Anti-GPNMB chimeric antigen receptors and methods of use thereof

Abstract

A glycoprotein NMB (GPNMB) binding chimeric antigen receptor (CAR) is provided. In some embodiments, the CARs of the present disclosure comprise an extracellular GPNMB binding domain (e.g., a single chain antibody, such as scFv), a transmembrane domain, and one or more intracellular signaling domains. Nucleic acids and expression constructs encoding such CARs are also provided. Also provided are cells (e.g., immune cells, such as T cells) comprising the nucleic acids and the expression constructs. Aspects of the disclosure further include compositions comprising a population of such cells that express GPNMB binding CARs on their surfaces; and methods of administering such compositions to treat conditions associated with GPNMB expression and / or activity in a subject in need thereof. Non-limiting examples of conditions associated with GPNMB expression and / or activity include cancer (e.g., sarcoma, such as soft tissue sarcoma), neurodegenerative disease, and the like.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 423,405, filed on November 7, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation of sequence listing XML by reference

[0004] The sequence listing is provided hereby as the sequence listing XML created on November 6, 2023, and having a size of 59,559 bytes, i.e., “UTIP - 001WO_SEQ_LIST”. The content of the sequence listing XML is incorporated herein by reference in its entirety.

[0005] Introduction

[0006] Glycoprotein NMB (GPNMB) is a glycoprotein consisting of a large extracellular domain (ECD) and a short 53 - amino - acid cytoplasmic tail connected by a single - pass transmembrane domain. The ECD contains an N - terminal signal peptide, an RGD motif that binds to integrin receptor subunits, several N - glycosylation sites, a proline - rich region, and an Ig - like polycystic kidney disease (PKD) domain. The ECD can be shed from the plasma membrane by ADAM10, and then ADAM10 can act as a paracrine factor to activate multiple signaling pathways in a variety of cells. The intracellular domain contains a hemITAM (hem - immunoreceptor tyrosine - based activation motif) and a di - leucine motif, which serves as an endosome / lysosome sorting motif in QNR - 71. Functionally relevant domains within GPNMB include the extracellular RGD motif, which is crucial for integrin receptor engagement and promoting cell adhesion. In addition, after GPNMB engagement, the cytoplasmic hemITAM domain can be phosphorylated at tyrosine residue 525, which leads to the activation of several downstream signaling pathways.

[0007] GPNMB is highly expressed in a variety of tumors, including glioblastoma and astrocytoma, breast cancer, hepatocellular carcinoma, gastric adenocarcinoma, uveal melanoma, lung cancer, malignant cutaneous melanoma, and sarcoma. Since its discovery, both in vitro and in vivo studies have revealed that GPNMB is a key promoter of tumor growth and an enhancer of malignant phenotypes such as tumor invasion and metastasis in prostate cancer, breast cancer, lung cancer, and pancreatic cancer. Summary of the invention

[0008] Provided are glycoprotein NMB (GPNMB)-binding chimeric antigen receptors (CARs). In some embodiments, the CARs of the present disclosure comprise an extracellular GPNMB-binding domain (e.g., a single-chain antibody, such as an scFv), a transmembrane domain, and one or more intracellular signaling domains. Also provided are nucleic acids and expression constructs encoding such CARs. Also provided are cells (e.g., immune cells) comprising the nucleic acids and expression constructs. Aspects of the present disclosure further include compositions comprising a population of cells expressing a GPNMB-binding CAR on the surface of such cells; and methods of administering such compositions to treat conditions associated with GPNMB expression and / or activity in a subject in need thereof. Non-limiting examples of conditions associated with GPNMB expression and / or activity include cancer, neurodegenerative diseases, tissue remodeling, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1: Genomic evaluation of cell surface genes on alveolar soft part sarcoma (ASPS). Bioinformatics pipeline for identifying potentially overexpressed cell surface proteins in ASPS versus normal tissue from microarray data in GSE13433 (a). Expression of 11 candidate cell surface ASPS genes derived from this pipeline across three published ASPS microarray datasets (b). GPNMB expression across different pediatric and AYA tumors from two separate publicly available datasets. Dashed line = median housekeeping gene expression; dotted line = one standard deviation of the mean (c). Surgical time course of an ASPS patient (d; not shown is the initial stereotactic radiosurgery for a right parietal lesion that presented at diagnosis, February 2016). GPNMB staining of each of these resections (e), where each Roman numeral corresponds to the surgery listed above.

[0010] Figure 2 : GPNMB and TFE3 staining of GPNMB and TFE on three primary ASPS tumor resections. Primary 1 is a resection from a primary tumor located in the upper thigh of a 14-year-old female. Primary 2 is a resection from a primary tumor located in the right upper shoulder of a male. Primary 3 is a resection from a primary tumor located in the thigh of a 30-year-old female.

[0011] Figure 3: Normal proteome DB of GPNMB. The Human Protein Atlas and Wang et al. were used to evaluate the expression levels of GPNMB (circles) across tissues in non-malignant samples. In addition, CD276 (triangles) and HER2 (crosses) were included as established CAR targets and 14 housekeeping genes (https: / / www.genomics-online.com / resources / 16 / 5049 / housekeeping-genes / ): ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPL13A, RPLP0, SDHA, TBP, TFRC, and YWHAZ. Since the range of mass spectrometry (MS) expression varies and can be extreme, MS values were limited to the 90th percentile across 17 proteins within the dataset. Then, the MS values were scaled from the [minimum, maximum] expression to [0, 1].

[0012] Figure 4 : Normal tissue histological score ≥2. Tissue images with staining ≥2 are represented on a 0 - 3 scale, including heart (a), tonsil (b), lymph node (c), skin (d), and placenta (e).

[0013] Figure 5: Adverse event profiles between the CDX - 011 and SGN - 035 trials. Charted results from Appendix II. All adverse events (a) and serious adverse events (≥3; b). 483 patients from 6 CDX - 011 trials (NCT00412828, NCT00704158, NCT01156753, NCT01997333, NCT02363283, NCT02302339). 356 patients from 6 SGN - 035 trials (NCT01100502, NCT00848926, NCT01807598, NCT01716806, NCT01352520, NCT01461538). All trials were evaluated using the NCI - CTCAE (v3 of v4) adverse event criteria.

[0014] Figure 6: GPNMB CAR and patient - derived cell lines targeting ASPS. Schematic of the GPNMB41bbζ (GPNMB) CAR construct (a). Immunofluorescence staining of patient - derived ASPS cell lines for GPNMB, TFE3, and DAPI (b). Flow cytometry of cell surface expression of GPNMB on patient - derived ASPS cell lines (c). In situ intramuscular implantation of NSG - MHC 35 days after tumor implantation KOGPNMB (left) and TFE3 (right) staining (d) of the ASPS cell line from mice. Representative flow cytometry analysis of GPNMB CAR or empty CAR vector into healthy donor 1 T cells (left), with corresponding transduction efficiencies (middle; 5 MOI) in healthy donor 1 (D1) and donor 2 (D2) T cells. Transduction efficiency measured by EGFP of CD4+ and CD8+ cells (right; e). Microscopy of 24-hour co-culture between patient-derived ASPS cell lines (red) and CAR T-cells (green), and quantification of cytotoxic activity measured by luminescence assay at the indicated CAR T to ASPS target cell ratios (f). IFNγ and IL-2 secretion of CAR T cells cultured at a 1:1 ratio in medium were measured after 24-hour co-culture. Solid lines are D1, and dashed lines are D2 (g).

[0015] Figure 7: Testing different GPNMB-binding scFvs in preclinical CAR constructs. Using the same CAR and plasmid backbone, four different GPNMB-binding scFvs were tested. Primary human T cells were transduced at 10 MOI and expanded for 8 days after transduction. Transduction efficiency was measured by flow cytometry and reported as the percentage of GFP positive (a). CAR surface expression was measured by flow cytometry using an anti-MYC antibody and reported as MFI (b). Representative images of GPNMB CAR T cells (GFP – green) and ASPS patient-derived xenograft (PDX)-derived cell lines (red – mCherry) after 20-hour co-culture (c). Quantification of cytotoxic activity measured by luminescence assay at the indicated CAR T to ASPS target cell ratios (d), and IFNγ and IL-2 secretion of CAR T cells cultured at a 1:1 ratio in medium were measured after 24-hour co-culture. All data are from the same healthy donor, where freshly harvested, activated, and transduced T cells were done three independent times.

[0016] Figure 8: GPNMB CAR in primary tumors in ASPS xenografts in vivo. Carrying 42-day ASPS mcherry / FLUCXenografts (day 0), and luciferase images of animals treated with 1e5, 1e6, or 5e6 GPNMB CAR T cells, 5e6 CD19 CART cells from healthy donor 1, or untreated (control) and imaged on the indicated dates (a; 1e4 CAR / mouse not shown). Kaplan-Meier curves (arrows) of animals treated from tumor initiation day to day 42 and survival tracked. Death was the result of the tumor reaching 17 mm in any direction (b). Expansion of CAR T cells in blood after CAR T treatment (c). Flow cytometry of CAR expression via the MYC tag on the surface of expanded CAR T cells in blood after 5e6 GPNMB CAR treatment. Representative histograms (left) and quantified MFI flow values (right). Black lines represent MYC expression, gray lines are EGFP expression, and the dashed line corresponds to MYC tag expression before implantation (d). Percentages of effector cells (Teff), effector memory cells (Tem), and central memory cells (Tcm) in blood of mice after 5e6 GPNMB CAR administration (e). Primary tumor ASPS treated with 1e6 GPNMB CAR T cells mcherry / FLUC Intravital microscopy of the ASPS model. ASPS xenografts (red), CD31 (vasculature; blue), and GPNMB CAR T cells (GFP; green) are the colors on a montage of multiple Z-stack images (approx. 100 uM depth; f).

[0017] Figure 9: Donor 2 - CAR intramuscular tumors. Luciferase images of animals carrying 42-day ASPSmc / FLUC xenografts (day 0), treated with 1e5, 1e6, or 5e6 GPNMB CAR, 5e6 CD19 CAR from healthy donor 1, or untreated and imaged on the indicated dates (a). CAR T cells in blood as measured by flow cytometry after CAR T treatment (b).

[0018] Figure 10: Systemic GPNMB CAR effectively targets ASPS CNS metastases. Carrying 42-day ASPS mcherry / FLUCBioluminescence images of xenografts (day 0) and animals treated with 1e4, 1e5, or 1e6 GPNMB-CAR T cells, 1e6 CD19 CAR T cells, or untreated and imaged on the indicated dates. Red arrows depict the putative spinal metastasis regions (a). Kaplan-Meier curves of treated animals from the tumor initiation day to day 42 and tracking survival. (b). CAR T cells in blood as measured by flow cytometry after CAR T treatment (c). Body weight of animals obtained after CAR T administration and expressed as a percentage of body weight on day 0 of treatment (d). Data are combined results of two experiments in independently harvested, transduced, and expanded healthy donor cells for control and 1e6 GPNMB and CD19 CAR T cells (n = 8). And one experiment for 1e5-GPNMB-CAR T cells (n = 4) (b-d).

[0019] Figure 11: Testing GPNMB CAR in ASPS patient T cells. ASPS patients had blood drawn during disease quiescence, and patient T cells were transduced with the GPNMB CAR construct and CAR expression was measured by flow cytometry (a). Microscopy of 24-hour co-cultures between patient-derived ASPS cell lines (red) and CAR T cells from the same patient (green; left), and quantification of cytotoxic activity as measured by luminescence assay at the indicated CAR T to ASPS target cell ratios (b; diamonds = untransduced cells; circles = CD19 CAR T; squares = GPNMB CAR). IFNγ and IL-2 secretion of CAR T cells cultured at a 1:1 ratio in media measured after 24-hour co-culture (circles = CD19; squares = GPNMB) (c). Carrying 42-day ASPS mcherry / FLUC Bioluminescence images of xenografts (day 0) and animals treated with 1e6 GPNMB CAR T cells or 1e6 CD19 CAR T cells, or untreated and imaged on the indicated dates (d), and CAR T cells in blood as measured by flow cytometry after CAR T treatment (e).

