus vector.

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