Oncostatin M-based chimeric antigen receptor (CAR) immune cells

Abstract

A chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, which comprises a polypeptide that binds to oncostatin M receptor (OSMR), leukemia inhibitory factor receptor (LIFR), a heterodimer between transmembrane glycoprotein 130 (gp130) receptor and OSMR, and / or a heterodimer between gp130 receptor and LIFR.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 487,919, filed March 2, 2023, the subject matter of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on March 4, 2024, has the file name CWR-032533WO ORD.st.26, and is 58,919 bytes in size. [Background technology]

[0003] Chimeric antigen receptor (CAR) T-cell therapy has become a mainstay of physicians' treatment regimens for liquid tumors. CAR-T cell therapies targeting both CD19 and BCMA have both been approved by the FDA. Early CD19 CAR-T trials in pediatric B-cell acute lymphoblastic leukemia (ALL) demonstrated a 93% "complete remission" rate and a 31.5% relapse rate. While CD19 and BCMA CAR-T cell therapies are not effective in all B-cell and plasma cell neoplasms, respectively, the magnitude and duration of responses they have achieved in highly relapsed patients is remarkable. Less than a year ago, CD19 CAR-T cell therapy received FDA approval as first-line therapy for adult patients with large B-cell lymphoma, demonstrating that these therapies are not a last resort for the relapsed / refractory population. However, while CAR-T therapy has been successful in liquid tumors, its impact in solid tumors has been limited, and no CAR-T therapy has been approved by the FDA to treat solid tumors.

[0004] There are two main reasons why CAR-T cells fail in solid tumors: their inability to penetrate the tumor microenvironment (TME) and their inability to kill the cancer itself. For example, mesothelin is overexpressed in mesothelioma cells, and CAR-T cells targeting mesothelin have been engineered. Combining the results of three completed Phase I human trials using mesothelin-targeting CAR-T cells for mesothelioma, only nine of 48 patients showed any response, and all nine were transient responses with stable disease. This disappointing response is not unique to mesothelioma, but has also been seen in many other solid tumor CAR-T trials. Summary of the Invention

[0005] Embodiments described herein relate to chimeric antigen receptors (CARs) containing an extracellular antigen-binding domain targeting the oncostatin M (OSM) receptor, engineered CAR immune cells expressing the CARs, and the use of CAR immune cells in CAR immunotherapy. The extracellular antigen-binding domain of the CAR comprises a polypeptide that binds to the oncostatin M receptor (OSMR), the leukemia inhibitory factor receptor (LIFR), a heterodimer between the transmembrane glycoprotein 130 (gp130) receptor and OSMR, and / or a heterodimer between the gp130 receptor and LIFR (expressed by cancer cells and surrounding cells that support these cancer cells in the tumor microenvironment). CARs can be expressed by immune cells, such as cytotoxic T lymphocytes (T cells), natural killer (NK) cells, and / or natural killer T (NKT) cells, and can target and kill cancer cells expressing OSMR and / or LIFR and surrounding cells that support these cancer cells in the tumor microenvironment.

[0006] In some embodiments, the polypeptide comprises an oncostatin M (OSM) ligand or a fragment thereof that binds to OSMR and / or LIFR and / or OSMR / gp130 receptor and / or LIFR / gp130 receptor heterodimers.

[0007] In some embodiments, OSM ligands comprise polypeptides having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:18.

[0008] In other embodiments, the polypeptide comprises a single chain variable fragment (ScFv) that binds OSMR and / or LIFR.

[0009] In some embodiments, the CAR further comprises one or more hinge domains.

[0010] In some embodiments, the hinge domain may comprise a hinge domain of CD8α having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:19.

[0011] In other embodiments, the hinge domain may comprise an IgG1 hinge domain having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:20.

[0012] In another embodiment, the CAR further comprises one or more transmembrane domains, which may include, for example, a CD8α transmembrane domain, a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0013] In some embodiments, the CD8α transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:21.

[0014] In some embodiments, the CD28 transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:22.

[0015] In other embodiments, the transmembrane domain of CD16a may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:23.

[0016] In other embodiments, the 2B4 transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:24.

[0017] In other embodiments, the NKG2D transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:25.

[0018] In another embodiment, the CAR further comprises one or more intracellular signaling domains, which may include, for example, a 41BB intracellular signaling domain, a CD28 intracellular signaling domain, a CD3ζ intracellular signaling domain, a CD16A intracellular signaling domain, a γc intracellular signaling domain, a 2B4 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and / or combinations thereof.

[0019] In some embodiments, the 41BB intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:26.

[0020] In some embodiments, the CD28 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:27.

[0021] In some embodiments, the CD3 zeta intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:28.

[0022] In some embodiments, the OX40 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:29.

[0023] In some embodiments, the CD16A intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:30.

[0024] In embodiments, the γc intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:31.

[0025] In some embodiments, the 2B4 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:32.

[0026] In some embodiments, the DAP10 intracellular signaling domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:33.

[0027] In some embodiments, the DAP12 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:34.

[0028] In some embodiments, the CAR may further comprise a spacer. The spacer may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 35.

[0029] In another embodiment, the CAR further comprises a signal peptide.

[0030] In some embodiments, the signal peptide comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:36 or SEQ ID NO:37.

[0031] In some embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a hinge domain, a transmembrane domain, a CD28 intracellular domain, and an OX40 intracellular domain.

[0032] In some embodiments, the hinge domain is an IgG1 hinge domain and the transmembrane domain is a CD28 transmembrane domain.

[0033] In other embodiments, the CAR comprises, from N- to C-terminus, an extracellular domain comprising an OSM ligand, an IgG1 hinge domain, a CD28 transmembrane domain, a CD28 intracellular domain, an OX40 intracellular domain, and a CD3ζ intracellular domain.

[0034] In some embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a CD16a intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0035] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a γc intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0036] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a CD28 intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0037] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a 2B4 intracellular signaling domain, a 41BB intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0038] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a 2B4 intracellular signaling domain, a DAP10 intracellular domain, a DAP12 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0039] Other embodiments relate to nucleic acids or nucleotides comprising a nucleotide sequence encoding a CAR described herein. The nucleotides can be operably linked to a promoter and provided in an expression construct. The expression construct can comprise a vector, such as a retroviral vector, a lentiviral vector, or an AAV vector.

[0040] In some embodiments, the expression construct may further comprise a nucleotide sequence encoding a cytokine, which may include, for example, IL-15, IL-12, IL-2, IL-18, IL-21, or a combination thereof.

[0041] In some embodiments, the expression construct can include a nucleotide sequence encoding a CAR and a cytokine. For example, the nucleotide sequence can be at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:6.

[0042] Still other embodiments relate to immune cells comprising the CARs described herein. The immune cells can be transduced or transduced with a vector comprising an expression construct described herein.

[0043] In some embodiments, the immune cells may comprise at least one of T cells, NK cells, or NKT cells.

[0044] In some embodiments, the T cells, NK cells, or NKT cells are isolated from a human.

[0045] In other embodiments, the T cells, NK cells, or NKT cells are autologous.

[0046] In yet other embodiments, the T cells, NK cells, or NKT cells are allogeneic.

[0047] Other embodiments relate to immunotherapeutic compositions comprising a plurality of CAR-expressing immune cells described herein.

[0048] Yet other embodiments relate to methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunotherapeutic composition described herein.

[0049] In some embodiments, the cancer comprises a solid tumor. For example, the cancer can comprise at least one of osteosarcoma, melanoma, mesothelioma, breast cancer, prostate cancer, gastric cancer, head and neck cancer, lung cancer, or brain cancer.

[0050] In other embodiments, the cancer comprises a hematological cancer or malignancy such as acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell malignancies, and multiple myeloma.