[0020] Figure 12: Comparison of preclinical and clinical GPNMB CAR vectors in patient T cells. In vitro testing of preclinical versus clinical GPNMB CAR T constructs on ASPS PDX-derived cell lines. Quantification of cytotoxic activity as measured by luminescence assay at the indicated CAR T to ASPS target cell ratios (a). IFNγ and IL-2 secretion of CAR T cells cultured at a 1:1 ratio in media measured after 24-hour co-culture (b).

[0021] Figure 13: CliniMAC validation runs were performed on two separate healthy donor apheresis products (HD1, HD2) or apheresis products from patients with epithelioid hemangioendothelioma (EHE). Runs on the CliniMAC utilized the listed volumes of lentiviral products produced according to GMP principles and meeting clinical use requirements. Total cells (a), viability, and CAR% (b) of each product at 12 days of manufacture. Potency of each product was measured by co-culture cytotoxicity assay (c) with ASPS PDX-derived cells and subsequent cytokine secretion (d). Carrying 42-day ASPS mc / FLUC intramuscular xenografts (day 0) and luminescence images of animals treated with 5e6 or 5e5 GPNMB CAR from the HD1 validation run (e). Number of HD1 CAR in blood after CAR T treatment (f). Controls carried subcutaneous tumors lacking GPNMB expression.

[0022] Figure 14: Representative images of GPNMB staining (a) and histological scores of slides scored by a renal cancer pathologist (0 - 3 intensity X % tumor positive = H index; b) of TSC / mTOR-altered renal cell carcinoma (RCC) subtypes low-grade oncocytoma (LOT), eosinophilic solid and cystic (ESC), and eosinophilic vacuolated tumor (EVT), translocation-related RCC (tRCC), and clear cell RCC. Scale bar = 50 μM. Groups of cell surface GPNMB expression of tRCC-related cell lines (FU-UR1, S-TFE3, UOK-109, UOK-120, UOK-124, UOK-145, UOK-146) and ccRCC cell lines (786-O, A498, ACHN) were tested by flow cytometry. Subcutaneous implantation of translocation-related RCC cell lines (GPNMB+) and ccRCC cell lines (GPNMB-) (d) when treated with 5e6 GPNMB CAR from the HD1 clinical run described in Figure 5. Expansion of the HD1 product in these models compared to GPNMB-non-responsive tumors (e). Including ASPS PDX as a positive control.

[0023] Figure 15: Scoring of 49 triple-negative breast cancer (TNBC) patients by a board-certified breast pathologist (0 - 3 intensity X % tumor positive = H-index). Representative images of high (150+), medium (40 - 149), and low (<50) scoring GPNMB in cores from individual patients (left) and scoring of the entire TMA (right;). Scale bar = 50 μm. Expression of GPNMB on the cell surface of TNBC cell lines MDA-MB-231 and Hs578T compared to positive control ASPS (b). Head-to-head testing of cytotoxicity and cytokine secretion on these cell lines when treated in co-culture experiments with HD1 clinical-grade GPNMB CAR (c). Luciferase-expressing Hs578T cells were implanted into the mammary fat pads of NSG-KO mice and treated with 1e6 GPNMB CAR-T from HD1 (d). Detailed Description

[0024] Before describing the CARs, compositions, and methods of the present disclosure in more detail, it is to be understood that the CARs, compositions, and methods are not limited to the particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the CARs, compositions, and methods will be defined only by the appended claims.

[0025] Where a range of values is provided, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated value or intervening value in that stated range, is encompassed within the CARs, compositions, and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the CARs, compositions, and methods, subject to any specific excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the CARs, compositions, and methods.

[0026] Certain ranges are given herein in which numerical values are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that it follows, as well as a number that is close to or approximates the number that follows. When determining whether a number is close to or approximates a specifically recited number, the unrecited number that is close to or approximates the specifically recited number may be a number that is substantially equivalent to the specifically recited number in the context in which it appears.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the CARs, compositions, and methods belong. Although any CARs, compositions, and methods similar or equivalent to those described herein may also be used in the practice or testing of the CARs, compositions, and methods, representative illustrative CARs, compositions, and methods are now described.

[0028] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and incorporated herein by reference to disclose and describe the materials and / or methods related to the cited publications. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the CARs, compositions, and methods of the present invention are not entitled to antedate such publication, as the provided publication dates may differ from the actual publication dates which may need to be independently confirmed.

[0029] It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended as a basis for use in connection with the recitation of claim elements of using exclusive terms such as "only", "solely", etc. or the use of "negative" limitations.

[0030] It should be understood that, for clarity, certain features of the CARs, compositions, and methods described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the CARs, compositions, and methods described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments are specifically included by this disclosure and are disclosed herein as if each combination was individually and explicitly disclosed, in the sense that such combinations include operable processes and / or compositions. Additionally, all sub-combinations listed in the embodiments describing such variables are also specifically included by the CARs, compositions, and methods of the present invention and are disclosed herein as if each such sub-combination was individually and explicitly disclosed herein.

[0031] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any other several embodiments without departing from the scope or spirit of the methods of the present invention. Any recited method can be performed in the order of the recited events or in any other order that is logically possible.

[0032] Anti-GPNMB chimeric antigen receptor

[0033] Aspects of the present disclosure include anti-glycoprotein NMB (GPNMB) chimeric antigen receptors (CARs). A CAR is a bioengineered molecule that typically comprises an antigen-binding (targeting) domain fused to a transmembrane domain and an intracellular signaling domain of the T cell receptor complex. A CAR functions by redirecting the cytotoxic activity of a T cell towards a tumor bearing a predetermined surface-expressed antigen. Historically, the efficacy of CAR T therapy in solid tumors has been poor. The widespread use of CAR T therapy for cancers such as soft tissue sarcoma (STS) faces several obstacles, including target selection. Targets for solid tumors such as sarcomas have two main requirements: ubiquitous, high-level, and stable cell surface expression and limited off-tumor targeting potential.

[0034] The CARs, CAR-T cells, and methods of the present disclosure are based in part on the inventors' identification of GPNMB as a candidate CAR target due to the high cell surface expression of GPNMB on cancers such as MITF family fusion-positive cancers such as alveolar soft part sarcoma (ASPS) and some renal cell carcinomas (RCC). However, the inventors determined that only an unpredictable subset of CARs with a GPNMB targeting binding domain (e.g., the ScFv "G1" and "G2" described herein) exhibit robust cell surface expression / localization. Similarly unpredictable is that among the CARs with robust cell surface localization, there is variable in vitro cytotoxicity and cytokine secretion activity. CAR T cells utilizing the CARs with the GPNMB targeting binding domain with the highest cell surface expression and in vitro activity exhibit clear in vivo efficacy in primary disease and metastasis models, with significant CAR T expansion and complete, durable tumor elimination. Similarly unpredictable is that some in vitro efficacy (e.g., "G4") was also observed despite low cell surface expression of the anti-GPNMB CAR. In addition, CAR T cells expressing the CAR exhibit efficacy at unexpectedly low doses, e.g., 1e5 in an orthotopic model of ASPS primary and metastatic disease, where the CAR comprises a subset of the GPNMB targeting binding domain that exhibits robust cell surface expression / localization. Details of the CARs of the present disclosure will now be described.

[0035] The CARs of the present disclosure comprise an extracellular GPNMB binding domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the extracellular GPNMB binding domain comprises a single-chain antibody (e.g., scFv) that specifically binds GPNMB.

[0036] The term "antibody" can include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of tetramers, which in turn are composed of two dimers of heavy and light chain polypeptides); single-chain antibodies (e.g., scFv); fragments of antibodies that retain specific binding to cell surface molecules of target cells (e.g., fragments of whole antibodies or single-chain antibodies), including but not limited to single-chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, such as humanized scFv); and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. In some embodiments, the antibody is selected from IgG, Fv, single-chain antibodies, scFv, Fab, (Fab’)2, F(ab’), or Fab’.

[0037] The immunoglobulin light or heavy chain variable region consists of "framework" regions (FRs) interrupted by three hypervariable regions (also called "complementary determining regions" or "CDRs"). The ranges of the framework regions and CDRs can be defined based on databases known in the art. See, e.g., "Sequences of Proteins of Immunological Interest", E. Kabat et al., Sequences of proteins of immunological interest, 4th ed., U.S. Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987); Lefranc et al., IMGT, the international ImMunoGeneTics information system Nucl. Acids Res., 2005, 33:D593-D597 (www.imgt.org / textes / IMGTScientificChart / ), and / or V Base at vbase.mrc-cpe.cam.ac.uk / . The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions that make up the light and heavy chains, are used to position and align the CDRs. The CDRs are primarily responsible for binding to the epitope of an antigen.

[0038] In certain embodiments, the extracellular GPNMB binding domain comprises an antibody that specifically binds to GPNMB. When referring to antibodies, the phrases "specific binding", "specific to ...", "immunoreactive" and "immunoreactive" and "antigen binding specificity" refer to a binding reaction with an antigen that is highly preferred to an antigen or a fragment thereof, thereby determining the presence of an antigen in the presence of a heterogeneous antigen population (e.g., proteins and other biological products, e.g., in a sample). Therefore, under specified immunoassay conditions, the specified antibody binds to the GPNMB antigen (e.g., human GPNMB-UniProtQ96F58) and will not bind to other antigens present in the sample in significant amounts. Specific binding to an antigen under such conditions may require the selection of an antibody that is specific to a particular antigen. For example, an anti-GPNMB antibody can specifically bind to GPNMB and does not exhibit binding comparable to other proteins present in the sample (e.g., does not exhibit detectable binding to other proteins present in the sample).

[0039] In some embodiments, if the antibody of the CAR of the present disclosure is, for example, greater than or equal to about 10 5 M -1 Affinity or K a An antibody "specifically binds" to GPNMB (e.g., human GPNMB) if it binds or associates with GPNMB with an equilibrium binding constant (i.e., a specific binding interaction with units of 1 / M). In certain embodiments, the antibody binds or associates with GPNMB with an equilibrium binding constant of greater than or equal to about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 K a Binds to GPNMB. "High affinity" binding means a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M-1 , at least 10 13 M -1 Alternatively, affinity can be defined as having a unit of M (e.g., 10 -5 M to 10 -13 M, or less) of the equilibrium dissociation constant (K D ). In some embodiments, specific binding means that the antibody binds to the -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, or less than or equal to about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M or smaller K D Binding to GPNMB. The binding affinity of an antibody to GPNMB can be readily determined using conventional techniques, such as by biolayer interferometry (BLI); competitive ELISA (enzyme-linked immunosorbent assay); equilibrium dialysis; surface plasmon resonance (SPR) technology (e.g., BIAcore2000 instrument, using the general procedures outlined by the manufacturer); by radioimmunoassay; and / or similar techniques.

[0040] According to some embodiments, the extracellular GPNMB binding domain comprises a single-chain antibody that specifically binds to GPNMB. In some cases, the single-chain antibody is a single-chain variable fragment (scFv). The single-chain antibodies (e.g., scFv) that can be used include but are not limited to single-chain antibodies, which comprise: the variable light chain (V) of antibody G1, G2 or G4 set forth in Table 1 below L ) polypeptide, wherein in some embodiments, such single-chain antibodies comprise V L Peptide, the V L The polypeptide comprises a V sequence of antibody G1, G2 or G4 produced in Table 1. L an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to an amino acid sequence of; and / or a variable heavy chain (V H ) polypeptide, wherein in some embodiments, such single-chain antibodies comprise V H Peptide, the V H The polypeptide comprises a V sequence similar to that of antibody G1, G2 or G4 described in Table 1. Han amino acid sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 100% identity to the amino acid sequence. Accessed on 2023.11.06 according to the CDR numbering of the AbRSA program (Li et al., (2019) Protein Sci 28(8):1524-1531).

