Chimeric Antigen Receptor Domain

Abstract

This application describes novel chimeric antigen receptor (CAR) constructs comprising novel spacer region sequences. Also provided are cells comprising the CARs, a nucleic acid or nucleic acids encoding the CARs, an expression vector or vectors comprising the nucleic acids, methods of making such molecules, and the use of such molecules in methods of drug treatment or prophylaxis.

Description

[Technical Field]

[0001] The present disclosure relates to the field of molecular biology, more specifically to chimeric antigen receptor (CAR) technology. The present disclosure also relates to methods of medical treatment and prevention. [Background technology]

[0002] The development of chimeric antigen receptor (CAR) T-cell therapy has significantly transformed the treatment of patients with certain B-cell leukemias and lymphomas (7), providing effective and durable clinical responses by utilizing T cells engineered to eliminate target cells expressing non-major histocompatibility complex (MHC)-restricted antigens. CARs are synthetic receptors that contain four major domains: an antigen-specific domain, typically a single-chain variable fragment (scFv) of an antibody, a spacer domain that connects the scFv to the third domain or transmembrane domain of the receptor, and finally, a signaling domain (8). The design and engineering of CAR receptors are critical to the efficacy and safety of CAR T-cell strategies.

[0003] The spacer, or hinge, domain plays a crucial role in CAR function, providing accessibility and flexibility to the scFv. Spacer flexibility allows the scFv to reach epitopes that would otherwise be sterically hindered, while the spacer length ensures optimal synaptic distance between the CAR T cell and the target cell for efficient delivery of granzymes and perforin for potent cytotoxicity (8). The most commonly used spacer domains are derived from antibody Fc domains, namely IgG1, IgG2, and IgG4-Fc, CD8α, and CD28. IgG-derived spacers can interact with Fc receptors on other immune cells, thus triggering the generation of an enhanced immune response against CAR-expressing T cells. Furthermore, this interaction can also lead to non-antigen-specific activation of CAR T cells, resulting in increased cytokine release, exhaustion, and activation of induced cell death (11). Alternatively, CARs possessing spacers derived from CD8α and CD28 have been shown to be highly effective and have been clinically approved for CAR T cell therapy. Furthermore, CARs with a CD28 spacer have been shown to lower the activation threshold of CD19 CAR T cells compared with those with a CD8α spacer. (12) CARs with a CD28 and CD8α spacer have recently been shown to promote heterodimerization of endogenous CD28, and with CARs containing CD28 transmembrane and endodomains (13), this has been shown to generate stronger activation signals that could potentially lower the threshold for CAR T activation and increase the risk of CAR T therapy-related toxicities, such as cytokine release syndrome and neurotoxicity. (14) Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (i) an antigen-binding domain that specifically binds to a target antigen; (ii) a spacer domain comprising, or consisting of, an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 100, 99, 101, and 102; (iii) a transmembrane domain; and (iv) a signaling domain comprising an amino acid sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).

[0005] In some embodiments, the CAR comprises a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 100. In some embodiments, the antigen-binding domain specifically binds to a target antigen selected from CD30, CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0006] In some embodiments, the antigen-binding domain specifically binds to CD30. In some aspects, the antigen binding domain comprises: (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2 HC-CDR2 having the amino acid sequence of SEQ ID NO: 3 HC-CDR3 having the amino acid sequence of SEQ ID NO: 4 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 10 LC-CDR2 having the amino acid sequence of SEQ ID NO: 11 LC-CDR3 having the amino acid sequence of SEQ ID NO: 12 Light chain variable (VL) region incorporating Includes:

[0007] In some aspects, the antigen binding domain comprises: a VH region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 1, 45, 52, 17, 21, 24, 26, and 28; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 9, 49, 54, 30, 35, 38, 41, 43, 147, 148, 149, 150, 151, or 152. Includes:

[0008] In some aspects, the antigen binding domain comprises: (i) a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 1, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 9; or (ii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 54; or (iii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 30; or (iv) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 21, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 35; or (v) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 38; or (vi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 41; or (vii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 43; or (viii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 147; or (ix) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 148; or (x) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 21, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 149; or (xi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 150; or (xii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 151; or (xiii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 152. Includes:

[0009] In some embodiments, the antigen-binding domain is or comprises a single-chain variable fragment (scFv) comprising a VH region and a VL region. In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 103, 104 or 105.

[0010] In some embodiments, the signaling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 106, 107, or 108. In some embodiments, the signaling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:111.

[0011] In some embodiments, the CAR is selected from the group consisting of SEQ ID NOs: 124, 144, 190, 210, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 145, 146, 183, 184, 185, 186, 187, 188, 189, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 85, 186, 187, 188, 189, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 211 or 212.

[0012] The present disclosure also provides a nucleic acid or nucleic acids, optionally isolated, encoding a CAR according to the present disclosure. The present disclosure also provides an expression vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure.

[0013] The present disclosure also provides a cell comprising a CAR, a nucleic acid or nucleic acids, or an expression vector or expression vectors according to the present disclosure. The present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for expression of a CAR by the cell.

[0014] The present disclosure also provides compositions comprising a CAR, a nucleic acid or nucleic acids, an expression vector or vectors, or a cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0015] The present disclosure also provides a CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition according to the present disclosure for use in a method of medical therapy or prophylaxis.

[0016] The present disclosure also provides a CAR, nucleic acid or nucleic acids, expression vector or vectors, cell, or composition according to the present disclosure for use in treating or preventing cancer.

[0017] In some embodiments, the cancer is a cancer that expresses a target antigen for the CAR, a CD30 positive cancer, an EBV-associated cancer, a hematological cancer, a myeloid hematological malignancy, a hematopoietic malignancy, a lymphoblastic hematological malignancy, a myelodysplastic syndrome, a leukemia, a T-cell leukemia, an acute myeloid leukemia, a chronic myeloid leukemia, an acute lymphoblastic leukemia, a lymphoma, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, a B-cell non-Hodgkin's lymphoma, a diffuse large B-cell lymphoma, a myelodysplastic syndrome ... Follicular lymphoma, primary mediastinal B-cell lymphoma, EBV-associated lymphoma, EBV-positive B-cell lymphoma, EBV-positive diffuse large B-cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection / AIDS, oral hairy leukoplakia, Burkitt lymphoma, post-transplant lymphoproliferative disorder, central nervous system lymphoma, anaplastic large cell lymphoma, T-cell lymphoma, AL ALK-positive anaplastic T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, NK-T-cell lymphoma, extranodal NK-T-cell lymphoma, thymoma, multiple myeloma, solid tumors, epithelial cell carcinoma, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, oropharyngeal cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, The cancer is selected from the group consisting of colorectal cancer, colon cancer, cervical cancer, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer, ovarian cancer, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor, or testicular embryonal carcinoma.