[0051] In another embodiment, the composition is co-administered with one or more chemotherapeutic agents. [Brief explanation of the drawings]

[0052] [Figures 1A-1H]Graphs showing that OSM CARs express OSM and selectively kill specific cell lines. A / B - OSM detected on the surface of transduced human CD3+ T cells from two donors. GFP- reflects CAR-T cells that do not express the CAR vector but were transduced with the CAR vector-containing virus, carrying the GFP reporter. C - OSMR surface expression levels of cell lines, displayed as a ratio to the IgG isotype control signal by flow cytometry. D / E - Cytotoxicity assay using a propidium iodide reporter by flow cytometry at a 7:1 ratio of CAR to target cells for 16 hours. F - Cytokine release assay obtained from supernatants after 16 hours of incubation with the listed cell lines. Results are shown as median fluorescence intensity and are relative values. G / HT CD107a staining was performed to determine the degree of cell degranulation. Intra (G) is a comparison between CAR-transduced wells distinguishing between CAR-expressing cells (GFP+) and non-transduced T cells (GFP-). Inter(H) is the comparison between CAR wells and untransduced control wells. UT means untransduced T cells. Student's unpaired t-test was performed for two-group analysis. One-way ANOVA and Tukey's post-hoc test for multiple comparisons was used for three or more groups. NS - not significant. P values ​​are shown in the graphs. [Figures 2A-2C] Images and graphs show that intratumoral injection of OSM-CAR reduces solid tumor burden. A-AGS-luciferase-labeled tumors were injected subcutaneously and allowed to grow until the luminescence signal reached e8. One mouse was then injected intratumorally with 4,000,000 CAR-T cells. Radiance values ​​for mice 7 days after injection are shown. n=2 for the PBS group, n=1 for the CAR group. B-Detroit 562 cells were injected subcutaneously and allowed to grow until palpable. Then, 4,000,000 OSM CAR-T cells, untransduced T cells (UT), or PBS (vehicle) were injected into the tumor. 12–14 days later, tumors were excised and measured. PBS (n=6), UT (n=5), OSM (n=7). One-way ANOVA with Tukey's post-hoc test for multiple comparisons was performed. NS=not significant. *=p<0.05. **=p<0.01 [Figures 3A-3E] Schematics, graphs, and plots showing that intravenously (IV) infused OSM CAR-T cells are cytotoxic to subcutaneous SAOS2 tumors. A - Schematic of the experiment. Endpoint defined as tumor volume exceeding 1700 mm3. B - Tumor volume measured with calipers in two dimensions using the length x width formula at 21 days after SAOS2 infusion and 11 days after T cell IV infusion. C - Tumor volume over time is shown. D - Mouse weight at 7 days after CAR-T cell infusion is shown. E - Kaplan-Meier survival curve showing the number of days mice survived after SAOS2 infusion. Student's independent samples t-test was performed. Kaplan-Meier survival analysis was performed. P values ​​are shown as numbers. [Figures 4A-4D] Schematics, graphs, and images showing that high doses of OSM CAR-T cells increase tumor-killing efficacy and toxicity. A - Schematic of the experiment. B - Mass of SAOS2 tumors excised 5 days after intravenous infusion of 6,000,000 T cells or vehicle. C - Volume of excised tumors measured as LxWxD 5 days after administration. D - Photograph of excised tumors. One-way analysis of variance (ANOVA) performed with Tukey's post-hoc test for multiple comparisons. P values ​​are shown as numbers. Red boxes indicate tumors that were not included in the analysis due to systemic abdominal tumor presence due to subcutaneous injection failure. L, LL, R, RR, etc. are individual mouse identifiers. [Figures 5A-5E] Schematic and graph showing that intravenously injected OSM CAR is cytotoxic to subcutaneous 143B tumors. A - Schematic of the experiment. B - Toxicity results in weight loss in mice immediately after injection (weights 7 days after injection are shown). C - Tumor volumes are shown at 7, 14, and 20 days after intravenous injection of D / ET cells. One-way analysis of variance (ANOVA) performed with Tukey's post-hoc test for multiple comparisons. P values ​​are shown as numbers. X indicates mice euthanized due to weight loss exceeding IACUC guidelines. [Figures 6A-6E]Schematics, images, plots, and graphs showing that intravenously injected OSM CAR does not exhibit significant cytotoxicity against subcutaneous Jeko cells. A - Schematic of the experiment. Jeko cells were grown for 14 days before injection of T cells or vehicle. B - Fluorescence intensity of luciferase-labeled Jeko cells was measured using a Spectrum Imaging System before intravenous T cell injection (day 0) and on day 7. C - Luciferase imaging values ​​(radiance) shown over time. Radiance of tumor 14 days after DT cell injection. E - Excised tumors are shown. L, R, and NC are identifiers of individual mice. Three mice were used per group. Student's unpaired t-test was performed. NS = not significant. DETAILED DESCRIPTION OF THE INVENTION

[0053] Methods involving conventional molecular biology techniques are described herein. These techniques are generally known in the art and are described in detail in methodology treatises such as "Current Protocols in Molecular Biology" ed. Ausubel et al. Greene Publishing and Wiley-Interscience, New York, 1992 (as updated from time to time). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Commonly understood definitions of molecular biology terms can be found, for example, in Rieger et al., "Glossary of Genetics: Classical and Molecular," 5th Ed., Springer-Verlag: New York, 1991, and Lewin, "Genes V," Oxford University Press: New York, 1994. The definitions provided herein are intended to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present invention.

[0054] It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a "drug carrier" includes mixtures of two or more such carriers, and the like. "Optional" or "optionally" means that the described event or circumstance may or may not occur, and that the statement includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0055] As used herein, the term "about" means that amounts, sizes, formulations, parameters, and other values ​​and characteristics are not precise and need not be exact, but may, as desired, be approximate and / or larger or smaller, reflecting tolerances, conversion factors, rounding errors, measurement errors, and the like, as well as other factors well known to those of ordinary skill in the art. When the term "about" is used in describing a value or the endpoint of a range, the disclosure should be understood to include the specific value or endpoint referred to. Whether or not a numerical value or an endpoint of a range in the specification is described as "about," the numerical value or endpoint is intended to include two embodiments: one modified by "about," and the other not. It is further understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint.

[0056] The term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0057] As used herein, the terms "substantial," "substantially," and variations thereof are intended to indicate that a described characteristic is equal to or approximately equal to a value or description. For example, a "substantially planar" surface is intended to indicate a flat or nearly flat surface. Furthermore, "substantially" is intended to indicate that two values ​​are equal or approximately equal. In some embodiments, "substantially" can indicate values ​​within about 10% of each other, such as within about 5% of each other, or within about 2% of each other.

[0058] It should be noted that the terms "substantially" and "about" may be used herein to express the degree of uncertainty inherent in any quantitative comparison, value, measurement, or other expression. These terms are also used herein to indicate the extent to which a quantitative expression may vary from a stated standard without resulting in a change in the basic function of the subject matter. Thus, for example, "T cell-free" or "substantially free of T cell contamination" cells are cells to which T cells are not actively added or batched into cell culture, but which may be present in trace amounts as contamination from natural cell progression during expansion. Similarly, other components may be similarly characterized as "free" or "substantially free." Furthermore, as used herein, the term "consisting essentially of" allows for elements not expressly recited, but excludes elements that affect the basic or novel characteristics of the invention. As described herein, the term "consisting of" excludes elements not expressly recited.

[0059] As used herein, the term "engineered" refers to an entity that is artificially produced, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered entity is synthetic and contains elements that do not naturally occur or are not constructed in the manner utilized in this disclosure. In certain embodiments, the vector is engineered by recombinant nucleic acid techniques and the cell is engineered by transfection or transduction of the engineered vector.

[0060] The terms "nucleic acid," "nucleic acid molecule," "nucleotide," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to the phosphate polymeric forms of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or their phosphate analogs (such as phosphorothioates and thioesters), in either single-stranded form or double-stranded helices. Single-stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to a particular tertiary structure. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having sequence homology to mRNA). A "recombinant DNA molecule" refers to a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, semi-synthetic DNA, and the like. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids described in this disclosure.