[0041] Table 1 - Amino acid and nucleotide sequences of exemplary anti-GPNMB antibody variable regions

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] The present disclosure provides each of the polypeptides provided in Table 1 and each of the individual domains therein, as well as nucleic acids encoding such polypeptides and individual domains. Cells comprising such polypeptides and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any polypeptide and individual domains therein, where in some cases, the variant polypeptide or its domain comprises an amino acid sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher amino acid sequence identity to the parental / reference sequence or a functional fragment thereof, where the variant retains the functionality of the parental / reference sequence (e.g., GPNMB binding, cell surface expression / localization, intracellular signaling when included in a CAR, etc.).

[0049] In certain embodiments, CAR variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs, framework regions (FRs), one or more intracellular domains (e.g., one or more intracellular signaling domains), etc. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions". More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the CAR of interest and the products screened for desired activities or characteristics (e.g., retained / improved antigen binding, reduced immunogenicity, improved cell surface expression / localization, improved intracellular signaling, etc.).

[0050] Table 2 - Amino acid substitutions

[0051]

[0052]

[0053] Amino acids can be grouped according to common side chain properties:

[0054] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0055] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0056] (3) Acidic: Asp, Glu;

[0057] (4) Basic: His, Lys, Arg;

[0058] (5) Residues affecting chain orientation: Gly, Pro;

[0059] (6) Aromatic: Trp, Tyr, Phe.

[0060] Non-conservative substitutions require replacement of a member of one of these classes with another class.

[0061] According to some embodiments, the anti-GPNMB antibodies of the present disclosure are humanized antibodies. As used herein, a humanized antibody is a recombinant polypeptide that is derived from a non-human (e.g., rabbit, rodent, etc.) antibody and has been modified to contain at least a portion of the framework and / or constant region of a human antibody. Humanized antibodies also encompass chimeric antibodies and CDR-grafted antibodies, where different regions can be derived from different species. A chimeric antibody can be an antibody that comprises a variable region from any source linked to a human constant region (e.g., a human Fc domain). Thus, in a chimeric antibody, the variable region can be non-human and the constant region is human. A CDR-grafted antibody is an antibody that comprises CDRs from a non-human "donor" antibody that are linked to a framework region from a human "recipient" antibody.

[0062] The CARs of the present disclosure can include one or more linker sequences between various domains. A "variable region linker sequence" is an amino acid sequence that links the heavy chain variable region to the light chain variable region and provides a spacer function that is compatible with the interaction of the two sub-binding domains such that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody comprising the same light and heavy chain variable regions. Non-limiting examples of variable region linker sequences are serine-glycine linkers such as the serine-glycine linker comprising the amino acid sequence GGGGSGGGGSGGGGS (G4S)3 (SEQ ID NO:54). In some embodiments, the linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, co-stimulatory domains, and / or primary signaling domains. In certain embodiments, the CAR includes one, two, three, four, or five or more linkers. In certain embodiments, the linker has a length of from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any amino acid length therebetween. In some embodiments, the linker has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.

[0063] In certain embodiments, the antigen-binding domain of the CAR is followed by one or more spacer domains that move the antigen-binding domain away from the effector cell surface (e.g., the surface of a CAR-expressing T cell) to enable proper cell / cell contact, antigen binding, and / or activation. The spacer domain (and any other spacer domains, linkers, and / or analogs described herein) can be derived from natural sources, synthetic sources, semi-synthetic sources, or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes CH2 and / or CH3 of IgG1, IgG4, or IgD. Illustrative spacer domains suitable for the CARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8α and CD4, which can be wild-type hinge regions from these molecules or variants thereof. In certain aspects, the hinge domain includes the CD8α hinge region. In some embodiments, the hinge is the PD-1 hinge or the CD152 hinge.

[0064] The "transmembrane domain" (Tm domain) is the part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the plasma membrane of a cell (e.g., an immune effector cell). The Tm domain can be derived from natural sources, synthetic sources, semi-synthetic sources, or recombinant sources. In some embodiments, the Tm domain is derived from the α or β chain of a T cell receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1 (e.g., including at least the transmembrane region or a functional portion thereof).

[0065] In one embodiment, the CAR includes a Tm domain derived from CD8α. In certain aspects, the CAR includes a Tm domain derived from CD8α and a short oligopeptide or polypeptide linker, such as a short oligopeptide or polypeptide linker between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the Tm domain of the CAR and the intracellular signaling domain. For example, a glycine-serine linker can be used as such a linker.

[0066] The "intracellular signaling" domain of a CAR refers to the part of the CAR that is involved in transducing signals from the CAR that binds to a target molecule / antigen into the interior of an immune effector cell to initiate effector cell functions, such as activation, cytokine production, proliferation, and / or cytotoxic activity, including the release of cytotoxic factors to target cells bound by the CAR, or other cellular responses triggered by a target molecule / antigen that binds to the extracellular CAR domain. Thus, the term "intracellular signaling domain" refers to the part of a protein that transduces signals for effector functions and directs the cell to perform specialized functions. In terms of using truncated portions of the intracellular signaling domain, such truncated portions can be used in place of the full-length intracellular signaling domain so long as it transduces signals for effector functions. The term intracellular signaling domain means any truncated portion of the intracellular signaling domain that is sufficient to transduce signals for effector functions.

[0067] Signals generated solely through the T cell receptor (TCR) are not sufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Thus, T cell activation is mediated by two classes of distinct intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex) and a co-stimulatory signaling domain that functions in an antigen-independent manner to provide secondary or co-stimulatory signals. Thus, the CARs of the present disclosure can include such an intracellular signaling domain that includes one or more "co-stimulatory signaling domains" and "primary signaling domains".

[0068] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. A primary signaling domain that functions in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM"). Non-limiting examples of ITAM-containing primary signaling domains suitable for the CARs of the present disclosure include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, the CAR includes a CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling domain and co-stimulatory signaling domain are operably linked to the carboxyl terminus of the transmembrane domain.

[0069] In some embodiments, the CAR includes one or more co-stimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) expressing the CAR. As used herein, the term "co-stimulatory signaling domain" or "co-stimulatory domain" refers to the intracellular signaling domain of a co-stimulatory molecule or an active fragment thereof. Exemplary co-stimulatory molecules contemplated for use in the CAR in certain embodiments include TLR1 to TLR10 (and downstream signaling molecules), CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. In some embodiments, the CAR includes one or more co-stimulatory signaling domains selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and a CD3ζ primary signaling domain.

[0070] The CARs of the present disclosure can include any suitable type of domain, including but not limited to a leader sequence; a hinge, spacer, and / or linker domain; a transmembrane domain; a co-stimulatory domain; a signaling domain (e.g., a CD3ζ domain); a ribosome skipping element; a restriction enzyme sequence; a reporter protein domain (e.g., a domain containing a tag (e.g., a MYC tag)); and / or the like.

[0071] According to some embodiments, the CARs of the present disclosure include an extracellular domain that binds to GPNMB (e.g., a single-chain antibody, such as any scFv described herein); a transmembrane domain from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1; one or more intracellular co-stimulatory signaling domains from a polypeptide selected from the group consisting of 4-1BB (CD137), CD28, and CD134; and an intracellular signaling domain from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. Such CARs can further include a spacer domain, such as a CD8α hinge, between the antigen-binding portion and the transmembrane domain.

[0072] In certain embodiments, the CARs of the present disclosure comprise a CD8α hinge, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ primary signaling domain. According to some embodiments, there is provided a CAR that, from the N-terminus to the C-terminus, comprises the variable light chain (V L ) polypeptide of the antibody described herein, a linker, the variable heavy chain (V H)), a CD8α hinge region (which in some embodiments is an extended CD8 hinge region), a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. According to certain embodiments, a CAR is provided that comprises, from the N-terminus to the C-terminus, the variable heavy chain (V H ) polypeptide of the antibody described herein, a linker, the variable light chain (V L ) of the antibody, a CD8α hinge region (which in some embodiments is an extended CD8 hinge region), a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. Any of the CARs of the present disclosure may include a domain at the N-terminus of the V H polypeptide. For example, a leader sequence (e.g., a CD8α or GM-CSFR leader sequence) may be present at the N-terminus of the CARs of the present disclosure.

[0073] Using the information provided herein, the anti-GPNMB CARs of the present disclosure can be expressed using techniques well known to those of skill in the art. For example, a nucleic acid sequence encoding the amino acid sequence of the CARs of the present disclosure can be used to express the CAR. The amino acid sequences provided herein (see, for example, Table 1 and the Experimental section below (e.g., Table 6)) can be used to determine a suitable nucleic acid sequence encoding the CAR and then the nucleic acid sequence for use in expressing the CAR. The nucleic acid sequence can be optimized to reflect the particular codon "preference" of the various expression systems according to standard methods well known to those of skill in the art. Using the provided sequence information, the nucleic acid can be synthesized according to many standard methods known to those of skill in the art.

[0074] Once the nucleic acid encoding the subject CAR is synthesized, it can be amplified and / or cloned according to standard methods. Molecular cloning techniques for achieving these purposes are known in the art. A variety of cloning and in vitro amplification methods suitable for constructing recombinant nucleic acids are known to those of skill in the art and are the subject of many textbooks and laboratory manuals.

[0075] Nucleic acids, expression vectors, and cells

[0076] In view of the foregoing section regarding methods of expressing the CARs of the present disclosure, it should be understood that the present disclosure also provides nucleic acids, expression vectors, and cells.

[0077] In certain embodiments, nucleic acids encoding any of the CARs of the present disclosure (e.g., any of the such CARs described above) are provided. Since the codons corresponding to the various amino acids are known, the availability of the amino acid sequence of a polypeptide of interest provides a description of all polynucleotides capable of encoding the polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative or synonymous codons, allows for the production of a vast number of nucleic acids, all of which encode the enzymes disclosed herein. Thus, after identifying a particular amino acid sequence, one of ordinary skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates every possible variant of the polynucleotide that can be generated by selecting combinations based on possible codon choices, and all such variants are considered to be specifically disclosed for any polypeptide disclosed herein, including any amino acid sequence of the G1, G2, or G4 scFv set forth in Tables 1 and 6 herein, and CARs comprising these amino acid sequences.

[0078] The nucleotide sequence of the nucleic acids of the present disclosure can be codon-optimized. "Codon-optimized" means that the codons of a polynucleotide encoding a polypeptide are changed to codons preferentially used in a particular organism, such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons (termed "synonym" or "synonymous" codons), it is well known that codon usage by a particular organism is non-random and biased towards specific codon triplets. This codon usage preference can be higher for a given gene, genes with a common function or ancestral origin, highly expressed proteins versus low-copy number proteins, and the aggregated protein-coding regions of the genome of an organism. In some embodiments, the nucleic acids of the present disclosure encoding a polypeptide can be codon-optimized for optimal production from a host organism selected for expression, such as human cells, such as human immune cells (e.g., human T cells).

[0079] An expression vector comprising any nucleic acid of the present disclosure is also provided. Expression of a natural or synthetic nucleic acid encoding the CAR of the present disclosure can be achieved by operably linking the nucleic acid encoding the CAR to a promoter (which is constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that are functionally oriented appropriately and can be used to regulate the expression of the nucleic acid encoding the antibody. The vector optionally contains a universal expression cassette containing at least one independent terminator sequence, sequences that permit the cassette to replicate in both eukaryotes and prokaryotes (such as those found in shuttle vectors), and selectable markers for both prokaryotic and eukaryotic systems.

[0080] Cells comprising any nucleic acid and / or expression vector of the present disclosure are also provided. In some aspects, cells are provided in which the CAR is expressed on the surface of the cell. "Expressed on the surface of the cell" means that the CAR has been transported to the cell membrane such that, in the case of the CAR, the extracellular binding domain is displayed on the cell surface, the transmembrane portion traverses the cell membrane, and one or more intracellular signaling domains are disposed on the intracellular side of the cell membrane. After the extracellular binding domain binds to GPNMB, the intracellular signaling domain of the CAR participates in transducing a signal from the binding into the interior of the cell (e.g., an effector cell, such as a T cell, to initiate effector cell function).

[0081] In some embodiments, the cell is a eukaryotic cell. Eukaryotic cells of interest include, but are not limited to, yeast cells, insect cells, mammalian cells, etc. Mammalian cells of interest include, for example, murine cells, non-human primate cells, human cells, etc.