[0018] The present disclosure also provides a CAR, nucleic acid or nucleic acids, expression vector or vectors, cell, or composition according to the present disclosure for use in treating or preventing a disease or condition characterized by an alloreactive immune response.

[0019] In some aspects, the disease or condition characterized by an alloreactive immune response is selected from the group consisting of a disease or condition associated with allogeneic transplantation, graft-versus-host disease (GVHD), or graft rejection.

[0020] The present disclosure also provides for the use of a CAR, nucleic acid or nucleic acids, expression vector or vectors, cell, or composition according to the present disclosure to deplete or increase killing of cells that express a target antigen for the CAR.

[0021] The present disclosure also provides in vitro complexes, optionally isolated, comprising a CAR or cell according to the present disclosure bound to a target antigen for the CAR. DETAILED DESCRIPTION OF THE INVENTION

[0022] explanation The present disclosure provides novel chimeric antigen receptor (CAR) constructs that comprise novel spacer region sequences.

[0023] Unexpectedly, T cells expressing the novel CAR constructs are shown to expand / proliferate similarly, have an improved safety profile, exhibit similar cytotoxicity to cells expressing the target antigen for the CAR, have improved persistence in vivo, and exhibit improved tumor growth inhibition of cancers containing cells expressing the target antigen for the CAR when compared to equivalent CAR constructs that instead have the known IgG1 CH2-CH3 spacer region. Chimeric antigen receptor (CAR) Aspects of the present disclosure relate to chimeric antigen receptors (CARs). CARs are recombinant receptors that provide both antigen binding and T cell activation functions. The structure and operation of CARs are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1), the entire contents of which are incorporated herein by reference. CARs comprise an antigen-binding domain linked to a signaling domain via a transmembrane domain. An optional hinge or spacer domain can separate the antigen-binding domain and the transmembrane domain and act as a flexible linker. When expressed by a cell, the antigen-binding domain is provided in the extracellular space, and the signaling domain is intracellular.

[0024] The antigen-binding domain mediates binding of the CAR to its specific target antigen. The antigen-binding domain of the CAR may be based on the antigen-binding region of an antibody specific for the antigen to which the CAR is targeted. For example, the antigen-binding domain of the CAR may comprise the amino acid sequence of the complementarity-determining region (CDR) of an antibody that specifically binds to the target antigen. The antigen-binding domain of the CAR may comprise or consist of the light and heavy chain variable region amino acid sequences of an antibody that specifically binds to the target antigen. The antigen-binding domain may be provided as a single-chain variable fragment (scFv) comprising the light and heavy chain variable region amino acid sequences of the antibody. The antigen-binding domain of the CAR may also target antigens based on other protein:protein interactions, such as ligand:receptor binding; for example, an IL-13Rα2-targeting CAR was developed using an antigen-binding domain based on IL-13 (see, e.g., Kahlon et al., 2004 Cancer Res 64(24):9160-9166).

[0025] The spacer domain provides separation between the antigen-binding domain and the transmembrane domain and can act as a flexible linker. Such domains can be or include flexible regions that allow the binding moieties to be oriented in various directions. The spacer domain is sometimes derived from the constant region of an immunoglobulin molecule.

[0026] The transmembrane domain is provided between the antigen binding domain and the signal transduction domain of CAR.The transmembrane domain, together with the antigen binding domain in the extracellular space and the signal transduction domain inside the cell, causes the CAR to be fixed to the cell membrane of the cell that expresses CAR.The transmembrane domain of CAR can be derived from the transmembrane region sequence for cell membrane-associated proteins (such as CD28, CD8, CD4, CD3-ζ, etc.).

[0027] The signaling domain contains an amino acid sequence required for activation of immune cell function. The CAR signaling domain may contain the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of cells expressing the CAR. Signaling domains containing sequences from other ITAM-containing proteins have also been employed in CARs, such as the ITAM-containing domain containing the FcγRI region (Haynes et al., 2001 J Immunol 166(1):182-187). CARs containing a signaling domain derived from the intracellular domain of CD3-ζ are often referred to as first-generation CARs.

[0028] The signaling domain of a CAR typically also contains the signaling domain of a costimulatory protein (e.g., CD28, 4-1BB, etc.) to provide the costimulatory signal necessary to enhance immune cell activation and effector function. CARs with signaling domains containing additional costimulatory sequences are often referred to as second-generation CARs. In some cases, CARs are engineered to generate costimulation of different intracellular signaling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1BB costimulation initiates signaling through TNF receptor-associated factor (TRAF) adaptor proteins. Therefore, the signaling domain of a CAR sometimes contains costimulatory sequences from the signaling domains of more than one costimulatory molecule. CARs containing signaling domains with multiple costimulatory sequences are often referred to as third-generation CARs.

[0029] Throughout this specification, polypeptides, domains and amino acid sequences "derived from" a reference polypeptide / domain / amino acid sequence have at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of the reference polypeptide / domain / amino acid sequence. Polypeptides, domains and amino acid sequences "derived from" a reference polypeptide / domain / amino acid sequence preferably retain functional and / or structural properties of the reference polypeptide / domain / amino acid sequence.

[0030] By way of example, an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the intracellular domain of CD28, for example as set forth in SEQ ID NO: 106. Furthermore, the amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional property of the amino acid sequence of SEQ ID NO: 106, i.e., the ability to activate signaling mediated by CD28.

[0031] The amino acid sequence of a given polypeptide or domain thereof may be retrieved from, or determined from, nucleic acid sequences retrieved from, databases known to those of skill in the art, such as GenBank, EMBL, and UniProt.

[0032] By engineering them to express CARs specific for a particular target antigen, immune cells (typically T cells, but also other immune cells, such as NK cells) can be directed to kill cells expressing the target antigen. Binding of a CAR-expressing T cell (CAR-T cell) to its specific target antigen initiates intracellular signaling, resulting in T cell activation. Activated CAR-T cells are stimulated to divide and produce factors that result in the killing of cells expressing the target antigen. antigen-binding domain An "antigen-binding domain" refers to a domain capable of binding to a target antigen. The target antigen may be, for example, a peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. An antigen-binding domain according to the present disclosure may be derived from an antibody (i.e., immunoglobulin (Ig)) and its antigen-binding fragment. As used herein, "antibody" encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as antigen-binding molecules derived from antibodies, such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, and single-domain antibodies (e.g., VhH). Antigen-binding fragments of antibodies include, for example, Fv, Fab, F(ab')2, and F(ab')2 fragments. In some embodiments, an antigen-binding domain according to the present disclosure may comprise or consist of an antibody or an antigen-binding fragment thereof.