[0061] As used herein, a "coding region" or "coding sequence" refers to a portion of a polynucleotide consisting of codons translatable into amino acids. A "stop codon" (TAG, TGA, or TAA) is not normally translated into an amino acid, but it can be considered part of the coding region. However, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are usually determined by a start codon at the 5'-terminus, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3'-terminus, which encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Thus, a single vector can contain only a single coding region, or it can contain two or more coding regions.

[0062] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence refers to a sequence following the start of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0063] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence relates to a coding region or a sequence located 5' to the start of transcription. For example, most promoters are located upstream of the start site of transcription.

[0064] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, e.g., an RNA or polypeptide. It includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or other RNA products, and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, lipid addition, conjugation to other protein subunits, or proteolytic cleavage. As used herein, the term "yield" refers to the amount of polypeptide produced by expression of a gene.

[0065] A "vector" refers to any vehicle for cloning and / or introducing a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached, thereby enabling replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Many vectors, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses, are known and used in the art. Insertion of a polynucleotide into an appropriate vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector having complementary cohesive termini.

[0066] The vector can be modified to encode a selectable marker or reporter that allows for the selection or identification of cells that have incorporated the vector. The expression of the selectable marker or reporter allows for the identification and / or selection of host cells that incorporate and express other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, sulfonamides, puromycin, etc., and genes used as phenotypic markers, such as anthocyanin regulatory genes and isopentanyl transferase genes. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered reporters.

[0067] The term "heterologous" means derived from a genotypically different entity from the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering into a different cell type is a heterologous polynucleotide (and may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a nucleotide sequence heterologous to the vector.

[0068] The term "heterologous gene" or "heterologous nucleic acid" refers to a gene that does not naturally occur as part of the viral genome. For example, the heterologous gene can be a mammalian gene, e.g., a therapeutic gene, e.g., a mammalian gene encoding a therapeutic protein. In some embodiments, the heterologous gene encodes a protein or portion thereof that is defective or absent in the target cell and / or subject. In some embodiments, the heterologous gene comprises one or more exons that encode a protein that is defective or absent in the target cell and / or subject. For example, in some embodiments, the heterologous gene comprises one or more trans-splicing molecules, e.g., as described in WO2017 / 087900 (incorporated herein by reference in its entirety). In some embodiments, the heterologous gene comprises a therapeutic nucleic acid, such as a therapeutic RNA (e.g., a microRNA).

[0069] The term "promoter" refers to a sequence that regulates the transcription of a heterologous gene operably linked to the promoter. A promoter provides sequences sufficient to direct transcription and / or their recognition sites for RNA polymerase and other transcription factors required for efficient transcription, and can direct cell-specific expression. In addition to sequences sufficient to direct transcription, the promoter sequence of the present invention may also contain sequences of other regulatory elements involved in regulating transcription (e.g., enhancers, Kozak sequences, and introns).

[0070] The terms "homology" and "identity" are used synonymously throughout this specification and refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that are aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous or identical at that position. The degree of homology or identity between sequences is a function of the number of matching or homologous positions shared between the sequences.

[0071] "Mutant" refers to any change in the genetic material of an organism, and particularly to any change in the wild-type polynucleotide sequence (i.e., deletion, substitution, addition, or alteration) or any change in the wild-type protein. The term "variant" is used interchangeably with "mutant." While it is often assumed that changes in genetic material result in changes in the function of the protein, the terms "mutant" and "variant" refer to changes in the sequence of a wild-type protein, regardless of whether the change alters the function of the protein (e.g., increases, decreases, confers a new function) or does not affect the function of the protein (e.g., the mutation or change is silent).

[0072] The term "target cell" refers to any cell that expresses a target gene and that is infected or intended to be infected with a vector. A vector can infect target cells present within a subject (in situ) or in culture.

[0073] The term "host cell," as used herein, refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of ssDNA or vectors. The term includes the progeny of the original cell that has been transduced. Thus, as used herein, "host cell" generally refers to a cell that has been transduced with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in genomic DNA or overall DNA makeup to the original parent cell due to natural, accidental, or deliberate mutation. In some embodiments, the host cell may be an in vitro host cell.

[0074] The term "subject" generally refers to an individual having a biological sample undergoing processing or analysis, and in particular to an individual having or suspected of having cancer. A subject can be any organism or animal subject that is the object of a method or material, including mammals (e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), domestic pets (e.g., dogs, cats, rodents), horses, and transgenic non-human animals. A subject can be a patient, e.g., having or suspected of having a disease (which may be referred to as a condition), such as a benign or malignant neoplasm or cancer. A subject can be undergoing or have undergone treatment. A subject can be asymptomatic. A subject can be healthy but desire cancer prevention.

[0075] The term "treatment" or "treating" includes a beneficial or desired effect on the symptoms or pathology of a disease or condition, and may further include a minimal reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can optionally include either a reduction or amelioration of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or associated symptoms.

[0076] The term "cancer" refers to any type of cancer, neoplasm, or malignant tumor found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumor, carcinoma, and sarcoma. Cancer may be either a liquid tumor type or a solid tumor type. Examples of cancers that may be treated with the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), Examples of the cancer include ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head and neck, esophageal), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, and multiple myeloma. Additional examples include thyroid cancer, endocrine system cancer, brain tumor, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer, or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioblastoma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic cancer, These include islet cell tumors, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the breast, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0077] The term leukemia broadly refers to progressive, malignant diseases of the blood-forming organs, generally characterized by the abnormal proliferation and development of white blood cells and their precursor cells in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and nature of the disease (acute or chronic), (2) the type of cells involved: bone marrow (myeloid), lymphoid (lymphotropic), or monocytic; and (3) the increased or absent number of abnormal cells in the blood: leukemic or non-leukemic (subleukemic). Exemplary leukemias that may be treated with the compounds, pharmaceutical compositions, or methods provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, germ cell leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemoblastic leukemia, hemopoietic blastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, Leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloblastic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or anaplastic cell leukemia.

[0078] The terms metastasis and metastatic cancer are used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ to another, non-adjacent organ or body site. Cancer originates at a site of origin, e.g., the breast, which is called a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to invade and infiltrate surrounding normal tissue in the local area and / or penetrate the walls of the lymphatic or vascular systems and circulate through these systems to other sites or tissues in the body. A clinically detectable second tumor formed from cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to the cells of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the breast site will be composed of abnormal lung cells, not abnormal breast cells. A secondary tumor in the breast is called "metastatic lung cancer." Thus, the term "metastatic cancer" refers to a disease in which a subject has or has had a primary tumor and has one or more secondary tumors. The phrase "non-metastatic cancer" or "subject with non-metastatic cancer" refers to a disease in which a subject has a primary tumor but does not have one or more secondary tumors. For example, "metastatic lung cancer" refers to a disease in a subject who has or has a history of a primary lung tumor and has one or more secondary tumors at a second site or sites, such as the breast.

[0079] In the context of a substance or the activity or function of a substance associated with a disease (e.g., cancer, such as osteosarcoma, melanoma, mesothelioma, breast cancer, prostate cancer, head and neck cancer, lung cancer, brain cancer, etc.), the term associated with means that the disease (e.g., osteosarcoma, melanoma, mesothelioma, breast cancer, prostate cancer, head and neck cancer, lung cancer, brain cancer, etc.) is caused (in whole or in part) by the substance or the activity or function of the substance, or that a symptom of the disease is caused (in whole or in part) by the substance or the activity or function of the substance.

[0080] The term "chimeric antigen receptor" or "CAR" refers, in its simplest embodiment, to a pair of polypeptides, usually two, which, when present in an immune cell, such as a T cell, provides the cell with specificity for a target cell, usually a cancer cell, and the ability to generate an intracellular signal. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some embodiments, the pair of polypeptides are present on the same polypeptide chain (e.g., comprising a chimeric fusion protein). In some embodiments, the pair of polypeptides are not contiguous with each other, e.g., present on different polypeptide chains. In some embodiments, the pair of polypeptides comprises a dimerization switch, allowing the polypeptides to associate with each other in the presence of a dimerization molecule, e.g., linking the antigen-binding domain to the intracellular signaling domain. In one embodiment, the stimulatory molecule of the CAR is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains of at least one costimulatory molecule as defined below.