[0082] Such terms as "recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other terms denoting a microbial or higher eukaryotic cell line refer to cells that can or have been used as recipients of a recombinant vector or other transferred DNA and include progeny of the transfected cells. Host cells can be cultured as single cell entities or multicellular entities (e.g., tissues, organs, or organoids) comprising the expression vector of the present disclosure.

[0083] In one aspect, the cells provided herein include immune cells. Non-limiting examples of immune cells that can comprise any nucleic acid and / or expression vector of the present disclosure include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, eosinophils, and hematopoietic stem cells. In some embodiments, the immune cells include T cells. Examples of T cells include naive T cells (T N ), cytotoxic T cells (TCTL ) Memory T cells (T MEM ) T memory stem cells (T SCM ) Central memory T cells (T CM ) Effector memory T cells (T EM ) Tissue-resident memory T cells (T RM ) Effector T cells (T EFF ) Regulatory T cells (T REG ) Helper T cells (T H T H T1 H T2 H 17), CD4+ T cells, CD8+ T cells, virus-specific T cells, αβ T cells (T αβ ) and γδ T cells (T γδ ).

[0084] In some cases, the cells expressing the CARs of the present disclosure are stem cells. Non-limiting examples of the stem cells of the present disclosure include hematopoietic stem cells (HSC), induced pluripotent stem cells (iPSC), or their derivatives, etc.

[0085] Methods for making the cells of the present disclosure are also provided. In some embodiments, such methods include transfecting or transducing cells with the nucleic acids or expression vectors of the present disclosure. The terms "transfection" or "transduction" are used to refer to the introduction of foreign DNA into cells. When foreign DNA has been introduced into the interior of the cell membrane, the cell has been "transfected". Many transfection techniques are well known in the art, see for example Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd ed., Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd ed., McGraw-Hill and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign DNA moieties into suitable host cells. The term refers to both stable and transient uptake of genetic material.

[0086] In some embodiments, the cells of the present disclosure are produced by transfecting cells with a viral vector encoding a CAR. In some embodiments, the cells are T cells, thus providing a method for producing CAR T cells. In some embodiments, such methods include activating a population of T cells (e.g., T cells obtained from an individual to whom CAR T cell therapy will be administered), stimulating the population of T cells to proliferate, and transducing the T cells with a viral vector encoding a CAR. In some embodiments, the T cells are transduced with a retroviral vector encoding a CAR, such as a gamma-retroviral vector or a lentiviral vector. In some embodiments, the T cells are transduced with a lentiviral vector encoding a CAR.

[0087] According to some embodiments, the cells of the present disclosure are produced by directed integration via genome editing techniques such as those employing CRISPR / Cas9 nucleases, TALEN nucleases, ZFNs, etc. For example, a CRISPR-Cas protein (such as, for example, the Cas9 protein), or a polynucleotide encoding a CRISPR-Cas protein and a guide RNA (gRNA), or a polynucleotide encoding a gRNA can be employed. As used herein, unless inappropriate and / or otherwise indicated, the term "gRNA" generally encompasses both two-component guide systems (e.g., two gRNAs) as well as single-guide RNA (sgRNA) systems. In some cases, the gRNA or multiple gRNAs can be configured to target and used to target a desired locus or one or more elements thereof as described herein, such as one of the multiple exons of a gene present at the locus. For example, in some cases, the gRNA or multiple gRNAs can be configured to target and used to target a locus or one or more elements thereof, such as, for example, one or more exons of a locus.

[0088] In some embodiments, targeted integration can include using Cas9 nucleases, including native Cas9 nucleases and engineered Cas9 nucleases, and nucleic acid sequences encoding the same. Useful Cas9 nucleases include, but are not limited to, for example, Streptococcus pyogenes Cas9 and its variants, Staphylococcus aureus Cas9 and its variants, Actinomyces naeslundii Cas9 and its variants, and Cas9 nucleases also include those discussed in PCT Publication Nos. WO 2013 / 176772 and WO 2015 / 103153 and those reviewed in, for example, Makarova et al. (2011) Nature Reviews Microbiology 9:467-477, Makarova et al. (2011) Biology Direct 6:38, Haft et al. (2005) PLOS Computational Biology 1:e60, and Chylinski et al. (2013) RNA Biology 10:726-737, the disclosures of which are incorporated herein by reference in their entirety. In some cases, non-Cas9 CRISPR nucleases (or their engineered variants) can be employed, including but not limited to, for example, Cpf1 or Cpf1 variants.

[0089] The CRISPR system provides significant versatility in gene editing, in part because of the small size and high frequency of the essential sequence targeting elements within the host genome. CRISPR-directed Cas9 nucleases require the presence of a protospacer adjacent motif (PAM), the sequence of which depends on the bacterial species from which the Cas9 is derived (e.g., for Streptococcus pyogenes, the PAM sequence is "NGG"), but such sequences are common in various target nucleic acids. The PAM sequence directly downstream of the target sequence is not part of the guide RNA, but is essential for cleavage of the DNA strand. Synthetic Cas9 nucleases with new PAM recognition have been generated, further increasing the targeting versatility and can be used in the methods described herein. Cas9 nickases that cleave only one strand of the target nucleic acid (e.g., Cas9(D10A), etc.) and nuclease-deficient (i.e., "dead") dCas9 variants with additional enzymatic activities added by attached fusion proteins have also been developed.

[0090] In certain embodiments, targeted integration can be performed by a base editing strategy (e.g., as described in U.S. Patent Application Publication No. US2022 / 0220462) or a prime editing strategy (e.g., as described in U.S. Patent No. 11,447,770); the disclosures of these patents are incorporated herein by reference in their entirety for all purposes.

[0091] The cells of the present disclosure can be autologous / autogeneic (“self”) or allogeneic (“non-self”, e.g., allogeneic, syngeneic, or xenogeneic). As used herein, “autologous” refers to cells from the same individual. As used herein, “allogeneic” refers to cells of the same species that are genetically different from the cells being compared. As used herein, “syngeneic” refers to cells of different individuals that are genetically identical to the cells being compared. In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0092] T cells can be obtained from a variety of sources, including but not limited to peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of known techniques, such as sedimentation (e.g., FICOLL TM separation), purification, or any other convenient method.

[0093] In some embodiments, a population of isolated or purified T cells is used. In some embodiments, T CTL and T H lymphocytes are purified from PBMCs. In some embodiments, T CTL and T H lymphocytes are sorted into naive (T N ), memory (T MEM ), and effector (T EFF ) T cell subsets before or after activation, expansion, and / or genetic modification. Suitable methods for such sorting are known and include, for example, magnetic-activated cell sorting (MACS), where TN is CD45RA + CD62L + CD95 – ; TSCM is CD45RA + CD62L + CD95 + ; TCM is CD45RO +CD62L + CD95 + ; and TEM is CD45RO + CD62L – CD95 + 。An exemplary method for such sorting is described in Wang et al. (2016) Blood 127(24):2980-90. According to some embodiments, the population of isolated or purified T cells is T cells purified from PBMC REG population.

[0094] Specific subsets of T cells expressing one or more of the following markers can be further isolated by positive or negative selection techniques: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR. In some embodiments, specific subsets of T cells expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38 are further isolated by positive or negative selection techniques. In some embodiments, the manufactured T cell composition does not express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3. In some embodiments, the manufactured T cell composition substantially does not express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0095] To achieve a therapeutically effective dose of the T cell composition, the T cells can be subjected to one or more rounds of stimulation, activation, and / or expansion. T cells can generally be activated and expanded using methods described in, for example, U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the T cells are activated and expanded for about 1 to 21 days, such as about 5 to 21 days. In some embodiments, the T cells are activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introducing the nucleic acid encoding the polypeptide (e.g., expression vector) into the T cells.

[0096] In some embodiments, the T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours prior to introducing the nucleic acid encoding the CAR (e.g., expression vector) into the T cells. In some embodiments, the T cells are activated while the nucleic acid encoding the CAR (e.g., expression vector) is being introduced into the T cells.

[0097] In some embodiments, conditions suitable for T cell culture include a suitable culture medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) and one or more factors required for proliferation and viability, including but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to those skilled in the art that are suitable for the growth of cells. Additional illustrative examples of cell culture media include but are not limited to RPMI 1640, Clicks, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate, and vitamins, without serum or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in an amount sufficient to allow T cell growth and expansion.

[0098] In some embodiments, a nucleic acid encoding a CAR (e.g., an expression vector) is introduced into a cell (e.g., a T cell) by microinjection, transfection, genome editing techniques (e.g., using CRISPR / Cas9 nuclease, TALEN nuclease, ZFN, etc.), lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, etc. In some embodiments, a nucleic acid encoding a CAR (e.g., an expression vector) is introduced into a cell (e.g., a T cell) by AAV transduction. The AAV vector may comprise ITRs from AAV2 and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10. In some embodiments, the AAV vector comprises ITRs from AAV2 and a serotype from AAV6. In some embodiments, a nucleic acid encoding a CAR (e.g., an expression vector) is introduced into a cell (e.g., a T cell) by lentiviral transduction. The lentiviral vector backbone may be derived from HIV-1 virus, HIV-2 virus, visna-maedivirus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or simian immunodeficiency virus (SIV). The lentiviral vector may be an integrative or integrase-deficient lentiviral vector (TDLV). In one embodiment, an IDLV vector comprising an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0099] Viruses comprising any nucleic acid and / or expression vector of the present disclosure are also provided.

[0100] Composition

[0101] In other aspects, the present disclosure provides compositions comprising any nucleic acid, expression vector, and / or cell of the present disclosure.

[0102] In some embodiments, the composition comprises any nucleic acid, expression vector, and / or cell of the present disclosure present in a liquid medium. The liquid medium can be an aqueous liquid medium such as water, buffer solutions, etc. One or more additives such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., nonionic detergents such as Tween-20, etc.), nuclease inhibitors, glycerol, chelating agents, etc. can be present in such compositions.

[0103] Compositions are also provided that comprise any cell of the present disclosure suitable for administration to a human subject. Such compositions generally comprise a therapeutically effective amount of the cells. "Therapeutically effective amount" means an amount of cells sufficient to produce the desired result, e.g., an amount sufficient to achieve a beneficial or desired therapeutic (including prophylactic) result compared to a control, such as a reduction in the symptoms of a condition associated with GPNMB expression and / or activity. The effective amount can be administered in one or more administrations.

[0104] The cells of the present disclosure can be incorporated into various formulations for therapeutic administration. In some embodiments, the cells of the present disclosure are formulated into a pharmaceutical composition by combination with a suitable pharmaceutically acceptable excipient or diluent.

[0105] Formulations of cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient depending on the selected route of administration.

[0106] The cells can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0107] Kit

[0108] Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in the production of the cells of the present disclosure, non-limiting examples of which include CAR T cells expressing the anti-GPNMB CAR of the present disclosure on their surface.

[0109] Thus, in certain embodiments, the kits of the present disclosure include any nucleic acid and / or expression vector of the present disclosure, and instructions for transducing cells (e.g., T cells) with the nucleic acid and / or expression vector. As will be appreciated, the kits of the present disclosure may include any of the features described above in the section related to the subject nucleic acids and expression vectors, which are not repeated herein for the sake of brevity.

[0110] The components of the kit may be present in separate containers, or multiple components may be present in a single container. Suitable containers include a single tube (e.g., vial), one or more wells of a plate (e.g., 96-well plate, 384-well plate, etc.), and the like.

[0111] The instructions included in the kit may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, on the label of the container of the kit or its components (i.e., associated with the packaging or sub-packaging), and the like. In other embodiments, the instructions are present as an electronic storage data file on a suitable computer-readable storage medium, such as a portable flash drive, DVD, CD-ROM, disk, etc. In still other embodiments, the actual instructions are not present in the kit, but means are provided for obtaining the instructions, for example, from a remote source via the Internet. An example of this embodiment is a kit that includes a web address at which the instructions can be viewed and / or downloaded. Like the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0112] Methods of Use

[0113] Aspects of the present disclosure further include methods of using cells expressing a CAR of the present disclosure. The methods can be used in a variety of situations, including in vitro and / or in vivo studies and / or clinical applications.