[0033] In some embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specifically binding to a target antigen. The antigen-binding domain formed by the VH and VL may also be referred to herein as Fv.

[0034] In some embodiments, the domain capable of binding to a target antigen comprises or consists of an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e., single domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin, e.g., as reviewed in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0035] The antigen-binding domain of the present disclosure generally comprises the VH and VL of an antibody capable of specifically binding to a target antigen. An antibody generally comprises six complementarity-determining regions (CDRs): three in the heavy chain variable region (VH): HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable region (VL): LC-CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the antibody paratope, which is the part of the antibody that binds to the target antigen. The VH and VL regions comprise framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and VL comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0036] The VH and VL sequences can be provided in any suitable format where the antigen-binding domain can be linked to other domains of the CAR. Formats contemplated for the antigen-binding domains of the present disclosure include those described in Carter, Nat. Rev. Immunol (2006), 6:343-357, such as scFv, dsFv, (scFv)2, diabody, triabody, tetrabody, Fab, minibody, and F(ab)2 formats.

[0037] In some embodiments, the antigen-binding domain comprises the CDRs of an antibody / antibody fragment capable of binding to a target antigen. In some embodiments, the antigen-binding domain comprises the VH and VL regions of an antibody / antibody fragment capable of binding to a target antigen. The portion of an antibody consisting of the VH and VL may also be referred to herein as a variable fragment (Fv). The VH and VL may be provided on the same polypeptide chain and connected via a linker sequence; such a portion is referred to as a single-chain variable fragment (scFv). Linker sequences suitable for preparing scFvs are known to those skilled in the art and may contain serine and glycine residues.

[0038] In some embodiments, the antigen-binding domain comprises or consists of an Fv capable of binding to the target antigen. The antigen-binding domain can be provided in any suitable format, such as an scFv, scFab, etc. In some embodiments, the antigen-binding domain comprises or consists of an scFv capable of binding to the target antigen.

[0039] The target antigen for which the antigen-binding domain is specific (and therefore the CAR) can be any target antigen. In some embodiments, the target antigen is an antigen whose expression / activity, or its upregulated expression / activity, is positively associated with a disease or disorder (e.g., cancer, infectious disease, or autoimmune disease). The target antigen is preferably expressed on the cell surface of the cell that expresses the target antigen. It will be understood that the CAR induces the effector activity of the cell that expresses the CAR against the cell / tissue that expresses the target antigen for which the CAR contains the specific antigen-binding domain.

[0040] In some embodiments, the target antigen may be a cancer cell antigen. A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of the cancer cell antigen may be associated with cancer. A cancer cell antigen may be aberrantly expressed by cancer cells (e.g., the cancer cell antigen may be expressed with abnormal localization) or may be expressed in an abnormal structure by cancer cells. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen that binds to the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e., is extracellular). A cancer cell antigen may be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of cancer symptoms. Cancer-associated antigens may be associated with the cause or pathology of cancer or may be aberrantly expressed as a result of cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells, for example, when compared with the expression level by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer-associated antigens may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer-associated antigens may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, cancer-associated antigens may be the product of an overexpressed cellular protein, a cancer antigen produced by a tumor virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0041] Cancer cell antigens are reviewed in Zarour HM, DeLeo A, Finn OJ, et al.: Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors, Holland-Frei Cancer Medicine. 6th ed. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include carcinoembryonic antigen (CEA), immature laminin receptor, TAG-72, and HPV. Oncoviral antigens such as E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; mutated antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; post-translationally modified antigens: MUC1; idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetoacinar pancreatic protein (FAPP), alkaline phosphatase placenta-like 2 (ALPPL-2), Siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.

[0042] In some embodiments, the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10:2250, which is incorporated by reference in its entirety. In some embodiments, the cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0043] In some embodiments, the cancer cell antigen is an antigen expressed by cells of hematological malignancies. In some embodiments, the cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, and BCMA. In some embodiments, the cancer cell antigen is an antigen expressed by cells of solid tumors. In some embodiments, the cancer cell antigen is selected from mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0044] In some embodiments, the antigen-binding domain (and thus the CAR) is multispecific. "Multispecific" means that the antigen-binding domain exhibits specific binding to more than one target. In some embodiments, the antigen-binding domain is a bispecific antigen-binding domain. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding moieties (i.e., at least two antigen-binding moieties, for example, comprising non-identical VH and VL). The individual antigen-binding sites of the multispecific antigen-binding domain may be linked, for example, via a linker sequence.

[0045] In some embodiments, the antigen-binding domain binds to at least two non-identical target antigens, and is therefore at least bispecific. The term "bispecific" means that the antigen-binding domain is capable of specifically binding to at least two distinct antigenic determinants. In some embodiments, at least one of the target antigens for the multispecific antigen-binding domain / CAR is CD30.

[0046] Each target antigen may independently be a target antigen as described herein. In some aspects, each target antigen is independently a cancer cell antigen as described herein.

[0047] It will be understood that an antigen-binding domain (e.g., a multispecific antigen-binding domain) according to the present disclosure comprises an antigen-binding portion capable of binding to the target(s) for which the antigen-binding domain is specific. For example, an antigen-binding domain capable of binding to CD30 and an antigen other than CD30 may comprise (i) an antigen-binding portion capable of binding to CD30, and (ii) an antigen-binding portion capable of binding to a target antigen other than CD30.

[0048] In aspects and embodiments of the present disclosure, the target antigen is CD30. Thus, in some aspects and embodiments of the present disclosure, the antigen-binding domain is a CD30-binding domain. CD30 (also known as TNFRSF8) is a protein identified at UniProt:P28908. CD30 is a type I transmembrane glycoprotein of the single-pass tumor necrosis factor receptor superfamily. The structure and function of CD30 are described, for example, in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack Immunol. Res. (2013) 57(1-3):151-8, both of which are incorporated herein by reference in their entirety.