[0081] In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain of a costimulatory molecule and a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains of one or more costimulatory molecules and a functional signaling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains of one or more costimulatory molecules and a functional signaling domain of a stimulatory molecule.

[0082] The terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that completes maturation in the thymus and has various roles in the immune system, including identifying specific foreign antigens in the body and activating and inactivating other immune cells. T cells can be any T cell, such as cultured T cells (e.g., primary T cells), T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell and at any stage of development, including CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 cells and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells, etc. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0083] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and participate in cellular immune responses. They are characterized by their secretory profile after stimulation, which may include secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as the relevant recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, they define helper / inducer subsets.

[0084] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic / suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the relevant recognition element in major histocompatibility complex class I-restricted interactions.

[0085] The term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the lack of a T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are used interchangeably and refer to a subset of NK cells that are phenotypically CD3-negative and CD56-positive, express at least one of NKG2C and CD57, and optionally express CD16, but lack expression of one or more of the following: PLZF, SYK, FcRγ, and EAT-2. In some embodiments, an isolated subpopulation of CD56+ NK cells contains expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and / or DNAM-1. CD56+ may be weakly or strongly expressed.

[0086] The term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which recognize peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the nonclassical MHC molecule, CD1d. Two types of NKT cells have been recognized. Invariant or type I NKT cells express a very limited TCR repertoire—a canonical α chain (Vα24-Jα18 in humans) combined with a limited spectrum of β chains (Vβ11 in humans). A second population of NKT cells, termed nonclassical or non-invariant type II NKT cells, exhibits more diverse TCRαβ utilization. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, CD56.

[0087] All parts and ratios used herein are by weight unless otherwise specified.

[0088] Embodiments described herein relate to chimeric antigen receptors (CARs) that include an extracellular antigen-binding domain that targets the receptor for oncostatin M (OSM), engineered CAR immune cells that express the CARs, and the use of CAR immune cells in CAR immunotherapy. The extracellular antigen-binding domain of the CAR comprises a polypeptide that binds to the oncostatin M receptor (OSMR), the leukemia inhibitory factor receptor (LIFR), a heterodimer between the transmembrane glycoprotein 130 (gp130) receptor and OSMR, and / or a heterodimer between the gp130 receptor and LIFR, which are expressed by cancer cells and surrounding cells that support these cancer cells in the tumor microenvironment.

[0089] In some embodiments, CARs can be expressed from immune cells, such as cytotoxic T lymphocytes (T cells), natural killer (NK) cells, and / or natural killer T (NKT) cells, to target and kill cancer cells that express OSMR and / or LIFR and surrounding cells that support these cancer cells in the tumor microenvironment. In one example according to the present disclosure, CAR-T cells can be engineered to target the receptor for OSM using OSM as a ligand. In another example according to the present disclosure, natural killer (NK) cells or natural killer T (NKT) cells are engineered to express a CAR.

[0090] Oncostatin M (OSM) is a soluble IL-6 family cytokine that binds to heterodimers between the transmembrane gp130 receptor and either LIFR or OSMR. These heterodimers are hereafter referred to as LIFR and OSMR. OSM has a higher binding preference for OSMR over LIFR. OSMR is upregulated in many cancers, including lung cancer, triple-negative breast cancer, head and neck cancer, and gastric cancer. OSM is secreted by fibroblasts in the tumor microenvironment (TME), and when OSM is knocked out only in surrounding fibroblasts, tumor growth can be significantly reduced. OSM-OSMR binding in these cells, encompassing the TME, creates a positive feedback loop that supports cancer growth. LIFR, on the other hand, is generally downregulated in cancer, with rare exceptions, including osteosarcoma. Given the upregulation of OSMR not only in cancer cells themselves but also in the surrounding tumor microenvironment, we engineered OSM-liganded CAR immune cells that express OSM on their surface and found that targeting OSMR with OSM-liganded CAR-T cell therapy uniquely resulted in both effective tumor penetration and killing. Thus, OSMR, along with LIFR, offers a targeting opportunity for such cancer treatments.

[0091] Thus, one embodiment described herein relates to a method of treating or preventing a disease or condition by targeting a receptor for OSM. Another embodiment relates to a method of treating or preventing a disease or condition by targeting cells that express or overexpress a receptor for OSM, such as OSMR or LIFR. In an exemplary embodiment, the disease or condition is cancer, such as a solid tumor or a hematological malignancy.

[0092] In some embodiments, the solid tumor can be osteosarcoma, melanoma, mesothelioma, breast cancer, prostate cancer, gastric cancer, head and neck cancer, lung cancer, or brain cancer.

[0093] In other embodiments, the hematological malignancy is any hematological malignancy in which the cancer cells express or overexpress a receptor for OSM, including, but not limited to, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell malignancies, and multiple myeloma.

[0094] In some embodiments, the polypeptide of the extracellular antigen-binding domain of the CAR comprises an oncostatin M (OSM) ligand, which binds to OSMR and / or LIFR and / or OSMR / gp130 receptor and / or LIFR / gp130 receptor heterodimers.

[0095] In some embodiments, "OSM ligand" or "OSM" refers to either a recombinant or naturally occurring form of OSM as set forth in SEQ ID NO: 7, or a variant to a homolog thereof that maintains OSM activity (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or within 100% of the activity of OSM).

[0096] In some embodiments, the OSM ligand comprises a polypeptide having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:7.

[0097] In other embodiments, the OSM ligand may be a fragment of the OSM ligand and may have a "subsequence" or "OSM subsequence." A "subsequence" or "OSM subsequence" refers to a portion of SEQ ID NO: 7 that maintains OSM activity similar to that of the entire sequence, and in particular refers to the extracellular region of OSM that is responsible for binding to the OSM receptor. In one example of a subsequence, the sequence may comprise at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the naturally occurring OSM sequence. Also contemplated are sequences that have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the subsequence.

[0098] In another embodiment, the OSM ligand is a mutant OSM ligand which has a stronger affinity for LIFR than native or wild-type OSM. The stronger affinity of the mutant OSM ligand is possible due to mutations in the BC alpha helix region. A variant OSM ligand may have an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:18.

[0099] In other embodiments, the polypeptide of the extracellular antigen-binding domain of the CAR can comprise a single-chain variable fragment (scFv) that binds to OSMR and / or LIFR. The scFv can include humanized and chimeric antibody fragments, or antigen-binding fragments thereof, that selectively bind to OSMR and / or LIFR. These antibody fragments and antigen-binding fragments thereof can be used in immunotherapy to treat cancer in humans or other mammals.

[0100] Methods for producing humanized scFv from non-human forms are well known in the art. Humanization is essentially performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. The selection of human variable regions, both light and heavy, used in producing humanized antibodies can sometimes be important to reduce antigenicity and / or human anti-mouse antibody (HAMA) responses.

[0101] The variable region of an antibody refers to a specific portion of the variable region whose sequence differs between antibodies. The diversity in antibodies and their antigen-binding fragments is concentrated in three CDR segments located in both the light chain and heavy chain variable regions. The highly conserved portion of the variable region is called the framework (FR) region. In the antibodies described herein, there are four FR regions connected by three CDRs, which may comprise the variable chain. The CDRs in the light chain and heavy chain are held together in close proximity by the FR regions and may contribute to the formation of the target binding site of the antibody together with the CDRs from the other chain.

[0102] Antibody humanization is a process that generates modified human antibodies with variable region ("V region") sequences substantially similar to the actual human germline sequences while retaining the binding specificity and affinity of the reference antibody. This process can, for example, incorporate the CDR1, CDR2, and CDR3 regions of heavy and light chain sequences into a humanized framework that is optimized and pre-specified before the transplantation process begins. The variable regions containing the humanized framework can be engineered into single-chain antigen-binding fragments. The resulting modified humanized antibody fragments can retain the binding specificity of the parent mouse antibody against OSMR or LIFR and have binding affinity for a particular antigen that is comparable to or higher than that of the parent antibody. The modified antigen-binding fragments can have heavy and light chain V regions with a high degree of amino acid sequence identity compared to the closest human germline antibody gene. For example, additional maturation changes can be introduced into the CDR3 region of each chain during the construction process to identify antibodies with optimal binding kinetics.