[0114] In certain embodiments, there is provided a method of treating a condition associated with GPNMB expression and / or activity in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising cells expressing a CAR (e.g., CAR T cells, CAR NK cells, etc.).

[0115] According to some embodiments, the condition associated with GPNMB expression and / or activity is cancer. The subject methods can be used to treat a variety of cancers. As used herein, "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign), as well as all pre-cancerous cells and tissues and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth / proliferation. Cancers can be characterized by cancer cells that express GPNMB on their surface (GPNMB+ cancer cells). In certain embodiments, the cancer includes solid tumors. According to some embodiments, the solid tumor is a sarcoma, carcinoma, lymphoma, or blastoma. In some embodiments, when the cancer includes a solid tumor, the cancer is characterized by non-cancerous cells in the tumor microenvironment (TME) that express GPNMB on their surface. Non-limiting examples of non-cancerous cells that display cell surface expression of GPNMB in the TME include immune cells (e.g., macrophages), endothelial cells, fibroblasts (e.g., cancer-associated fibroblasts (CAF)), and the like.

[0116] Examples of cancers that can be treated using the methods of the present disclosure include, but are not limited to, sarcomas, carcinomas, lymphomas, and blastomas. More specific examples of such cancers include: renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast cancer; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; epidermal cancer; leukemias, such as, but not limited to, acute leukemias, acute lymphocytic leukemia, acute myelogenous leukemia, such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia leukemia, and myelodysplastic syndromes, chronic leukemias, such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease; multiple myelomas, such as, but not limited to, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenström macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas, such as, but not limited to, osteosarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors, such as, but not limited to, gliomas, astrocytomas, brainstem gliomas, ependymomas, oligodendrogliomas, non-gliomas, acoustic neuromas, craniopharyngiomas, medulloblastomas, meningiomas, pineocytomas, pineoblastomas, and primary brain lymphoma; breast cancer, including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease), and inflammatory breast cancer; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, such as, but not limited to, insulinoma, gastrinoma, glucagonoma, vasoactive intestinal peptide tumor, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancer, such as, but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancer, such as, but not limited to, ocular melanoma, such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, such as squamous cell carcinoma, adenocarcinoma, and melanoma;Vulvar cancer, such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, such as but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, such as but not limited to endometrial cancer and uterine sarcoma; ovarian cancer, such as but not limited to ovarian epithelial cancer, borderline tumor, germ cell tumor, and stromal tumor; cervical cancer; esophageal cancer, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous cell carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, such as but not limited to adenocarcinoma, fungating (polypoid), ulcerative, superficially spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; colorectal cancer, KRAS-mutated colorectal cancer; colon cancer; rectal cancer; liver cancer, such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancer, such as adenocarcinoma; bile duct cancer, such as but not limited to papillary bile duct cancer, nodular bile duct cancer, and diffuse bile duct cancer; lung cancer, such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; pulmonary cancer; testicular cancer, such as but not limited to embryonal carcinoma, seminoma, anaplastic seminoma, classical (typical) seminoma, spermatocytic seminoma, nonseminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma (yolk sac tumor), prostate cancer, such as but not limited to androgen-independent prostate cancer, androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penile cancer; oral cancer, such as but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, such as but not limited to squamous cell carcinoma and verrucous carcinoma; skin cancer, such as but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficially spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma; kidney cancer, such as but not limited to renal cell carcinoma, adenocarcinoma, oncocytoma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter); renal cancer; Wilms' tumor; and bladder cancer, such as but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelioma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinoma.;

[0117] In certain embodiments, the cancer is a sarcoma. Non-limiting examples of sarcomas treatable using the methods of the present disclosure include soft tissue sarcoma (STS). In some embodiments, when the sarcoma is STS, the STS is alveolar soft part sarcoma (ASPS). In certain embodiments, the subject comprises metastatic cancer. For example, the subject may comprise central nervous system (CNS) metastasis.

[0118] According to some embodiments, the cancer is a carcinoma. In some cases, when the cancer is a carcinoma, the carcinoma is basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma.

[0119] In certain embodiments, the cancer is renal cell carcinoma (e.g., translocation renal cell carcinoma (tRCC) or TSC / mTOR-altered RCC (see Salles et al. (2022) J Pathol. 257(2):158 - 171; FIGS. 14a, b)), breast cancer (e.g., triple-negative breast cancer (TNBC)), melanoma, or soft tissue sarcoma (e.g., ASPS). In some cases, the cancer is tRCC. According to some embodiments, the cancer is GPNMB+TNBC. In certain embodiments, the cancer is ASPS.

[0120] In some cases, the subject has a cancer comprising a fusion protein involving a microphthalmia family transcription factor. Such transcription factors include MITF, TFEB, TFE3, and TFEC. GPNMB is known to be expressed on the surface of cancer cells comprising such fusions. See, e.g., Salles et al. (2022) J Pathol. 257(2):158 - 171. See also FIGS. 14a, b.

[0121] According to some embodiments, the subject has a cancer characterized by the activation of one or more microphthalmia family transcription factors (e.g., MITF, TFEB, TFE3, and / or TFEC). GPNMB is known to be expressed on the surface of cancer cells, including the activation of such transcription factors. See, e.g., Hong et al. (2010) PLoS One 5(12):e15793; and Salles et al. (2022) J Pathol. 257(2):158 - 171). See also FIGS. 14a, b.

[0122] In certain cases, the condition associated with GPNMB expression and / or activity is a neurodegenerative disease. Non-limiting examples of neurodegenerative diseases treatable by the methods of the present disclosure include Alzheimer's disease (AD), Gaucher's disease, Niemann-Pick type C disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), amyloidosis, and the like.

[0123] Cells (e.g., CAR T cells) can be administered via any suitable route of administration, such as parenterally (e.g., by intravenous, intraarterial, subcutaneous, intramuscular, or epidural injection, intratumoral administration, intracerebral administration), etc.

[0124] Cells (e.g., CAR T cells) can be administered in a therapeutically effective amount in a composition. A "therapeutically effective amount" means a dose sufficient to produce a desired result, e.g., an amount sufficient to achieve a beneficial or desired therapeutic (including prophylactic) result as compared to a control, such as a reduction in the symptoms of cancer or a neurodegenerative disease. With respect to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow tumor growth, reduce tumor size, etc. The effective amount can be administered in one or more administrations.

[0125] As described above, aspects of the present disclosure include methods for treating conditions associated with GPNMB expression and / or activity. Treatment means at least improving one or more symptoms associated with a condition of an individual, where improvement is used in a broad sense to mean at least reducing the magnitude of a parameter (e.g., a symptom) associated with the condition being treated. Thus, treatment also includes situations where the condition or at least one or more symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated, such that the individual no longer has the condition or at least no longer has the symptoms that characterize the condition.

[0126] Cells (e.g., CAR T cells) can be administered to an individual alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for the treatment of cancer (e.g., sarcoma, such as soft tissue sarcoma, e.g., ASPS). In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are not limited to, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed cell death-1 (PD-1) inhibitors, programmed cell death ligand-1 (PD-L1) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, T cell immunoglobulin and mucin domain 3 (TIM-3) inhibitors, indoleamine 2,3-dioxygenase (IDO) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, T cell activation V domain Ig inhibitor (VISTA) inhibitors, B7-H3 inhibitors, and any combination thereof.

[0127] When cells (e.g., CAR T cells) are administered with a second agent, the cells can be administered to an individual according to any suitable administration regimen. According to certain embodiments, the cells and the second agent are administered according to a dosing regimen approved for use in the individual. In some embodiments, the administration of the cells permits the second agent to be administered according to a dosing regimen involving one or more lower and / or less frequent doses, and / or a reduced number of cycles, as compared to that utilized when the second agent is administered in the absence of the administration of the cells. In certain embodiments, the administration of the second agent permits the cells to be administered according to a dosing regimen involving one or more lower and / or less frequent doses, and / or a reduced number of cycles, as compared to that utilized when the cells are administered in the absence of the administration of the second agent.

[0128] In some embodiments, one or more doses of the cells and the second agent are administered to the subject simultaneously. "Simultaneously" means that the cells and the second agent are present in the same composition, or that the cells and the second agent are administered as separate pharmaceutical compositions within 1 hour or less, 30 minutes or less, or 15 minutes or less.

[0129] In some embodiments, one or more doses of the cells and the second agent are administered to the subject sequentially.

[0130] In some embodiments, the cells are administered to the subject in different compositions and / or at different times. For example, the cells can be administered before the second agent (e.g., in a particular cycle). Alternatively, the second agent can be administered before the cells (e.g., in a particular cycle). The second agent to be administered can be administered for a period of time that begins at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more after the administration of the first agent to be administered.

[0131] In some embodiments, the administration of one agent is specifically timed relative to the administration of the other agent. For example, in some embodiments, the cells are administered to observe a particular effect (or it is expected to observe a particular effect, e.g., based on population studies showing a correlation between a given dosing regimen and the particular effect of interest).

[0132] According to some embodiments, the desired relative dosing regimens of the agents administered in combination can be evaluated or determined empirically, such as by using in vitro, in vivo, and / or ex vivo models; in some embodiments, such evaluation or empirical determination is performed in vivo, in a patient population (e.g., to establish correlations), or alternatively in a particular individual of interest. The cells and the second agent can be administered together or independently via any suitable route of administration. The cells and the second agent can be administered via routes independently selected from oral, parenteral (e.g., by intravenous, intraarterial, subcutaneous, intramuscular, or epidural injection), topical, intranasal, intratumoral administration, and the like.

[0133] The following examples are provided by way of illustration and not by way of limitation.

[0134] Experimental Example 1 - ASPS gene expression profile

[0135] Soft tissue sarcoma (STS) is a rare and diverse cancer that derives from cells of primitive mesenchymal origin. Approximately 30% of STSs, but most pediatric and AYA cases, are molecularly characterized by recurrent, balanced chromosomal translocations that result in the formation of new oncogenes. These fusions involve transcription factors and / or chromatin remodeling complexes that are capable of driving transformation and tumorigenesis, often in the absence of a few other genetic abnormalities. Despite current understanding of how pathologic fusions drive STS etiology, many STS subtypes still have no standard of care treatment and are ultimately fatal, especially in the case of recurrent and / or metastatic disease.

[0136] Alveolar soft part sarcoma (ASPS) is cytogenetically characterized by the t(X:17)(p11;q25) translocation, which fuses the third or fourth exon of the TFE3 transcription factor with the first seven exons of the DNA-binding protein ASPSCR1. ASPS has a slow course and often presents as a painless mass in the lower extremities. The asymptomatic presentation delays diagnosis, resulting in metastasis in approximately half of patients, usually to the lungs, bone, and / or brain. Recurrence in patients with metastasis is inevitable, and limited systemic treatment leaves metastasectomy, if possible, as the only life-prolonging intervention. All patients with metastasis will ultimately die of their disease.

[0137] To analyze the potential surfaceome of ASPS, differential expression analysis was performed using the gene expression profile of ASPS (Figure 1a). Eleven genes were identified as "ASPS-specific genes" that may be expressed on the cell surface membrane (Table 3). GPNMB was reported as the most statistically significant gene (lowest FDR; FDR = 0.0001, Log2-FC = 0.6044), while SLC2A4 was the most highly regulated ASPS-specific gene (greatest fold change; FDR = 0.0003, Log2-FC = 1.1520). Using these 11 ASPS-specific genes from the cohort, the expression levels were investigated in multiple ASPS samples (Figure 1b). GPNMB was observed to be highly expressed in each ASPS sample from the three cohorts. NTSR2, CHRNA1, and STS appeared to be data-specific DE genes, while the remaining candidate genes were below or near median expression. Focusing on GPNMB, expression was detected across several publicly available gene expression datasets, comparing GPNMB expression across other pediatric and AYA tumor types. GPNMB was found to be highly expressed in all analyzed ASPS samples (Figure 1c). Since GPNMB expression is regulated by the microphthalmia family of basic helix-loop-helix leucine zipper transcription factors (MiTF / TFE) (TFE3 is a member of this family), the high and ubiquitous GPNMB expression in ASPS is likely driven by the ASPSCR1-TFE3 fusion. Indeed, this has been demonstrated in Xp11-fusion-driven renal cell carcinoma, where Xp11-fusion partners, including ASPSCR1, have been shown to drive GPNMB expression in these cancers (Tanaka et al. (2017) Cancer Res 77:897–907; Baba et al. (2019) Mol Cancer Res 17:1613–1626), as also shown in subsequent examples.