[0049] Alternative splicing of the mRNA encoded by the human TNFRSF8 gene generates three isoforms: isoform 1 (the "long" isoform; UniProt: P28908-1, v1; SEQ ID NO: 87), isoform 2 (the "cytoplasmic", "short" or "C30V" isoform, UniProt: P28908-2; SEQ ID NO: 88) (which lacks the amino acid sequence corresponding to positions 1 to 463 of SEQ ID NO: 87), and isoform 3 (UniProt: P28908-3; SEQ ID NO: 89) (which lacks the amino acid sequence corresponding to positions 1 to 111 and 446 of SEQ ID NO: 87). The N-terminal 18 amino acids of SEQ ID NO:87 form a signal peptide (SEQ ID NO:90), followed by a 367 amino acid extracellular domain (positions 19 to 385 of SEQ ID NO:87, shown in SEQ ID NO:91), a 21 amino acid transmembrane domain (positions 386 to 406 of SEQ ID NO:87, shown in SEQ ID NO:92), and a 189 amino acid cytoplasmic domain (positions 407 to 595 of SEQ ID NO:87, shown in SEQ ID NO:93).

[0050] As used herein, "CD30" refers to CD30 from any species and encompasses CD30 isoforms, fragments, variants, or homologs from any species. As used herein, a "fragment," "variant," or "homolog" of a reference protein may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of the reference protein (e.g., a reference isoform). In some embodiments, fragments, variants, isoforms, and homologs of a reference protein may be characterized by their ability to perform a function performed by the reference protein.

[0051] In some embodiments, the CD30 is from a mammal (e.g., a primate (rhesus, cynomolgus, or human) and / or rodent (e.g., rat or mouse) CD30). In preferred embodiments, the CD30 is human CD30. Isoforms, fragments, variants, or homologs may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature CD30 isoform from a given species, e.g., human. A fragment of CD30 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 590 amino acids and a maximum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 595 amino acids.

[0052] In some embodiments, CD30 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 87, 88, 89 or 91.

[0053] In some embodiments, CD30 comprises an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 92 or 95. In some embodiments, a fragment of CD30 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 92 or 95.

[0054] The CD30 binding domain of the CAR of the present disclosure preferably exhibits specific binding to CD30 or a fragment thereof. The CD30 binding domain of the CAR of the present disclosure preferably exhibits specific binding to the extracellular domain of CD30. The CD30 binding domain can be derived from an anti-CD30 antibody or other CD30 binding agent, such as a CD30-binding peptide or a CD30-binding small molecule.

[0055] The CD30 binding domain may be derived from the antigen-binding portion of an anti-CD30 antibody. Anti-CD30 antibodies include HRS3 and HRS4 (described, for example, in Hombach et al., Scand J Immunol (1998) 48(5):497-501), the HRS3 derivative described in Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12):847-860, BerH2 (MBL International Cat# K0145-3, RRID:AB_590975), SGN-30 (also known as cAC10, described, for example, in Forero-Torres et al., Br J Haematol (2009) 146:171-9), MDX-060 (described, for example, in Ansell et al., J Clin Oncol (2007) 25:2764-9; 5F11, also known as iratumumab), and MDX-1401 (described, for example, in Cardarelli et al., Clin Cancer Res. (2009) 15(10):3376-83), and the anti-CD30 antibodies described in WO2020 / 068764 A1, WO2003 / 059282 A2, WO 2006 / 089232 A2, WO 2007 / 084672 A2, WO 2007 / 044616 A2, WO 2005 / 001038 A2, US 2007 / 166309 A1, US 2007 / 258987 A1, WO 2004 / 010957 A2, and US 2005 / 009769 A1.

[0056] In some embodiments, a CD30 binding domain according to the present disclosure comprises the CDRs of an anti-CD30 antibody. In some embodiments, a CD30 binding domain according to the present disclosure comprises the VH and VL regions of an anti-CD30 antibody. In some embodiments, a CD30 binding domain according to the present disclosure comprises an scFv comprising the VH and VL regions of an anti-CD30 antibody.

[0057] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and Retter et al., Nucl. Acids Res. (2005) 33 (Suppl. 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database Edition): D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0058] In some embodiments, the antigen-binding domain comprises a polypeptide(s) comprising (i) a VH region comprising HC-CDR1, HC-CDR2, and HC-CDR3 as set forth in column A of Table A, and (ii) a VL region comprising LC-CDR1, LC-CDR2, and LC-CDR3 as set forth in column B of Table A, wherein the sequences in column A and column B are selected from the same row of Table A. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide(s) comprising (i) a VH region comprising HC-CDR1=SEQ ID NO:2, HC-CDR2=SEQ ID NO:3, and HC-CDR3=SEQ ID NO:4, and (ii) a VL region comprising LC-CDR1=SEQ ID NO:10, LC-CDR2=SEQ ID NO:11, and LC-CDR3=SEQ ID NO:12.

[0059] In some embodiments, the antigen-binding domain is selected from the group consisting of HRS3, VH1VK1, VH1VK2, VH1VK3, VH1VK4, VH1VK5, VH2VK1, VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1, VH3VK2, VH3VK3, VH3VK4, VH3 ... K5, VH4VK1, VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1, VH5VK2, VH5VK3, VH5VK4, VH5VK 5, HRS3Cys, VH1VK1Cys, VH1VK2Cys, VH1VK3Cys, VH1VK4Cys, VH1VK5Cys, VH2VK1Cys, V The antibody comprises a polypeptide(s) comprising a VH region comprising a heavy chain FR, and a VL region comprising a light chain FR, of an antibody selected from H2VK2Cys, VH2VK3Cys, VH2VK4Cys, VH2VK5Cys, VH3VK1Cys, VH3VK2Cys, VH3VK3Cys, VH3VK4Cys, VH3VK5Cys, VH4VK1Cys, VH4VK2Cys, VH4VK3Cys, VH4VK4Cys, VH4VK5Cys, VH5VK1Cys, VH5VK2Cys, VH5VK3Cys, VH5VK4Cys, VH5VK5Cys, VH1-5ConVK1-3Con, or VH3-5ConVK2-3Con. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide(s) comprising: (i) a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as shown in column A of Table B; and (ii) a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as shown in column B of Table B, wherein the sequences in columns A and B are selected from the same row of Table B.