[0103] In some embodiments, the CAR can further comprise one or more hinge domains.

[0104] In some embodiments, the hinge domain may comprise a hinge domain of CD8α having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:19.

[0105] In other embodiments, the hinge domain may comprise an IgG1 hinge domain having an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:20.

[0106] In yet another example, a CAR cell of the present disclosure comprises the hinge domain of CD28. In yet another example, a CAR cell of the present disclosure comprises the hinge domain of FcγRIII.

[0107] In another embodiment, the CAR may further comprise one or more transmembrane domains, such as a CD8α transmembrane domain, a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0108] In some embodiments, the CD8α transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:21.

[0109] In some embodiments, the CD28 transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:22.

[0110] In other embodiments, the transmembrane domain of CD16a may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:23.

[0111] In other embodiments, the 2B4 transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:24.

[0112] In other embodiments, the NKG2D transmembrane domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:25.

[0113] In another embodiment, the CAR further comprises one or more intracellular signaling domains, which may include, for example, a 41BB intracellular signaling domain, a CD28 intracellular signaling domain, a CD3ζ intracellular signaling domain, a CD16A intracellular signaling domain, a γc intracellular signaling domain, a 2B4 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and / or combinations thereof.

[0114] In some embodiments, the 41BB intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:26.

[0115] In some embodiments, the CD28 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:27.

[0116] In some embodiments, the CD3 zeta intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:28.

[0117] In some embodiments, the OX40 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:29.

[0118] In some embodiments, the CD16A intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:30.

[0119] In embodiments, the γc intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:31.

[0120] In some embodiments, the 2B4 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:32.

[0121] In some embodiments, the DAP10 intracellular signaling domain may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:33.

[0122] In some embodiments, the DAP12 intracellular signaling domain can comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:34.

[0123] Because the distance between T cells and target cells is an important factor affecting tumor recognition and cytotoxicity, a spacer domain can be incorporated between the extracellular antigen-binding domain and the hinge domain, or between the hinge domain and the transmembrane domain. In some embodiments, the spacer comprises a simple alkyl chain, such as -(CH2)nCH3 units, where n is the number of CH2 groups and can range from 1 to 100, preferably 1 to 50, 1 to 20, or 1 to 10. In another example, the spacer can be a peptide of 1 to 50 amino acids, e.g., 1 to 20 amino acids or 1 to 10 amino acids.

[0124] In some embodiments, the spacer may comprise an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35.

[0125] In another embodiment, the CAR further comprises a signal peptide.

[0126] In some embodiments, the signal peptide comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:36 or SEQ ID NO:37.

[0127] In some embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a hinge domain, a transmembrane domain, a CD28 intracellular domain, and an OX40 intracellular domain.

[0128] In some embodiments, the hinge domain is an IgG1 hinge domain and the transmembrane domain is a CD28 transmembrane domain.

[0129] In other embodiments, the CAR comprises, from N- to C-terminus, an extracellular domain comprising an OSM ligand, an IgG1 hinge domain, a CD28 transmembrane domain, a CD28 intracellular domain, an OX40 intracellular domain, and a CD3ζ intracellular domain.

[0130] In some embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a CD16a intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0131] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a γc intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0132] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a CD28 intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0133] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a 2B4 intracellular signaling domain, a 41BB intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0134] In other embodiments, the CAR may comprise, from N- to C-terminus, an extracellular domain comprising an OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a 2B4 intracellular signaling domain, a DAP10 intracellular domain, a DAP12 intracellular domain, and a CD3ζ intracellular domain. The transmembrane domain may be selected from a CD28 transmembrane domain, a CD16a transmembrane domain, a 2B4 transmembrane domain, or an NKG2D transmembrane domain.

[0135] Other embodiments relate to nucleic acids comprising a nucleotide sequence encoding a CAR described herein. Nucleic acids encoding a CAR can be easily prepared from a predetermined CAR amino acid sequence by any conventional method. The base sequence encoding the amino acid sequence can be obtained from the aforementioned NCBI RefSeq ID or GenBank accession number for the amino acid sequence of each domain, and the nucleic acids of the present disclosure can be prepared using standard molecular biological and / or chemical procedures. For example, polynucleotides can be synthesized based on the base sequence, and the polynucleotides of the present disclosure can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0136] The nucleotide sequence encoding CAR can be operably linked to a promoter and provided in an expression construct. The vector can be suitable for replication and integration into eukaryotes. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of desired nucleic acid sequences.

[0137] In some embodiments, the vector is a viral vector. Viral vector technology is known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. In some embodiments, viruses useful as vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In some embodiments, the vector is a lentiviral vector. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme cleavage sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0138] Vectors derived from viruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and their infection in daughter cells.Compared with vectors derived from retroviruses, such as murine leukemia viruses, lentivirus vectors have the additional advantage that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0139] The retroviral vector may also be, for example, a gammaretroviral vector. A gammaretroviral vector may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (for example, two) long terminal repeats (LTRs), and a gene encoding a desired transgene, for example, a gene encoding a CAR. A gammaretroviral vector may lack viral structural genes such as gag, pol, and env. Representative gammaretroviral vectors include murine leukemia virus (MLV), spleen focus-forming virus (SFFV), myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0140] In some embodiments, a vector can express two or more genes, each expressed separately under the control of a different promoter region, for example, by using a bicistronic or tricistronic promoter. Expression of two or more genes from the same vector can be achieved by using a multiple promoter plasmid, e.g., a bicistronic or tricistronic promoter. Examples of lentiviral vectors containing multiple promoters are known in the literature. For example, the vector pLENTI-bi-cistronic uses the PKG promoter and the mini-CMV promoter in opposite orientations to drive the expression of two genes (Applied Biological Material Inc., Richmond, BC, Canada). Similarly, the tricistronic vector pLENTI-tri-cistronic drives the expression of three genes. In this configuration, one gene can be driven by the mini-CMV promoter, while the second and third genes can be driven by the PGK promoter, with a T2A peptide cleavage site separating the two genes.

[0141] In another embodiment, bi- or tricistronic vectors may also be constructed utilizing an internal ribosome entry site (IRES), such as elements derived from the encephalomyocarditis virus (EMCV), for translation of two or more open reading frames (ORFs). Such vectors are designed to drive transcription of bi- or tricistronic messages under the control of a strong human promoter regulatory region, such as CMV or EF1alpha. An IRES is a relatively short DNA sequence that can initiate RNA translation in a 5′ cap-independent manner. The first cistron is translated in a cap-dependent manner driven by a strong mammalian promoter, while subsequent ones utilize virally derived intercistronic regions, such as the poliovirus internal ribosome entry site or the encephalomyocarditis virus cap-independent translation enhancer for enhanced translation.

[0142] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are usually located in the 30-110 bp region upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, allowing promoter function to be maintained even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

[0143] Other examples of promoters include the SFFV promoter and the cytomegalovirus (CMV) promoter. Other constitutive promoter sequences may also be used, including the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters (including, but not limited to, actin promoter, myosin promoter, elongation factor-1α promoter (EF1α), hemoglobin promoter, and creatine kinase promoter).

[0144] Furthermore, embodiments are not limited to the use of constitutive promoters, but may include, for example, inducible promoters. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, and can turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0145] Vectors may also include, for example, a signal sequence to facilitate secretion, a polyadenylation signal and a transcription terminator (e.g., from the bovine growth hormone (BGH) gene), elements allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1, or others known to those skilled in the art), and / or elements allowing selection (e.g., a puromycin resistance gene, an ampicillin resistance gene, and / or a zeocin marker).