[0138] Table 3: Eleven ASPS-specific candidate genes and cell surface proteins.

[0139]

[0140] Example 2 - Validation of GPNMB expression in clinical ASPS samples

[0141] To validate the expression of GPNMB in clinical ASPS samples, three primary ASPS resections were stained. These samples were clinically diagnosed as ASPS, which was confirmed by assessment of nuclear localization of TFE3. High and homogeneous expression of GPNMB was found in these primary tumor sections ( Figure 2)。One of the subjects was initially diagnosed with metastatic disease at presentation and has since undergone multiple metastasectomy procedures to eliminate recurrences in the lung, brain, spine, and gastrointestinal tract (Figure 1d). Stained GPNMB homogenous cell surface expression was observed in all surgical samples, demonstrating the spatial and temporal stability of the potential target (Figure 1e).

[0142] Example 3 - Evaluation of GPNMB as a CAR target

[0143] GPNMB has been described as having differential expression across several normal human tissues, but is primarily expressed in several subsets of skin, bone, and myeloid-derived cells. Additionally, GPNMB can be present in different cellular locations, such as the cell surface, intracellular vesicles, and as a secreted product. To deconvolute human and mouse protein expression data from the literature, two publicly available mass spectrometry-based proteomics databases were first evaluated (Kim et al. (2014) Nature 509:575–81; Wang et al. (2019) Mol Syst Biol 15, e8503), and GPNMB expression was evaluated against other known CAR targets for sarcoma (HER2 and CD276) for 14 housekeeping genes ( Figure 3 ). Housekeeping genes showed widespread expression at the protein level, and GAPDH and ACTB expression was high across tissues / databases. HER2 and CD276 showed relatively low expression as expected. GPNMB showed relatively low or no expression in all normal tissues, suggesting that GPNMB would be a safe CAR target. However, both databases were found to list “heart” as the highest tissue expresser. GPNMB expression was further examined in 34 normal tissues across two normal tissue microarrays. Most tissues had little or no expression, but significant staining (≥2 histological score) was observed in skin, heart, placenta, and lymphoid tissue (Table 4); however, except for placenta and tonsil, GPNMB expression in these tissues was restricted to the cytosol ( Figure 4 ).

[0144] To further evaluate the potential safety of GPNMB CAR, adverse events associated with glembatumumab vedotin (CDX-011) were studied. CDX-011 is a human GPNMB-targeted antibody-drug conjugate that was clinically evaluated in over 600 patients with melanoma, breast cancer, lung cancer, or osteosarcoma (Table 5). In all of these trials, including NCT02487979 (which mainly included pediatric and AYA patients), CDX-011 was considered well-tolerated or generally well-tolerated. CDX-011 binds to proteolytically cleaved monomethyl auristatin E (MMAE), similar to vedotin (SGN-35), which is an FDA-approved therapy for CD30 in Hodgkin's and T cell lymphoma. The adverse event profiles of CDX-011 and SGN-35 were directly compared to distinguish the cytotoxicity attributable to targeting normal GPNMB + cells from the non-specific toxicity attributable to the released MMAE. In six comparable trials with the same dosing strategy and toxicity grading, it was found that among all adverse events (AEs), in the case of CDX-011, there was a significant increase in skin-related toxicities (rash 53% vs. 19%; pruritus 36% vs. 10%; alopecia 49% vs. 7%) and neutropenia (40% vs. 19%). Isolating severe AEs (≥3) alone, only rash (11% vs. 4%) remained significantly different (Figure 5). Importantly, there was no cardiac toxicity in the CDX-011 trials.

[0145] Table 4: Summary of normal tissue arrays ≥2

[0146]

[0147] Table 5: GPNMB clinical trials

[0148]

[0149] Example 4 - Development of a CAR against GPNMB

[0150] Given the highly elevated nature of GPNMB in ASPS and the acceptable safety profile demonstrated in TMA and CDX-011 trials, a second-generation 41bbζ CAR T cell (CAR T) targeting GPNMB was developed. A MYC-epitope tag was included between the scFv and the post-linker used to measure CAR expression, and a P2A sequence that separates it from the EGFP protein was included to measure transduction efficiency (Figure 6a). The domains of the CAR and their amino acid sequences employed in this example are provided in the table below (Table 6).

[0151] Table 6: CAR domains and amino acid sequences

[0152]

[0153]

[0154] To test the efficacy of the GPNMB CAR, an ASPS patient-derived cell line cultured from a lung metastasis (Figure 1d) was used. This model contains the type II ASPSCR1-TFE3 fusion with nuclear localization of TFE3 and cell surface GPNMB expression in vitro (Figure 6b-c). In addition, this model retained the histological features of ASPS when grown orthotopically as a primary tumor in NSG mice with a consistent high level of GPNMB (Figure 6d). Four individual conjugates (G1-G4) were tested on the ASPS PDX-derived cell line. Unexpectedly, although having similar transduction efficiencies (Figure 7a), only conjugates G1 and G2 demonstrated robust CAR surface expression (Figure 7b). These CARs were tested head-to-head in in vitro co-culture experiments with the ASPS PDX-derived cell line, showing that G1 had excellent CAR-T activation / expansion, cytotoxicity, and cytokine secretion capabilities (Figure 7c-e). This G1 conjugate was used for the remainder of the preclinical studies and was thereafter referred to as the GPNMB CAR. GPNMB CAR activity was demonstrated in two groups of healthy donors, and good transduction efficiency of the GPNMB CAR at 5 MOI was shown in both the CD4 T cell population and the CD8 T cell population (Figure 6e). When co-cultured with ASPS mCherry / FLUC cells, the GPNMB CAR T cells were efficiently killed (Figure 6f) and secreted activation-related cytokines (Figure 6e).

[0155] Example 5 - In vivo efficacy of GPNMB CAR T product in an ASPS model

[0156] After demonstrating the function of the GPNMB CAR in vitro, its efficacy was tested in an orthotopic model of ASPS primary disease. The ASPS patient-derived cell line was implanted intramuscularly into NSG-MHC KOIn (NSG) mice, they were treated with various doses of GPNMBCAR T cells or CD19 CAR T cells. Immediate tumor reduction was observed starting 4 days after treatment with 5e6 and 1e6 GPNMB CAR T cells, with complete tumor elimination between 7 and 14 days after treatment (Figure 8a). Mice with cleared tumors did not relapse within 300 days after treatment (Figure 8b). Efficacy was also observed at the 1e5 dose, resulting in a maximum tumor size reduction of 68 ± 25%, as measured by bioluminescence on day 21. These mice eventually relapsed, with a slight overall survival advantage (Figure 8b), but retained GPNMB expression. 1e4 GPNMB CAR T cells per mouse did not result in a therapeutic benefit. Tumor efficacy correlated with CAR T cell expansion in the periphery (Figure 8c), where at the 5e6 CAR T cell dose, it averaged 5.92e7 ± 2.60e7 cells / mL at peak expansion on day 14, corresponding to a 1668 ± 733-fold expansion of GPNMB CAR T cells compared to non-targeted CD19 CAR T cells. Significant expansion was also observed at the 1e6 and 1e5 doses, although peak expansion of 1e5 CAR T cells was observed on day 21. Repeating GPNMB CAR T cell efficacy and expansion using separate healthy donor T cells yielded very similar results (Figure 9). A significant but transient loss of CAR expression on circulating CAR T cells in the periphery was also observed (Figure 8d). This was attributed to CAR internalization caused by target engagement, as the MFI of the EGFP transduction marker remained unchanged. However, 28 days after treatment, CAR levels recovered to near original levels and remained stable. Circulating CAR T cells in the periphery were initially polarized to an effector phenotype (CD45RO - CD62L - ), where the ratio of effector memory (CD45RO + CD62L - ) to effector cells increased until it approached pre-injection levels between days 28 and 42 (Figure 8e). Memory CAR T cells (CD45RO + CD62L +) appeared starting from day 42 and further increased on day 63. Finally, in order to measure and observe the expansion of GPNMB CAR T cells within the tumor, intravital microscopy was employed (Figure 8f). CAR T infiltration into the tumor could be seen as early as day 2, and a sharp expansion was observed in the following days. Four days after treatment, a significant negative correlation was observed between the area of the tumor and the area of the expanded CAR T cells. By day 7, no significant tumor area could be found within any microscopic field of view, and all areas were dominated by extravascular CAR T cells. In summary, these data demonstrate that the GPNMB CAR T product is a highly functional and effective systemic therapy in this proof-of-concept tumor model (primary ASPS tumor).

[0157] Example 6 - Eradication of CNS metastases by systemic administration of GPNMB CAR T cells

[0158] Among all sarcomas, ASPS has the highest incidence of brain metastases (11%-19%), which may be an important cause of morbidity and death in patients. To test whether GPNMB CAR T cells could be administered systemically to target ASPS CNS metastases, patient-derived cell lines of ASPS were implanted intracranially. Surprisingly, it was found that doses of 1e6 and 1e5 GPNMB CAR T cells completely eradicated CNS metastases by 7-14 days after treatment, resulting in a durable response with no tumor recurrence (Figure 10a-b). Additionally, in some cases, spinal metastases developed in several animals by the start of treatment (Figure 10a, white arrow). The lesions were completely eliminated in animals with bioluminescent signals in the spine treated with systemic GPNMB CAR T cells. Tumor regression was accompanied by the expansion of CAR T cells in the periphery (Figure 10c), although this was to a lesser extent than that observed using the primary tumor model (8.00e4 ± 1.4e5 vs. 2.23e7 ± 1.65e7 CAR / mL in 1e6 CAR T cells per mouse in the intracranial vs. intramuscular sites), reaching a maximum 16.5-fold increase compared to non-targeting CD19 CAR T cells 14 days after treatment.

[0159] Interestingly, systemic administration of GPNMB CAR T cells to intracranial ASPS tumors marked the first observation of any treatment-related toxicity of this therapy. Mice treated with 1e6 GPNMB CAR T cells rather than 1e5 GPNMB CAR T cells demonstrated immediate and rapid weight loss that began to resolve 7 days after treatment (Figure 10d). Neuropathological evaluation of the brains of treated mice showed no residual tumor and the absence of any obvious nerve damage. This observation is consistent with the fact that toxicity was only observed in animals using high doses of CAR T-cells, leading us to speculate that this toxicity was due to the rapid, simultaneous expansion of CART cells in the intracranial space resulting in transient intracranial edema.

[0160] Example 7 - Clinical translation of GPNMB CAR

[0161] Given the efficacy observed in the in vivo model of ASPS and the fact that metastatic ASPS is considered "incurable" with limited systemic therapy options, clinical translation of GPNMB CAR for ASPS was initiated. First, the GPNMB transduction efficiency and in vitro activity of ASPS patient T cells were tested on matched patient-derived cell lines. In vitro analysis demonstrated comparable transduction efficiency to healthy donor cells (Figure 11a), as well as equally potent cytotoxic activity and cytokine secretion (Figure 11b). Additionally, treatment of the ASPS mouse model with 1e6 CAR T cells per mouse resulted in complete regression of the primary tumor, as observed using GPNMB CAR T cells from healthy donor sources (Figure 11c).