[0060] In some aspects, the antigen binding domain comprises: a VH comprising or consisting of an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO: 1, 45, 52, 17, 21, 24, 26, 28; and a VL comprising or consisting of an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO: 9, 49, 54, 30, 35, 38, 41, 43, 147, 148, 149, 150, 151 or 152; Includes:

[0061] In some embodiments, the antigen-binding domain comprises a polypeptide(s) comprising: (i) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence as shown in column A of Table C; and (ii) an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence as shown in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0062] In some aspects, the antigen binding domain comprises: a VH region having an amino acid sequence that has at least 70% amino acid sequence identity with SEQ ID NO: 1, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity with SEQ ID NO: 9; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 45, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 49; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 54; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 17, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 30; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 21, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 35; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 38; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 26, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 41; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 28, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 43; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 1, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 147; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 17, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 148; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 21, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 149; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 150; or a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 26, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 151; or a VH region having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID NO: 28, and a VL region having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID NO: 152. Includes:

[0063] In some embodiments, the antigen-binding domain is selected from the group consisting of HRS3, VH1VK1, VH1VK2, VH1VK3, VH1VK4, VH1VK5, VH2VK1, VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1, VH3VK2, VH3VK3, VH3VK4, VH3 ... VH3VK5, VH4VK1, VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1, VH5VK2, VH5VK3, VH5VK4 , VH5VK5, HRS3Cys, VH1VK1Cys, VH1VK2Cys, VH1VK3Cys, VH1VK4Cys, VH1VK5Cys, VH2 The polypeptides include a polypeptide(s) comprising the VH and VL regions of an antibody selected from VK1Cys, VH2VK2Cys, VH2VK3Cys, VH2VK4Cys, VH2VK5Cys, VH3VK1Cys, VH3VK2Cys, VH3VK3Cys, VH3VK4Cys, VH3VK5Cys, VH4VK1Cys, VH4VK2Cys, VH4VK3Cys, VH4VK4Cys, VH4VK5Cys, VH5VK1Cys, VH5VK2Cys, VH5VK3Cys, VH5VK4Cys, VH5VK5Cys, VH1-5ConVK1-3Con or VH3-5ConVK2-3Con. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide(s) comprising (i) the amino acid sequence set forth in column A of Table C and (ii) the amino acid sequence set forth in column B of Table C, wherein the sequences in columns A and B are selected from the same row of Table C.

[0064] In some embodiments, the antigen binding domain is selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 23 71, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182.

[0065] In some embodiments, the antigen-binding domain does not comprise or consist of a polypeptide having the amino acid sequence of SEQ ID NO:59. In some aspects, a VH region according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 1. In some aspects, a VL region according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 9.

[0066] In some aspects, a VH region according to the present disclosure does not include an HC-FR1 having the amino acid sequence of SEQ ID NO: 5. In some aspects, a VH region does not include an HC-FR2 having the amino acid sequence of SEQ ID NO: 6. In some aspects, a VH region does not include an HC-FR3 having the amino acid sequence of SEQ ID NO: 7.

[0067] In some aspects, a VL region according to the present disclosure does not comprise LC-FR1 having the amino acid sequence of SEQ ID NO: 13. In some aspects, a VL region does not comprise LC-FR2 having the amino acid sequence of SEQ ID NO: 14. In some aspects, a VL region does not comprise LC-FR3 having the amino acid sequence of SEQ ID NO: 15.

[0068] In some embodiments, the antigen-binding domain comprises or consists of a CD30-binding Fv (i.e., a VH and VL pair) as described herein. In some embodiments, the antigen-binding domain comprises or consists of a CD30-binding Fv in which the VH and VL are covalently linked. In some embodiments, the VH and VL of the CD30-binding Fv sequence are linked by a flexible linker sequence, for example, a flexible linker sequence as described herein. The flexible linker sequence may be attached to the termini of the VH sequence and the VL sequence, thereby linking the VH sequence and the VL sequence.

[0069] In some embodiments, the CD30 binding domain may comprise or consist of a single-chain variable fragment (scFv) comprising a VH sequence and a VL sequence as described herein. The VH sequence and the VL sequence may be covalently linked. In some embodiments, the VH sequence and the VL sequence are linked by a flexible linker sequence, for example, a flexible linker sequence as described herein. The flexible linker sequence may be attached to the termini of the VH sequence and the VL sequence, thereby linking the VH sequence and the VL sequence.

[0070] In some embodiments, the VH and VL are joined via a linker sequence comprising one or more copies of the amino acid sequence according to SEQ ID NO: 57. In some embodiments, the linker sequence comprises at least 1, 2, 3, or 4 copies of the amino acid sequence according to SEQ ID NO: 57. In some embodiments, the VH and VL are joined via a linker sequence comprising or consisting of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 58.

[0071] In some aspects, the antigen-binding molecule is capable of binding to an epitope of CD30 to which the antibody HRS3 binds within the region of amino acids 185-335 of human CD30 numbered according to SEQ ID NO:87, e.g., as set forth in SEQ ID NO:95 (Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12):847-860, incorporated herein by reference in its entirety). In some embodiments, the antigen-binding domain is selected from the group consisting of HRS3, VH1VK1, VH1VK2, VH1VK3, VH1VK4, VH1VK5, VH2VK1, VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1, VH3VK2, VH3VK3, VH3VK4, VH3VK5, VH4VK1, VH4VK2 , VH4VK3, VH4VK4, VH4VK5, VH5VK1, VH5VK2, VH5VK3, VH5VK4, VH5VK5, HRS3Cys, VH1VK1C ys, VH1VK2Cys, VH1VK3Cys, VH1VK4Cys, VH1VK5Cys, VH2VK1Cys, VH2VK2Cys, VH2VK3Cys, The region of CD30 bound by an antibody comprising the VH and VL regions of one of VH2VK4Cys, VH2VK5Cys, VH3VK1Cys, VH3VK2Cys, VH3VK3Cys, VH3VK4Cys, VH3VK5Cys, VH4VK1Cys, VH4VK2Cys, VH4VK3Cys, VH4VK4Cys, VH4VK5Cys, VH5VK1Cys, VH5VK2Cys, VH5VK3Cys, VH5VK4Cys, VH5VK5Cys, VH1-5ConVK1-3Con or VH3-5ConVK2-3Con (see, e.g., Table C) can bind to the same region of CD30 or overlapping regions of CD30. spacer domain In some embodiments, CAR comprises a spacer domain. The spacer domain may be provided between the antigen binding domain and the transmembrane domain. The spacer domain may also be referred to as a hinge domain. The spacer domain is an amino acid sequence that provides a flexible connection between the antigen binding domain and the transmembrane domain of CAR.

[0072] The presence, absence, and length of a spacer domain have been shown to affect CAR function (e.g., Dotti et al., Immunol Rev (2014) 257(1) and Jayaraman et al., EBioMedicine (2020) 58:102931, reviewed above). Spacer domains include those derived from the CH2-CH3 region of human IgG1 (e.g., as set forth in SEQ ID NO: 96), the CH2-CH3 region of human IgG1 (e.g., as set forth in SEQ ID NO: 98), the CH1-CH2 hinge region of human IgG1, CD8α, as described, for example, in WO2012 / 031744 A1, and CD28, as described, for example, in WO2011 / 041093 A1. Hombach et al., Gene Therapy (2010) 17:1206-1213, describe a variant hIgG1 CH2-CH3 spacer region for reduced activation of FcγR-expressing cells such as monocytes and NK cells. The amino acid sequence of the variant CH2-CH3 region is shown in SEQ ID NO: 97.