[0146] The sequences encoding various elements of CAR can be placed on the same nucleic acid molecule, for example, the same plasmid or vector, for example, a viral vector, for example, a lentiviral vector. For example, (i) the sequence encoding the extracellular antigen-binding domain and (ii) the sequence encoding the intracellular signaling member can both be present on the same nucleic acid, for example, a vector. The production of corresponding proteins can be achieved, for example, by using separate promoters or by using bicistronic transcription products (which result in the production of two proteins by cleavage of a single translation product or translation of two separate protein products).

[0147] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected with a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection method. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as the neomycin resistance gene.

[0148] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and encode polypeptides whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with the minimal 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0149] Methods for introducing and expressing genes into cells are known in the art.In the case of expression vectors, the vector can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art.For example, the expression vector can be introduced into host cells by physical, chemical or biological means.

[0150] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistics, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is the calcium phosphate method.

[0151] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0152] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems (polymer complexes, nanocapsules, microspheres, beads, etc.) and lipid systems (including water-in-oil emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be combined with a lipid. Lipid-bound nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, bound to liposomes via linking molecules attached to both the liposomes and the oligonucleotides, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to a particular structure in solution. For example, they may exist in a bilayer structure as micelles or with a "collapsed" structure. They may also simply be dispersed in solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic. For example, lipids include the lipid droplets naturally occurring in the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives (fatty acids, alcohols, amines, amino alcohols, aldehydes, etc.).

[0153] Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine (DMPC) is obtained from Sigma (St. Louis, Mo.), dicetyl phosphate (DCP) is obtained from K & K Laboratories (Plainview, NY), cholesterol ("Choi") is obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the solvent of choice because it volatilizes more readily than methanol.

[0154] "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5;505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0155] Regardless of the method used to introduce exogenous polynucleotides into host cells or otherwise expose cells to the polynucleotides of this disclosure, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include, for example, "molecular biological" assays such as Southern blotting, Northern blotting, RT-PCR, PCR, etc., which are well known to those of skill in the art; and "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of this disclosure.

[0156] In another embodiment, the present disclosure provides modified cells that express the chimeric antigen receptor polypeptide or the polynucleotide encoding it.

[0157] "Modified cells" refers to any cell of any organism that has been modified, transformed, or engineered by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated cells, host cells, and genetically modified cells of the present disclosure include isolated immune cells, such as NK cells and T cells, that contain a DNA or RNA sequence encoding a chimeric antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on their cell surface. The isolated host cells and modified cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, to treat cancer, and to treat infectious diseases.

[0158] Any cell capable of expressing and / or incorporating into its membrane a chimeric antigen receptor polypeptide as disclosed herein may be used.

[0159] In some embodiments, CAR can be expressed in at least one immune cell. In certain embodiments, the immune cell is T cell, for example, CD8+ T cell (for example, CD8+ naive T cell, central memory T cell, or effector memory T cell), CD4+ T cell, natural killer T cell (NKT cell), regulatory T cell (Treg), stem cell memory T cell, lymphoid progenitor cell, hematopoietic stem cell, natural killer cell (NK cell), or dendritic cell. In certain embodiments, the cell is monocyte or granulocyte, for example, myeloid cell, macrophage, neutrophil, dendritic cell, mast cell, eosinophil, and / or basophil.

[0160] In some embodiments, immune cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof (e.g., defined by function, activation state, maturity, differentiation potential, proliferation potential, recirculation potential, localization potential, and / or persistence potential, antigen specificity, type of antigen receptor, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation). Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes (such as stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells (such as Th1 cells, Th2 cells, Th3 cells, Th17 cells, Th9 cells, Th22 cells, follicular helper T cells, α / β T cells, δ / γ T cells, etc.). In certain embodiments, any number of T cell lines available to one of skill in the art may be used.

[0161] The modified cells may be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, or thymus tissue. Host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. Cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. Cells may be obtained from established cell lines.

[0162] The cells may be obtained by any known method. The cells may be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the modified cells.

[0163] In certain embodiments, the T cells and NK cells are derived from human peripheral blood mononuclear cells (PBMCs), leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0164] In some embodiments, multiple modified CAR immune cells can be provided in an immunotherapy composition. The immunotherapy composition can be used in a method for treating cancer in a subject in need thereof. The method includes administering a therapeutically effective amount of an immunotherapy composition comprising modified CAR immune cells to the subject, thereby treating cancer in the subject.

[0165] In certain embodiments, the immunotherapeutic compositions may be used as a treatment for virtually all types of cancer and precancerous conditions (e.g., myelodysplastic syndromes), including but not limited to carcinomas, sarcomas, melanomas, lymphomas, and leukemias, and having sites of origin including but not limited to colon, prostate, brain, breast, liver, lung, pancreas, bone, ovaries, skin, pancreas, blood, etc. The methods disclosed herein contemplate treatment of both metastatic cancer sites and primary tumor sites.

[0166] The immunotherapeutic compositions described herein may optionally include a pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition comprises at least an immunotherapeutic agent, e.g., the CAR immune cells described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of the CAR immune cells described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of the CAR immune cells described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include saline and aqueous buffer solutions, Ringer's solution, and serum components (serum albumin, HDL, LDL, etc.). Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein.

[0167] In some embodiments, the immunotherapy compositions described herein can be in parenteral dosage forms.Since parenteral administration usually bypasses the patient's natural defense mechanism against contaminants, components other than the immunotherapeutic agent itself are preferably sterile or can be sterilized before administration to a patient.Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products that are immediately dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions for injection, and emulsions.Any of these can be added to the immunotherapy formulation before administration.

[0168] Suitable vehicles that can be used to provide parenteral dosage forms of the immunotherapies disclosed herein are well known to those skilled in the art, and include, but are not limited to, aqueous vehicles such as saline, dextrose solution, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; aqueous vehicles such as ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0169] In some embodiments, the immunotherapeutic compositions described herein can be administered as monotherapy, i.e., no other therapeutic agent for the disease is administered to the subject at the same time.

[0170] Methods of administration may include, for example, intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient intraarterially, intratumorally, intranodally, or intramedullarily. In some embodiments, the immunotherapeutic composition may be injected directly into a tumor, lymph node, or site of infection. In one embodiment, the compositions described herein are administered into a body cavity or fluid (e.g., peritoneal, pleural, ascites, or cerebrospinal fluid).

[0171] The dosage of the above-described treatments administered to a patient will vary depending on the precise nature of the disease being treated and the recipient of the treatment. Adjustment of dosages for human administration can be performed according to art-recognized practices.

[0172] In some embodiments, only one treatment regimen is required.In other embodiments, one or more subsequent doses or treatment regimens can be administered.For example, after 3 months of biweekly treatment, treatment can be repeated once a month for 6 months or for more than 1 year.In some embodiments, no additional treatment is administered after the first treatment.

[0173] The dosage of the compositions described herein can be determined by a physician and adjusted as necessary to meet the observed effects of treatment.Regarding the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment is producing a therapeutic effect and whether additional cells should be administered, treatment should be discontinued, treatment should be resumed, or other changes should be made to the treatment regimen.The dosage should not be so large as to cause harmful side effects, such as cytokine release syndrome.In general, the dosage will vary depending on the age, condition, and sex of the patient, and can be determined by those skilled in the art.The dosage can also be adjusted by an individual physician if any complications occur.

[0174] In some embodiments, the immunotherapy composition administered to a subject may comprise at least about 1 million modified CAR immune cells, at least about 2 million modified CAR immune cells, at least about 3 million modified CAR immune cells, at least about 4 million modified CAR immune cells, at least about 5 million modified CAR immune cells, or at least about 10 million modified CAR immune cells.

[0175] In another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an immunotherapeutic composition described herein, thereby treating the cancer in the subject. Optionally, the cancer is a solid tumor or a hematological malignancy, etc.

[0176] In some embodiments, the solid tumor can be osteosarcoma, melanoma, mesothelioma, breast cancer, prostate cancer, gastric cancer, head and neck cancer, lung cancer, or brain cancer.

[0177] In other embodiments, the hematological malignancy is any hematological malignancy in which the cancer cells express or overexpress a receptor for OSM, including, but not limited to, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell malignancies, and multiple myeloma.