[0162] Next, a clinical GPNMB CAR vector was constructed by placing the same GPNMB targeting sequence into the CD19-41BBζ-CLIC backbone of a current clinical trial in Canada (NCT03765177) and removing the EGFP protein. A direct head-to-head comparison of the preclinical CAR construct and the clinical CAR construct showed that the CARs behaved similarly when transduced into patient T cells (Figure 12). Under Good Manufacturing Practice (GMP), subsequent production of the clinical-grade lentiviral vector yielded 86 mL of virus at a titer of 1.0e7 IU / mL. Four mock clinical runs of prodigy were performed using different volumes of the clinical-grade lentivirus in apheresis materials from both healthy donors (HD) and cancer patients (EHE). In All runs on [[product name]] demonstrated robust T cell expansion of the CAR product (Figure 13a-b). The potency of these products was evaluated on ASPS PDX-derived cell lines and measured by cytotoxicity assay and cytokine secretion (Figure 13c-d). All products passed the quality assurance specifications (Table 7). The product from the HD1 run was tested in an in situ model of ASPS primary disease and demonstrated complete tumor clearance in all mice at a dose of 5e6 CAR / mouse and in 3 out of 5 mice at 5e5 CAR / mouse (Figure 13e). When compared to mice bearing GPNMB-negative tumors (Figure 14c, 786-O, ccRCC line) and treated with the same dose, significant CAR T expansion and persistence were also observed in the case of the clinical-grade product in the ASPS model (Figure 13f).

[0163] Table 7: Quality Assurance Specifications of the Products

[0164] Analytical methods Specifications HD1 (4 mL) HD2 (2 mL) EHE (2 mL) EHE (1 mL) Harvest product purity (CD3% of total cells) ≥90％ 94.94 97.45 92.81 100.00 Harvest product viability (live %) ≥70％ 96.70 99.80 100.00 98.00 Harvest product CAR expression (total cell %) To be reported 63.02 53.57 62.14 40.82 Drug product appearance Clear, no particles Clear, no particles Clear, no particles Clear, no particles Clear, no particles Drug product pH 7-7.5 7-7.5 7-7.5 7-7.5 7-7.5 Drug product sterility - anaerobic No growth No growth No growth No growth No growth Drug product sterility - aerobic No growth No growth No growth No growth No growth Drug product sterility - fungal No growth No growth No growth No growth No growth Drug product mycoplasma Not detected Not detected Not detected Not detected Not detected Drug product endotoxin (EU / mL) Run / formulation-dependent ＜0.050 ＜0.050 ＜0.500 ＜0.500 Drug product vector copy # ( / CAR+ T cell) ≤5 copies 1.55 1.01 1.35 1.40

[0165] Example 8 - Indication expansion of GCAR1

[0166] GPNMB is known to be regulated by members of the MiTF family at steady state and has previously been shown to be a transcriptional target of Xp11 translocation-positive (t)RCC fusion proteins, resulting in high and homogeneous upregulation of GPNMB (Tanaka et al. (2017) Cancer Res 77:897–907; Baba et al. (2019) Mol Cancer Res 17:1613–1626) and TSC / mTOR-altered RCC (Salles et al. (2022) J Pathol 257(2):158-171). GPNMB expression was evaluated in a cohort of patients with TSC / mTOR-altered RCC and translocation-positive tRCC, and high and homogeneous GPNMB expression was confirmed in the majority of these RCC subtypes (Figure 14a-b). Next, a panel of fusion-positive tRCC cell lines and fusion-negative RCC cell lines was tested, and only the tRCC cell lines were found to display cell surface GPNMB expression (Figure 14c). FU-UR1, UOK-124, and UOK-146 (tRCC) and 786-O (RCC) were subcutaneously implanted into mice and treated with 5e6 CAR / mouse of the clinical-grade HD1 GPNMB CAR product. The tRCC model showed a robust therapeutic response to GPNMB CAR, accompanied by significant product expansion in the blood. These responses were not observed in the RCC (GPNMB-) model (Figure 14d-e). Tissue microarrays of 49 TNBCs were also evaluated, showing variable GPNMB expression (Figure 15a), as has been shown by others. The cell surface expression of GPNMB in TNBC cell lines MDA-MB-231 and Hs578T was evaluated compared to the positive control ASPS (Figure 15b), showing GPNMB expression in the Hs578T cell line. Head-to-head tests of cytotoxicity and cytokine secretion were performed on TNBC cell lines when treated in co-culture experiments with the HD1 clinical-grade GPNMB CAR (Figure 15c), showing GPNMB CAR-specific cytotoxicity and cytokine responses against GPNMB+ TNBC. Luciferase-expressing Hs578T cells were implanted into the mammary fat pads of NSG-KO mice and treated with 1e6 GPNMB CAR-T cells from HD1, showing a robust and durable response in this model (Figure 15d).

[0167] Materials and methods

[0168] Differential expression in ASPS samples

[0169] Download pre-existing microarray-based gene expression profiles of ASPS (GSE13433, 14 ASPS samples and two universal RNAs) from GEO. First, map the log-transformed and normalized expression values to official gene symbols at the probe level using the R package biomaRt, and combine gene expression levels by taking the average of probes mapped to the same gene symbol, which yielded a total of 17,545 genes. Then, prior to assessing differential expression (DE), average the technical replicates (n = 7) for each ASPS tumor. Use the integrated database of the surfaceome to select 5,725 possible cell surface proteins (genes), in which four databases (Cell Surface Protein Atlas, COMPARTMENTS, Human Protein Atlas, SURFY) were integrated to prioritize them. Eleven DE genes identified by SurfaceGenie with a score > 2 were considered as final candidates. Obtain DE genes from Welch's t-test between the ASPS tumor group and the universal RNA group, and apply false discovery rate (FDR) correction. If FDR < 0.01 and log2 fold change > 0.5, the gene is considered DE (i.e., an ASPS-specific gene). Finally, download two evaluation datasets (GSE32569 and GSE49327) from GEO, in which only ASPS tumor profiles (i.e., pre-treatment or post-treatment, primary or metastatic) exist, and process them in the same manner as above to confirm the expression levels among the top candidates.

[0170] GPNMB expression across tissues

[0171] Two proteomics databases, namely the Human Protein Atlas and Wang et al., were located to examine the GPNMB expression levels across tissues in non-malignant samples. In addition, CD276 and HER2 were included as established CAR targets and 14 important housekeeping genes (https: / / www.genomics-online.com / resources / 16 / 5049 / housekeeping-genes / ): ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPL13A, RPLP0, SDHA, TBP, TFRC, and YWHAZ. Since the range of mass spectrometry (MS) expression varies and can be extreme, MS values were limited to the 90th percentile across 17 proteins within the dataset. Then, scale the MS values from the [minimum, maximum] expression to [0, 1].

[0172] CAR generation, preclinical vector manufacturing, and in vitro testing

[0173] The lentiviral plasmid containing the CAR construct was generated by standard molecular cloning methods. The preclinical GPNMB CAR construct was assembled from a human scFv sequence recognizing GPNMB, a MYC epitope tag, a CD8α hinge and transmembrane domain, and 4-1BB and CD3ζ intracellular signaling domains. The scFv used in this example contains an antibody-drug conjugate (ADC), i.e., the V of the antibody present in gemtuzumab vedotin (CDX-011-MMAE) L and V H . See, e.g., WO2006071441A2.

[0174] The construct was cloned into the pULTRA-EGFP vector (Addgene #24129) downstream of EGFP and separated by the P2A site. The UbC promoter was replaced with the full-length EF1α promoter. The same CD19 (FMC63) targeting construct was also prepared in this vector.

[0175] Preclinical lentiviral particles were packaged in LentiX 293T (Takara) cells using the packaging plasmid pCMV-dR8.91, the envelope plasmid pMD2.G, and the CAR construct (at a ratio of 5:1:5). The supernatant containing lentiviral particles was collected 48 hours after transfection and concentrated by ultracentrifugation. The viral titer in transducing units per milliliter was determined by flow cytometry analysis of transduced Lenti-X 293 cells.

[0176] Human PBMCs were isolated from healthy donors or patient blood by Ficol-paque density centrifugation. CD3-positive T cells were sorted from PBMCs using a CD3 isolation kit from Miltenyi Biotec. The isolated CD3-positive cells were cultured in TexMACs T cell expansion medium (BioLegend; 10 ng / mL) supplemented with IL7 and IL15 and activated with CD3 / 28 Transact beads (Miltenyi Biotec). Twenty-four hours after activation, the T cells were transduced with the CAR-containing lentivirus at a multiplicity of infection of 5. The cells were then expanded for an additional 7 to 9 days.

[0177] ASPS patient-derived cell lines were transduced with a lentiviral construct containing mCherry and firefly luciferase. The ASPS mC / herryFLUCPlated in 96-well plates and treated with CAR T cells at the specified ratio for 24 hours. Cytotoxic activity was quantified by adding fluorescein (GoldBio) to a final concentration of 150 μg / mL and measured on a SpectraMax i3. IL2 and IFNγ were measured using an ELISA kit (BioLegend) and read on a SpectraMax i3.

[0178] In vivo CAR T testing

[0179] The ASPS patient-derived cell line was obtained from a clinical biopsy collected from a 17-year-old female with ASPS who was undergoing surgery for lung metastasis. Fresh tumor tissue was implanted into the flanks of SCID mice and established as a patient-derived xenograft (PDX). The established PDX tissue was removed, formed into a single-cell suspension by gentle grinding, and plated in complete OptiMEM medium to establish the patient-derived ASPS cell line.

[0180] Eight- to ten-week-old female NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab1 em1Mvw H2-D1 tm1Bpe Il2rg tm1Wjl / SzJ (Jackson Laboratory; strain #025216) were used for this study. To model the primary disease, 1e6 ASPS were injected into the gastrocnemius muscle. mC / FLUC . Intracranial metastasis was modeled by aseptically implanting 1e6 ASPS mC / FLUC into the right striatum of the mice. 35 - 42 days after implantation, the mice were treated intravenously with the specified number of CAR T cells, and tumor burden was evaluated by bioluminescence imaging using the Xenogen system and processed with LivingImage software. Blood was drawn weekly for measuring CAR T in peripheral blood by bleeding the saphenous vein into an EDTA tube. For other animal models, 5e6 cells from each RCC cell line were subcutaneously injected into mice of the same strain in Matrigel. When the tumors reached an average volume of 150 - 250 mm 3 CAR treatment was initiated. 5e6 Hs578T cells were implanted into the mammary fat pad of this strain and treated 47 days after implantation. The overall health of the animals was monitored daily until they reached the humane or experimental endpoint. Animal experiments were approved by the University of Calgary Conjoint Health Research Ethics Board.

[0181] Flow cytometry

[0182] Single cells from cell cultures were resuspended in PBS + 2% FBS + 0.25 mM EDTA (flow buffer). Alternatively, blood derived from saphenous vein bleeding was processed in ACK and resuspended in flow buffer. For GPNMB staining of ASPS patient-derived cell lines, anti-GPNMB (R&D MAB25501) with anti-mouse AF647 (BioLegend) antibody was used. For staining of in vitro and in vivo-derived T cells, antibodies from BioLegend were used. Samples were run on an ATTUNE flow cytometer and data were analyzed with Kaluza software.

[0183] Immunohistochemistry and immunofluorescence

[0184] Tissue sections were deparaffinized with xylene and rehydrated through a graded ethanol series. Heat-induced epitope retrieval was performed on slides in 1X antigen retrieval buffer (10 mM citrate buffer pH 6.0 + 0.05% Tween 20) using a microwave at 95 °C for 20 minutes. Endogenous peroxidase activity was blocked by adding 1 drop of peroxidase blocking solution (Dako, S202386-2) to each slide and incubating for 15 minutes. Nonspecific binding was blocked with 200 μL of protein blocker (Agilent, X090930-2) containing 0.2% Triton X-100 (Sigma, Oakville) and incubated for 20 minutes. Each section was incubated with goat polyclonal anti-human GPNMB antibody (R&D systems, AF2550, 1:250) or rabbit polyclonal anti-human TEF3 (Sigma-Cellmarque 354R-15 1:200) for 1 hour or overnight. After washing each section with TBS, 1 drop of the appropriate polymer-HRP secondary antibody (Vector Laboratories or DAKO Envision) was applied, incubated for 30 minutes, washed with TBS, and detected using DAB reagent (DAKO, 1 drop of DAB in 1 mL DAB substrate solution). Sections were counterstained with hematoxylin, then dehydrated through a graded ethanol series, and sections were mounted with Entellan (Electron Microscopy Sciences). Stained slides were scanned at 20× or 40× resolution using an Aperio XT (Aperio Inc.) slide scanner and images were acquired using Imagescope v12.2.2.5015 software.