[0073] The spacer domain can provide flexibility to the antigen-binding domain, thus enabling recognition of target epitopes that are otherwise sterically inaccessible. For example, Wilkie et al., J Immunol. (2008) 180:4901-4909, replaced the spacer region of CD28 with the highly flexible IgD spacer, improving binding of CAR to the sterically hindered epitope of MUC1.

[0074] Spacer domains can also be engineered to regulate synaptic cleft distance and thus modulate signal transduction. To maintain optimal synaptic distance, membrane-proximal epitopes typically require shorter spacers, while membrane-distal epitopes require longer spacers (Hudecek et al., Clin Cancer Res. (2013) 19:3153-3164). Increasing the epitope-paratope distance can allow interference by inhibitory phosphatases, potentially impairing the delivery of perforin and granzymes to target cells, thereby reducing the efficiency of cell lysis. A smaller immune synapse reduces the diffusion of lytic granules and enhances perforin pore formation and granzyme delivery (Woodsworth et al., Biophys J. (2015) 109:477-488).

[0075] Spacer length has been shown to affect cytolytic activity and signal transduction by CAR-T cells. The introduction of an IgG1-Fc spacer into a first-generation anti-CEA CAR was found to reduce IFNγ secretion without affecting lytic efficiency (Guest et al., J Immunother. (2005) 28:203-211). Spacer length has also been shown to affect mechanotransduction of ligand recognition. Chang et al., Nat Chem Biol. (2018) 14:317-324, found that cells expressing a soluble homodimeric TGF-β-specific CAR with a longer IgG4-Fc spacer had a reduced activation profile compared to cells expressing a CAR with a shorter IgG4 hinge-only spacer.

[0076] If T cells are overactivated, the persistence and function of CAR-T cells in vivo may be impaired due to activation-induced cell death (AICD) (Kuunkele et al., Cancer Immunol. Res. (2015) 3:368-379). Excessive activation of CAR-T cells may also contribute to unwanted side effects such as cytokine release syndrome. Engineering the spacer domain may be useful for fine-tuning the activation of CAR-expressing immune cells to optimal levels.

[0077] Human IgG1 Fc, IgG4 Fc, and IgG2 Fc spacer domains have been used in CARs, and variants of the IgG1 Fc and IgG4 Fc spacers designed to reduce / eliminate binding to FcγR have been shown to reduce depletion of CAR-expressing cells and improve persistence in vivo.

[0078] Non-IgG Fc-based spacers, such as CD8 and CD28 spacer domains, have also been used in clinically approved CAR T cell therapies. CD28-derived spacers have been shown to result in increased CAR activation compared to CD8α-derived spacers, which may be the result of the increased propensity of CD28-derived spacers to participate in homotypic associations (Alabanza et al., Mol. Ther. (2017) 25:2452-2465).

[0079] Spacer domains are also commonly used in the detection of CAR-expressing cells in vitro and in vivo. Fc-specific antibodies can be used to detect CARs containing spacer domains containing appropriate Fc regions, and specific epitope tags have also been incorporated into CAR spacer domains for such purposes (e.g., Liu et al., Nat. Biotechnol. (2016) 4:430-434).

[0080] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence derived from the amino acid sequence of one of 41BB, OX40, CD96, CD44, IgG2, or IgG1. In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence derived from the amino acid sequence of one of 41BB, OX40, CD96, or CD44. In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence derived from the amino acid sequence of one of 41BB.

[0081] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:96.

[0082] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:97.

[0083] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:98.

[0084] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:99.

[0085] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100.

[0086] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 101.

[0087] In some embodiments, a spacer domain according to the present disclosure comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 102. Transmembrane domain The CAR of the present disclosure comprises a transmembrane domain.Transmembrane domain refers to any three-dimensional structure formed by the sequence of thermodynamically stable amino acids in biological membrane, for example, cell membrane.In the context of the present disclosure, transmembrane domain can be the amino acid sequence that spans the cell membrane of the cell that expresses CAR.

[0088] The transmembrane domain may comprise or consist of a sequence of amino acids that form a hydrophobic alpha helix or beta barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be or be derived from the amino acid sequence of the transmembrane domain of a protein that contains a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001 J Mol Biol 305:567-580).

[0089] In some embodiments, the amino acid sequence of the transmembrane domain of a CAR of the present disclosure may be or may be derived from the amino acid sequence of the transmembrane domain of a protein expressed on the cell surface. In some embodiments, the protein expressed on the cell surface is a receptor or ligand, for example, an immunoreceptor or ligand. In some embodiments, the amino acid sequence of the transmembrane domain is selected from the group consisting of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I alpha, MHC class II alpha, MHC class II beta, CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, TCR alpha, TCR beta, CD4, CD8 alpha, CD8 beta, CD40, CD40L, PD-1, PD-L1, PD-L2, 4-1BB, 4-1BBL, OX40, OX40L, GITR, GITRL, TIM-3, and Gale. The transmembrane domain may be or may be derived from the amino acid sequence of one of the following: cutin-9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1, ICAM1, LFA-1, LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1, CD2, CD48, 2B4, SLAM, CD30, CD30L, DR3, TL1A, CD226, CD155, CD112, and CD276. In some embodiments, the transmembrane domain is or is derived from the amino acid sequence of the transmembrane domain of CD28, CD3-zeta, CD8α, CD8β, or CD4. In some embodiments, the transmembrane domain is or is derived from the amino acid sequence of the transmembrane domain of CD28.

[0090] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 103.

[0091] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 104.

[0092] In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 105. Signaling domains The chimeric antigen receptors of the present disclosure comprise a signaling domain that provides a sequence for initiating intracellular signaling in cells expressing the CAR. ITAM-containing sequences: Signaling domains include ITAM-containing sequences. ITAM-containing sequences contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs include the amino acid sequence YXXL / I (SEQ ID NO: 109), where "X" represents any amino acid. In ITAM-containing proteins, sequences according to SEQ ID NO: 109 are often separated by 6 to 8 amino acids; YXXL / I(X) 6-8 YXXL / I (SEQ ID NO: 110). When a phosphate group is added to the tyrosine residue of an ITAM by a tyrosine kinase, a signal transduction cascade within the cell is initiated.