[0178] Optionally, the method further comprises administering to the subject an additional therapeutic agent in combination with the immunotherapeutic composition. The additional treatment may include therapies for other types of cancer, such as chemotherapy, surgery, radiation therapy, gene therapy, etc. Such treatments can be administered simultaneously with the immunotherapeutic composition described herein or sequentially (in any order). When co-administered with an additional therapeutic agent, the appropriate therapeutically effective dose of each agent may be reduced due to additive or synergistic effects.

[0179] Non-limiting examples of other anti-cancer therapeutics useful for combination with the CAR immune cells described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1, CTLA4 inhibitors), anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitor of metalloproteinases, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental growth factor-related protein); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); chemotherapeutic agents. Exemplary chemotherapeutic agents include pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine, cytarabine), purine analogs (e.g., fludarabine), folate antagonists (e.g., mercaptopurine, thioguanine), antiproliferative or mitotic inhibitors (e.g., vinca alkaloids); microtubule disrupting agents, such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, navelbine, epidipodophyllotoxins; DNA damaging agents (e.g., actinomas, These include: isin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylethylamine oxaliplatin, ifosfamide, melphalan, merchlorthamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, etoposide).

[0180] In some embodiments, radiation, or radiation and chemotherapy, are used in combination with the cell populations comprising CAR immune cells described herein. Additional useful drugs and treatments can be found in the following references: Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy, 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0181] The following examples are illustrative and are not intended to limit the scope of the invention as claimed.

[0182] Example We engineered oncostatin M (OSM) ligand-based chimeric antigen receptor (CAR) T cells, which have the ligand OSM displayed on the surface of the CAR construct to bind to the oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) to target and kill cancer cells and surrounding cells that support these cancer cells in the tumor microenvironment. The following examples show that OSM-CAR-T cells express OSM on their surface and exhibit greater cytotoxicity than non-transduced T cells against various solid cancer cell lines that all express OSMR, both in vitro and in vivo. They do not exhibit this cytotoxicity against JeK0 cells, which lack OSMR, either in vitro or in vivo.

[0183] Furthermore, mutant CARs that bind LIFR with higher affinity are possible by mutating the B-Cα helical region. These mutant OSM molecules can be displayed as ligands, and their sequences are described herein.

[0184] Experimental Note: All experiments were performed with Nod-Scid-Gamma (NSG) mice aged 9–12 weeks at the time of injection.

[0185] Example 1 Figure 1 (A-H) shows that OSM CARs express OSM and selectively kill specific cell lines. OSM ligand was added to our third-generation lentiviral CAR-T vector (the pHR backbone and the existing CAR-T internal structure of BAFF-CAR-T cells, described in U.S. Patent Application Publication No. 2020 / 0376032A1, incorporated by reference in its entirety) containing a GFP reporter measurable in the FITC channel of flow cytometry. We utilized an APC channel flow cytometry-compatible antibody that recognizes OSM to determine whether transduced CAR-T cells presented the OSM ligand on their surface. To confirm OSM expression on the CAR-T cell surface, we searched for cells expressing OSMR (the binding partner of the OSM ligand) on their surface. Importantly, we used the mouse melanoma cell line B16F10 as a specificity control, demonstrating that our human OSMR flow-based antibody does not bind mouse OSMR. Results were normalized to an IgG control antibody; a value of 1 indicates no expression above the control antibody. Jeko cells, a mantle cell lymphoma, do not express OSMR. Other solid tumors express OSMR at varying levels. We next tested cytotoxicity against these human cancer cell lines at a 7:1 (CAR:tumor cell) ratio for 16 hours. We found significantly increased cytotoxicity in all OSMR-expressing cell lines, but not in Jeko cells, which do not express OSMR. Furthermore, comparison with our BAFF-CAR-T cells (which use the same lentiviral CAR vector except for the external ligand) targeting a receptor on the Jeko cell surface showed that OSM-CAR-T cells did not kill Jeko cells, whereas BAFF-CAR-T cells did. We next examined cytokine release after 16 hours of coculture of OSM-CAR-T cells with cancer cells. We observed increases in many cytotoxic and T cell activity-mediating cytokines, with a preferential increase in specific mesothelioma (H2052) and osteosarcoma (SAOS2) cell lines. Comparing CD107a-mediated degranulation, we observed a dramatic increase in CAR-T cell degranulation upon exposure to OSMR-expressing cancer cells.This was true when comparing CAR-T cells with separately expanded untransduced T cells (UT) or when sorting the GFP-positive population of T cells within the transduced CAR wells themselves by flow cytometry. Importantly, this controls T cells exposed to increased cytokines secreted by the CAR, indicating that OSM-CAR-T cells are primarily responsible for responding to cancer cells. Note that the same number of T cells was added to each well in all assays. This means that if 175,000 "OSM-CAR-T cells" were added to a plate to match 175,000 untransduced T cells, only 40–90% of these were OSM-CAR-T cells, depending on transduction efficiency.

[0186] Example 2 Figure 2 (A-C) shows images and graphs demonstrating that intratumoral injection of OSM-CAR reduces solid tumor burden. Two pilot studies were conducted to demonstrate whether OSM-CAR-T cells exhibit similar cytotoxicity in solid tumors in vivo as in vitro. AGS-luciferase-labeled cells were injected subcutaneously into mice. Their growth was tracked by intraperitoneal D-luciferin injection and imaging with an IVIS Spectrum imager. One mouse received 4,000,000 T cells (approximately 70% CAR cells) injected intratumorally, and two mice received PBS intratumorally. We observed complete regression of tumors treated with OSM-CAR-T cells at the time of excision. Importantly, we expanded these pilot results using Detroit 562 cells, a pharyngeal carcinoma, because this model grew better subcutaneously. Once tumors became palpable, intratumoral injection of 4,000,000 T cells (approximately 70% CAR cells) resulted in a statistically significant reduction in tumor volume and mass at harvest 12–14 days post-injection compared to both PBS injection and untransduced T cell injection.

[0187] Example 3 Figure 3 (AE) shows a schematic, graph, and plot demonstrating the cytotoxicity of intravenously (IV) infused OSM CAR-T cells against subcutaneous SAOS2 tumors. To determine whether intravenously infused OSM-CAR-T cells are cytotoxic to subcutaneous solid tumors, we utilized a pediatric osteosarcoma cell line called SAOS2. We intravenously injected 4,000,000 T cells (approximately 50% CAR cells) after subcutaneous SAOS2 tumors (maintained in Matrigel) became palpable (day 7). There was statistically significant weight loss that correlated with the level of response observed in the mice. As the response waned, the mice regained weight and the tumors grew. No special interventions (gel, diet, etc.) were administered to address the weight loss in the mice. All mice eventually recovered, demonstrating a survival benefit of approximately 10 days. Overall, there was a cytotoxic effect of OSM-CAR-T cells against established SAOS2 subcutaneous tumors.

[0188] Example 4 Figure 4 (AD) shows a schematic, graph, and image demonstrating that higher doses of OSM CAR-T cells increase tumor-killing efficacy and toxicity. We investigated whether the cytotoxic effect of our OSM-CAR-T cells could be enhanced by increasing the number of T cells and the proportion of CAR-T cells injected. After SAOS2 tumors (with Matrigel support) became palpable, we intravenously injected 6,000,000 T cells (approximately 90% of CAR-T cells). Significant reductions in tumor burden were observed, including the complete disappearance of detectable tumors after just 5 days. The difference in the SAOS2 tumor-reducing efficacy of OSM-CAR-T cells compared with untransduced T cells (UT) and PBS (physiological saline) was highly significant. Untransduced T cells did not produce statistically significant tumor shrinkage compared with PBS alone. Importantly, these mice exhibited more severe weight loss / toxicity than conventionally injected mice, which was lethal for some mice. We collected serum from these mice and analyzed it for cytokines after only 5 days, halting the experiment. Serum showed a significant increase in many human cytokines only in OSM-CAR-T mice, including elevated IFNγ, a common cytokine associated with CAR-T cell cytotoxicity against solid tumors.