[0185] The ASPS cell line was cultured in OPTI-MEM + 10% fetal bovine serum for 48 h. Sections were fixed with 4% PFA and permeabilized with 0.5% Triston X-100. Each section was incubated ON at 4 °C with goat polyclonal anti-human GPNMB antibody (R&D systems, AF2550, 20 μg / mL) and rabbit monoclonal anti-TFE3 antibody (Sigma-Cellmarque, 354R-15, 1:25). Secondary antibody Alexa 647 goat anti-mouse IgG (Biolegend, 405322) was added for GPNMB detection, and DyLight TM 488 donkey anti-rabbit IgG (Biolegend, 406404) was added for TFE3 detection. Both were added at 1 μg / mL for 30 min. Sections were stained with one drop of ProLong TM Gold antifade reagent (Invitrogen, P36935). Stained slides were digitized at 40× resolution using an ECHO Revolve (ECHO, RVL-100-M).

[0186] Production of clinical vectors and CAR T

[0187] The clinical GPNMB plasmid was assembled with the same GPNMB targeting sequence and cloned into the CD19-41BBζ-CLIC backbone currently in clinical use (NCT03765177). Plasmids were manufactured using GMP-grade reagents and strict quality control at the BC Cancer Agency. GMP manufacturing of lentiviral particles was completed by the Ottawa Hospital Research Institute Biotherapeutics manufacturing Center. Standard T cell transduction (TCT) process protocols and CliniMACS TS 520 tubing sets were used, and mock clinical runs were performed on Miltenyi Biotec CliniMACS according to the manufacturer's protocols and the SOPs of the Alberta Cell Therapy Program. Apheresis materials from healthy donors were purchased from StemCell Technologies. Apheresis materials from a patient with epithelioid hemangioendothelioma (EHE; HREBA.CC-22-0367) were also utilized. This EHE patient had a confirmed YAP1-EHE fusion and was a candidate for GPNMB CAR T therapy.

[0188] Accordingly, only the principles of this disclosure have been illustrated above. It should be understood that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Moreover, all of the examples and conditional language recited herein are principally intended to assist the reader in understanding the principles of the invention and the concepts contributed by the inventor to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Further, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof are intended to encompass both structural and functional equivalents thereof. Moreover, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function regardless of structure. Accordingly, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. A chimeric antigen receptor (CAR) comprising: An extracellular glycoprotein NMB (GPNMB) binding domain; A transmembrane domain; and One or more intracellular signaling domains.

2. The CAR according to claim 1, wherein the extracellular GPNMB binding domain comprises a single-chain antibody that specifically binds GPNMB.

3. The CAR according to claim 2, wherein the single-chain antibody is a single-chain variable fragment (scFv).

4. The CAR according to claim 2 or claim 3, wherein the single-chain antibody competes with an antibody comprising: Variable light chain (V L ) polypeptide, comprising: V comprising the amino acid sequence QSVDNN (SEQ ID NO:2) L CDR1 V containing the amino acid sequence GAS L CDR2, and V containing the amino acid sequence QQYNNWPPWT (SEQ ID NO:3) L CDR3; and Variable heavy chain (V H ) polypeptide, comprising: V containing the amino acid sequence GGSISSFNYY (SEQ ID NO:7) H CDR1 V containing the amino acid sequence IYYSGST (SEQ ID NO:8) H CDR2, and V containing the amino acid sequence ARGYNWNYFDY (SEQ ID NO:9) H CDR3; Variable light chain (V L ) polypeptide, comprising: V containing the amino acid sequence QSLLHSNGYNY (SEQ ID NO:16) L CDR1 V containing the amino acid sequence LGS L CDR2, and V containing the amino acid sequence MQGLQTPIT (SEQ ID NO:17) L CDR3; and Variable heavy chain (V H ) polypeptide, comprising: V containing the amino acid sequence GFAFSSYG (SEQ ID NO:21) H CDR1 V containing the amino acid sequence ISYDGNNK (SEQ ID NO:22) H CDR2, and V comprising the amino acid sequence ARDLVVRGIRGYYYYFGMDV (SEQ ID NO:23) H CDR3, or Variable light chain (V L ) polypeptide, comprising: V containing the amino acid sequence QSLLHSNGYNY (SEQ ID NO:16) L CDR1 V containing the amino acid sequence LGS L CDR2, and V containing the amino acid sequence MQALQTHPT (SEQ ID NO:44) L CDR3; and Variable heavy chain (V H ) polypeptide, comprising: V containing the amino acid sequence GGTFSSYA (SEQ ID NO:46) H CDR1 V containing the amino acid sequence IIPIFGTA (SEQ ID NO:47) H CDR2, and V containing the amino acid sequence ARGPNT (SEQ ID NO:48) H CDR3 Wherein the CDRs are numbered according to IMGT.

5. The CAR according to claim 4, wherein the single-chain antibody comprises: Variable light chain (V L ) polypeptide, comprising: V containing the amino acid sequence QSVDNN (SEQ ID NO:2) L CDR1 V containing the amino acid sequence GAS L CDR2, and V containing the amino acid sequence QQYNNWPPWT (SEQ ID NO:3) L CDR3; and Variable heavy chain (V H ) polypeptide, comprising: V containing the amino acid sequence GGSISSFNYY (SEQ ID NO:7) H CDR1 V containing the amino acid sequence IYYSGST (SEQ ID NO:8) H CDR2, and V containing the amino acid sequence ARGYNWNYFDY (SEQ ID NO:9) H CDR3; or Variable light chain (V L ) polypeptide, comprising: V comprising the amino acid sequence QSLLHSNGYNY (SEQ ID NO:16) L CDR1 V containing the amino acid sequence LGS L CDR2, and V containing the amino acid sequence MQGLQTPIT (SEQ ID NO:17) L CDR3; and Variable heavy chain (V H ) polypeptide, comprising: V containing the amino acid sequence GFAFSSYG (SEQ ID NO:21) H CDR1 V containing the amino acid sequence ISYDGNNK (SEQ ID NO:22) H CDR2, and V containing the amino acid sequence ARDLVVRGIRGYYYYFGMDV (SEQ ID NO:23) H CDR3, or variable light chain (V L ) polypeptide, comprising: V containing the amino acid sequence QSLLHSNGYNY (SEQ ID NO:16) L CDR1 V containing the amino acid sequence LGS L CDR2, and V containing the amino acid sequence MQALQTHPT (SEQ ID NO:44) L CDR3; and Variable heavy chain (V H ) polypeptide, comprising: V comprising the amino acid sequence GGTFSSYA (SEQ ID NO:46) H CDR1 V comprising the amino acid sequence IIPIFGTA (SEQ ID NO:47) H CDR2, and V containing the amino acid sequence ARGPNT (SEQ ID NO:48) H CDR3 Wherein the CDRs are numbered according to IMGT.

6. The CAR according to claim 4 or claim 5, wherein the single-chain antibody comprises: a variable light chain (V L ) polypeptide, which comprises an amino acid sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO:

43.

7. The CAR according to any one of claims 4 to 6, wherein the single-chain antibody comprises: a variable heavy chain (V H ) polypeptide comprising an amino acid sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher or 100% identity to the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 20 or SEQ ID NO:

45.

8. The CAR according to any one of claims 1 to 7, wherein the transmembrane domain comprises a CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD35, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 or PD-1 transmembrane domain.

9. The CAR according to any one of claims 1 to 8, further comprising a hinge domain disposed between the extracellular GPNMB binding domain and the transmembrane domain.

10. The CAR according to claim 9, wherein the hinge domain comprises a CD8α hinge domain, a CD28 hinge domain, an IgG4(CH3) hinge domain, a PD-1 hinge domain or a CD152 hinge domain.

11. The CAR according to any one of claims 1 to 10, wherein the one or more intracellular signaling domains comprise a CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, CD66δ, FcRγ or FcRβ primary signaling domain.

12. The CAR according to any one of claims 1 to 11, wherein the one or more intracellular signaling domains comprise one or more co-stimulatory domains.

13. The CAR according to claim 12, wherein the one or more co-stimulatory domains comprise a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD278(ICOS), DAP10, LAT, KD2C, SLP76, TRIM or ZAP70 co-stimulatory domain.

14. A nucleic acid encoding the CAR according to any one of claims 1 to 13.

15. An expression construct comprising the nucleic acid according to claim 14 operably linked to a promoter.

16. A cell comprising the expression construct according to claim 15, wherein the cell expresses the CAR on its surface.

17. The cell according to claim 16, wherein the cell is a human cell.

18. The cell according to claim 16 or claim 17, wherein the cell is an immune cell.

19. The cell according to claim 18, wherein the cell is a T cell.

20. The cell according to claim 18, wherein the cell is a natural killer (NK) cell or a macrophage.

21. The cell according to claim 16 or claim 17, wherein the cell is a stem cell.

22. The cell according to claim 21, wherein the cell is a hematopoietic stem cell (HSC).

23. The cell according to claim 21, wherein the cell is an induced pluripotent stem cell or a derivative thereof.

24. A composition comprising a population of cells as defined in any one of claims 16 to 23.

25. The composition according to claim 24, wherein the composition is formulated for administration to a subject in need thereof.

26. A method of treating a condition associated with GPNMB expression and / or activity in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition according to claim 25.

27. The method according to claim 26, wherein the condition associated with GPNMB expression and / or activity is cancer.

28. The method according to claim 27, wherein the cancer is characterized by cancer cells that express GPNMB (GPNMB+).

29. The method according to claim 27 or 28, wherein the cancer is carcinoma, lymphoma, blastoma or sarcoma.

30. The method according to claim 29, wherein the sarcoma is soft tissue sarcoma (STS).

31. The method according to claim 30, wherein the STS is alveolar soft part sarcoma (ASPS).

32. The method according to any one of claims 27 to 29, wherein the cancer is renal cell carcinoma (RCC), breast cancer or melanoma.

33. The method according to claim 32, wherein the RCC is translocation RCC (tRCC) or TSC / mTOR-altered RCC (Figure 14a, b; Salles et al. (2022) Journal of Pathology 257(2):158-171).

34. The method according to claim 32, wherein the breast cancer is GPNMB+ triple-negative breast cancer (TNBC).

35. The method according to any one of claims 27 to 29, wherein the cancer comprises a fusion protein involving a transcription factor of the microphthalmia family (MITF, TFEB, TFE3 or TFEC) (Figure 14a, b; Salles et al. (2022) Journal of Pathology 257(2):158-171).

36. The method according to any one of claims 27 to 29, wherein the cancer is characterized by the activation of one or more microphthalmia-associated transcription factors (MITF, TFEB, TFE3, and TFEC) (Figure 14a, b; Salles et al. (2022) Journal of Pathology 257(2):158-171; Hong et al. (2010) PLoS One 5(12):e15793).

37. The method according to any one of claims 27 to 36, wherein the subject has metastatic cancer.

38. The method according to claim 37, wherein the subject has central nervous system (CNS) metastases.

39. The method according to claim 27, wherein the cancer cells of the cancer are GPNMB-negative, and wherein the cancer comprises a tumor microenvironment containing GPNMB-positive cells.

40. The method according to claim 39, wherein the GPNMB-positive cells in the tumor microenvironment are macrophages, fibroblasts, or a combination thereof.

41. The method according to claim 26, wherein the condition associated with GPNMB expression and / or activity is a neurodegenerative disease.

42. The method according to claim 41, wherein the neurodegenerative disease is Alzheimer's disease (AD), Gaucher's disease, Niemann-Pick type C disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), or a neurodegenerative disease associated with amyloidosis.

43. The method according to any one of claims 26 to 42, wherein the composition is administered parenterally.

44. The method according to claim 43, wherein the composition is administered intravenously.

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