[0093] In some embodiments, the signaling domain comprises one or more copies of the amino acid sequence according to SEQ ID NO: 109 or SEQ ID NO: 110. In some embodiments, the signaling domain comprises at least 1, 2, 3, 4, 5, or 6 copies of the amino acid sequence according to SEQ ID NO: 109. In some embodiments, the signaling domain comprises at least 1, 2, or 3 copies of the amino acid sequence according to SEQ ID NO: 110.

[0094] In some embodiments, the signaling domain comprises an ITAM-containing sequence that is or is derived from the amino acid sequence of an ITAM-containing sequence of a protein having an ITAM-containing amino acid sequence. In some embodiments, the signaling domain comprises an ITAM-containing sequence that is or is derived from the amino acid sequence of the intracellular domain of one of CD3-zeta, FcγRI, CD3ε, CD3δ, CD3γ, CD79α, CD79β, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIV, or DAP12. In some embodiments, the signaling domain comprises an ITAM-containing sequence that is or is derived from the amino acid sequence of the intracellular domain of CD3-zeta.

[0095] In some embodiments, the signaling domain comprises an ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111. Costimulatory sequence: The signaling domain may additionally comprise one or more costimulatory sequences. A costimulatory sequence is an amino acid sequence that provides costimulation of cells expressing a CAR of the present disclosure. Costimulation promotes the proliferation and survival of cells expressing a CAR upon binding to a target antigen, and can also promote cytokine production, differentiation, cytotoxic function, and memory formation by cells expressing a CAR. The molecular mechanisms of T cell costimulation are reviewed in Chen and Flies (2013) Nat Rev Immunol 13(4):227-242.

[0096] The costimulatory sequence may be or may be derived from the amino acid sequence of a costimulatory protein, hi some embodiments, the costimulatory sequence is or is derived from the amino acid sequence of the intracellular domain of a costimulatory protein.

[0097] When a CAR binds to a target antigen, the costimulatory sequence, when ligated with its cognate ligand, provides costimulation to a cell expressing the CAR of the type predicted to be provided by the costimulatory protein from which the costimulatory sequence is derived. As an example, in the case of a CAR that includes a signaling domain that includes a costimulatory sequence derived from CD28, binding to the target antigen initiates signaling in a cell expressing the CAR of the type predicted to be initiated by binding of CD80 and / or CD86 to CD28. Thus, the costimulatory sequence is capable of delivering the costimulatory signal of the costimulatory protein from which the costimulatory sequence is derived.

[0098] In some embodiments, the costimulatory protein domain may be a member of the B7-CD28 superfamily (e.g., CD28, ICOS) or a member of the TNF receptor superfamily (e.g., 4-1BB, OX40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the costimulatory sequence is or is derived from the intracellular domain of one of CD28, 4-1BB, ICOS, CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226, and LIGHT. In some embodiments, the costimulatory sequence is or is derived from the intracellular domain of CD28.

[0099] In some embodiments, the signaling domain comprises more than one costimulatory sequence. In some embodiments, the signaling domain comprises 1, 2, 3, 4, 5, or 6 costimulatory sequences. Multiple costimulatory sequences may be provided in tandem.

[0100] Whether a given amino acid sequence is capable of initiating signal transduction mediated by a given costimulatory protein can be investigated, for example, by analyzing correlations in costimulatory protein-mediated signal transduction (e.g., expression / activity of factors whose expression / activity is up-regulated or down-regulated as a result of costimulatory protein-mediated signal transduction).

[0101] Costimulatory proteins upregulate the expression of genes that promote cell growth, effector function, and survival through numerous pathways. For example, CD28 and ICOS signal through phosphatidylinositol 3-kinase (PI3K) and AKT, and upregulate the expression of genes that promote cell growth, effector function, and survival through NF-κB, mTOR, NFAT, and AP1 / 2. CD28 also activates AP1 / 2 through CDC42 / RAC1 and ERK1 / 2 through RAS, and ICOS activates C-MAF. 4-1BB, OX40, and CD27 recruit TNF receptor-associated factors (TRAFs) and signal through PI3K in addition to the MAPK pathway.

[0102] In some embodiments, the signaling domain comprises a costimulatory sequence that is or is derived from CD28. Kofler et al., Mol. Ther. (2011) 19:760-767, describe a variant CD28 intracellular domain in which the lck kinase binding site is mutated to reduce induction of IL-2 production upon CAR ligation in order to minimize regulatory T cell-mediated suppression of CAR-T cell activity. The amino acid sequence of the variant CD28 intracellular domain is set forth in SEQ ID NO: 107.

[0103] In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 108.

[0104] In some embodiments, the signaling domain comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:115.

[0105] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 116. Linkers and additional sequences The CAR of the present disclosure may further comprise additional amino acids or amino acid sequences.

[0106] The amino acid sequences described herein (e.g., amino acid sequences of domains / regions of a CAR described herein) may comprise one or more additional amino acids at one or both termini (i.e., N- or C-terminus) of the reference amino acid sequence. In some embodiments, the peptide / polypeptide comprises, for example, 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 20 to 30, 20 to 40, or 20 to 50 additional amino acids at one or both termini of the reference amino acid sequence. In some embodiments, the additional amino acids correspond to the amino acids provided at that position relative to the reference amino acid sequence in the context of the protein from which the reference amino acid sequence is derived. As an example, if a reference amino acid sequence corresponds to amino acid positions 20 to 30 of the amino acid sequence of the protein from which the reference amino acid sequence is derived, and the amino acid sequence has five additional amino acids at its N-terminus, those five additional amino acids may correspond to positions 15 to 19 of the amino acid sequence of the protein from which the reference amino acid sequence is derived.

[0107] The CAR of the present disclosure may comprise one or more linker sequences between amino acid sequences. For example, a linker sequence may be provided between the domains of the CAR (e.g., between the antigen-binding domain and the spacer domain, and / or between the spacer domain and the transmembrane domain, and / or between the transmembrane domain and the signaling domain). By way of further example, a linker sequence may be provided between subsequences of the domains of the CAR (e.g., between the VH and VL of the antigen-binding domain, and / or between the costimulatory sequence and the ITAM-containing sequence of the signaling domain).

[0108] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0109] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm, where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5 or 6) copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0110] In some embodiments, the linker sequence comprises one or more copies of the amino acid sequence according to SEQ ID NO: 57. In some embodiments, the linker sequence comprises at least 1, 2, 3, or 4 copies of the amino acid sequence according to SEQ ID NO: 57.