[0189] Example 5 Figure 5(AE) shows a schematic and graph demonstrating that intravenously injected OSM-CAR exhibits cytotoxicity against subcutaneous 143B tumors. We repeated the outline of the previous SAOS2 experiment using another osteosarcoma cell line, 143B, in which subcutaneous tumors were established using Matrigel support. Six million T cells (70% OSM-CAR-T cells) were intravenously injected seven days after the 143B tumors were palpable. Again, we observed a reduction in tumor burden in OSM-CAR-treated mice while maintaining the mice on dietary and hydration gel. While some OSM-CAR-treated mice plateaued in weight loss, others continued to lose tumor burden. All mice showed significantly reduced tumor burdens 21 days after OSM-CAR-T cell injection, at which point the experiment was discontinued, and most mice in the PBS and UT groups had tumors of 2000 mm. 3 Tumor volumes were compared because tumors were approaching the experimental cutoff of 10. Two of the 10 mice in the OSM-CAR-T treatment group had to be euthanized due to weight loss / toxicity.

[0190] Example 6 Figure 6 (AE) provides schematics, images, plots, and graphs demonstrating that intravenously infused OSM-CARs do not exhibit significant cytotoxicity against subcutaneous Jeko cells. To determine the specificity of OSM-CAR toxicity, a subcutaneous Jeko-luciferase model was created by intravenously injecting 5,000,000 T cells (approximately 50% OSM-CAR-T). These mice did not exhibit a reduction in tumor burden similar to that observed in the SAOS2 and 143B experiments. Importantly, imaging performed with IP injection of D-luciferin and analysis with an IVIS Spectrum imager showed that tumor burden was unchanged in the presence of OSM-CARs. Experiments were performed at a tumor burden of 2000 mm, the experimental cutoff value. 3 The infusion was discontinued 14 days after administration due to the lack of efficacy of OSM-CAR T cells against Jeko cells.

[0191] array [ka] TIFF2026507180000003.tif212159TIFF2026507180000004.tif212159TIFF202 6507180000005.tif214159TIFF2026507180000006.tif212159TIFF20265071800 00007.tif212159TIFF2026507180000008.tif210159TIFF2026507180000009.t if208159TIFF2026507180000010.tif208159TIFF2026507180000011.tif221159

[0192] From the above description of the invention, those skilled in the art will recognize improvements, changes, and modifications. Such improvements, changes, and modifications within the scope of the art are intended to be included within the scope of the appended claims. All references, publications, and patents cited in this application are hereby incorporated by reference in their entirety.

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a polypeptide that binds to oncostatin M receptor (OSMR), leukemia inhibitory factor receptor (LIFR), a heterodimer between a transmembrane glycoprotein 130 (gp130) receptor and OSMR, and / or a heterodimer between a gp130 receptor and LIFR.

2. The CAR of claim 1, wherein the OSMR and / or LIFR are expressed by cancer cells or other cells in the tumor microenvironment.

3. The CAR of claim 1 or 2, wherein the polypeptide is an oncostatin M (OSM) ligand that binds to OSMR and / or LIFR and / or OSMR / gp130 receptor and / or LIFR / gp130 receptor heterodimers.

4. 4. The CAR of claim 3, wherein the OSM ligand comprises a polypeptide comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:

18.

5. The CAR of claim 1 or 2, wherein the polypeptide is a single-chain variable fragment (ScFv) that binds to OSMR and / or LIFR.

6. The CAR according to any one of claims 1 to 5, further comprising a hinge domain.

7. The hinge domain of CD8α comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

19.

7. The CAR of claim 6, wherein the CAR is selected from the group consisting of a hinge domain of IgG1 comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

20.

8. The CAR according to any one of claims 1 to 7, further comprising one or more transmembrane domains.

9. The transmembrane domain of CD8α comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

21. a transmembrane domain of CD28 comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:22;a transmembrane domain of CD16a comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:23 a transmembrane domain of 2B4 comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:24;or a transmembrane domain of NKG2D comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

25.

10. The CAR according to any one of claims 1 to 9, further comprising one or more intracellular signaling domains.

11. The CAR according to any one of claims 1 to 10, wherein the one or more intracellular signaling domains are selected from a 41BB intracellular signaling domain, a CD28 intracellular signaling domain, a CD3ζ intracellular signaling domain, a CD16A intracellular signaling domain, a γc intracellular signaling domain, a 2B4 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and / or a combination thereof.

12. The intracellular signaling domain comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

26. an intracellular domain of CD28 comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:27;an intracellular domain of CD3ζ comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:28; an intracellular domain of OX40 comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:29;an intracellular domain of CD16A comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:30; an intracellular domain of γc comprising an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:31;an intracellular domain of 2B4 comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:32; or at least about 70%, at least about 71%, at least about 72%, at least about 73%, or at least about 74% identical to SEQ ID NO:

33. an intracellular domain of DAP10 comprising an amino acid sequence that is at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical;or an intracellular domain of DAP12 comprising an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

34.

13. The CAR according to any one of claims 1 to 12, further comprising a spacer.

14. 14. The CAR of claim 13, wherein the spacer comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

35.

15. The CAR according to any one of claims 1 to 14, further comprising a signal peptide.

16. The CAR of claim 15, wherein the signal peptide comprises an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 36 or SEQ ID NO:

37.

17. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, a hinge domain, a transmembrane domain, a CD28 intracellular domain, and an OX40 intracellular domain.

18. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, an IgG1 hinge domain, a CD28 transmembrane domain, the CD28 intracellular domain, the OX40 intracellular domain, and a CD3ζ intracellular domain.

19. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a CD16a intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain.

20. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a γc intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain.

21. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a CD28 intracellular signaling domain, a 2B4 intracellular domain, and a CD3ζ intracellular domain.

22. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a 2B4 intracellular signaling domain, a 41BB intracellular domain, and a CD3ζ intracellular domain.

23. The CAR of any one of claims 1 to 4 or 6 to 16, comprising, from N-terminus to C-terminus, an extracellular domain comprising the OSM ligand, a spacer, an IgG1 hinge domain, a transmembrane domain, a 2B4 intracellular signaling domain, a DAP10 intracellular domain, a DAP12 intracellular domain, and a CD3ζ intracellular domain.

24. A nucleic acid comprising a nucleotide sequence encoding the CAR according to any one of claims 1 to 23.

25. 25. The nucleic acid of claim 24, wherein the nucleotide is operably linked to a promoter.

26. 26. An expression construct comprising the nucleic acid of claim 24 or 25.

27. 27. The expression construct of claim 26, comprising a vector.

28. 28. The expression construct of claim 27, wherein the vector is a retroviral vector, a lentiviral vector, or an AAV vector.

29. 29. The expression construct of any one of claims 26 to 28, further comprising a nucleotide sequence encoding a cytokine.

30. 30. The expression construct of claim 29, wherein the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, or a combination thereof.

31. 31. The expression construct of claim 30, comprising a nucleotide sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:

6.

32. An immune cell comprising the CAR according to any one of claims 1 to 23.

33. An immune cell transduced or transduced with an expression construct according to any one of claims 26 to 31.

34. 34. The immune cell of claim 32 or 33, comprising at least one of a cytotoxic T lymphocyte, a natural killer cell, or a natural killer T cell.

35. An immunotherapeutic composition comprising a plurality of immune cells according to any one of claims 32 to 34.

36. 36. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immunotherapeutic composition of claim 35.

37. 37. The method of claim 36, wherein the cancer comprises a solid tumor.

38. 37. The method of claim 36, wherein the cancer comprises at least one of osteosarcoma, melanoma, mesothelioma, breast cancer, prostate cancer, head and neck cancer, lung cancer, or brain cancer.

39. 37. The method of claim 36, wherein the cancer comprises a hematological cancer or malignancy.

40. 40. The method of claim 39, wherein the cancer or malignant tumor is acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell malignancies, and multiple myeloma.

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