[0111] In some embodiments, the linker sequence comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:58.

[0112] The CARs of the present disclosure may comprise an amino acid sequence to facilitate expression, folding, trafficking, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids that forms a detectable moiety, for example, as described herein below.

[0113] The CAR of the present disclosure may additionally contain a signal peptide (also known as a leader sequence or signal sequence). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed on the cell surface often contain signal peptides. Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0114] Signal peptides may be present at the N-terminus of CAR or may be present in newly synthesized CARs.Signal peptides provide efficient transport of CARs to the cell surface.Signal peptides are often removed by cleavage, and therefore are not included in the mature CARs expressed on the cell surface.

[0115] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0116] In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113. In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:114. Labels and conjugates In some embodiments, the CAR of the present disclosure further comprises a detectable moiety.

[0117] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radiolabel, a chemical, a nucleic acid, or an enzyme label. The CAR may be covalently or non-covalently labeled with a detectable moiety.

[0118] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radiolabels include hydrogen 3 ,sulfur 35 ,carbon 14 , Phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 ,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 , fluorine 18 ,yttrium 90 ,palladium 100 , bismuth 217 and antimony 211Examples of the label include radioisotopes such as 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 13

[0119] In some embodiments, the CAR comprises an epitope tag, such as His (e.g., 6xHis), FLAG, c-Myc, StrepTag, hemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and a hapten (e.g., biotin, digoxigenin, dinitrophenol), optionally at the N- or C-terminus of the antigen-binding molecule / polypeptide / CAR.

[0120] In some embodiments, the CAR comprises a moiety with a detectable activity, such as an enzymatic moiety, including, for example, luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucuronidases, phosphatases (e.g., alkaline phosphatase), peroxidases (e.g., horseradish peroxidase), and cholinesterases.

[0121] In some embodiments, the CAR of the present disclosure is conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect, i.e., a drug moiety. The drug moiety may be a small molecule (e.g., an organic compound with a low molecular weight (<1000 daltons, typically about 300-700 daltons)). Drug moieties are described, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14 (incorporated herein by reference in its entirety). In some embodiments, the drug moiety may be or include a cytotoxic agent. In some embodiments, the drug moiety may be or include a chemotherapeutic agent. Drug moieties include, for example, calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD. Specific exemplary CAR In some embodiments, a CAR according to the present disclosure comprises or consists of: SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 23 an antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of 6, 177, 178, 179, 180, 181, or 182; a spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100, 98, 99, 101, or 102; a transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 103, 104, or 105; and Signaling domains containing: a costimulatory sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106, 107, or 108; and An ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111.

[0122] In some embodiments, the CAR comprises or consists of: SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 23 an antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of 6, 177, 178, 179, 180, 181, or 182; a spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 100, 98, 99, 101, or 102; a transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 103; and Signaling domains containing: a costimulatory sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106; and An ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111.

[0123] In some embodiments, the CAR is selected from the group consisting of SEQ ID NOs: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224.

[0124] In some embodiments, the CAR is selected from the group consisting of SEQ ID NOs: 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 97, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 or 212.

[0125] In some embodiments, the CAR comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119, 124, 129, 134, 139, 144, 185, 190, 195, 200, 205, or 210.

[0126] In some embodiments, the CAR comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 124, 144, 190, or 210. Nucleic acids and vectors The present disclosure provides a nucleic acid or multiple nucleic acids encoding a CAR according to the present disclosure. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0127] In some embodiments, the nucleic acid may be or be included in a vector or vectors. That is, the nucleotide sequence of the nucleic acid may be contained in a vector. A CAR according to the present disclosure may be produced intracellularly by transcription from a vector encoding an antigen-binding molecule, polypeptide, or CAR, followed by translation of the transcribed RNA.

[0128] Therefore, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate delivery of a nucleic acid encoding a CAR according to the present disclosure. The vector may be an expression vector containing the elements necessary to express a nucleic acid comprising / encoding a CAR according to the present disclosure.

[0129] The nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e., purified or isolated from other nucleic acids or naturally occurring biological materials.

[0130] The nucleotide sequence may be contained in a vector, e.g., an expression vector. As used herein, a "vector" refers to a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acids into cells. The vector may be a vector for expressing a nucleic acid in a cell. Such a vector may include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0131] The term "operably linked" can include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked such that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript can then be translated into a desired peptide / polypeptide.

[0132] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., retroviral vectors, such as gammaretroviral vectors (e.g., vectors derived from murine leukemia viruses (MLV) such as SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated by reference in their entireties.

[0133] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector that includes elements necessary for expression of a protein from the vector in a eukaryotic cell, hi some embodiments, the vector may be a mammalian vector, e.g., a vector that includes a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. Cells expressing the CAR of the present disclosure In some aspects and embodiments, the present disclosure provides cells comprising a CAR according to the present disclosure. A CAR according to the present disclosure can be used to generate a CAR-expressing cell, for example, a CAR-expressing immune cell (e.g., a CAR-T or CAR-NK cell).

[0134] It will be understood that where a cell is referred to in the singular herein (ie, "a / the cell"), a plural / population of such cells is also contemplated. A cell that expresses a CAR may contain or express a nucleic acid encoding a CAR according to the present disclosure. It will be understood that a cell that expresses a CAR contains the CAR that it expresses. It will also be understood that a cell that expresses a nucleic acid encoding a CAR also expresses and contains the CAR encoded by the nucleic acid.

[0135] The cell may be a eukaryotic cell, for example a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human), or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodents), cat, dog, pig, sheep, goat, cow (including cattle, e.g., dairy cows, or any animal in the order Bos), horse (including any animal in the family Equidae), donkey, and non-human primate).

[0136] The cell expressing the CAR is preferably an immune cell. The immune cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, for example, a T cell, a B cell, a NK cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor thereof. The immune cell may express, for example, a CD3 polypeptide (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), a TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the immune cell is a T cell, such as a CD3+ T cell. In some embodiments, the T cell is a CD3+ and a CD4+ T cell. In some embodiments, the T cell is a CD3+ and a CD8+ T cell. In some embodiments, the T cell is a T helper cell (T HIn some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0137] Aspects and embodiments of the present disclosure particularly relate to T cells comprising / expressing a CAR specific for CD30 according to the present disclosure. In some aspects and embodiments, the immune cells may be virus-specific immune cells. "Virus-specific immune cells," as used herein, refer to immune cells specific to a virus. Virus-specific immune cells express / contain a receptor (preferably a T cell receptor) capable of recognizing a viral antigen peptide (e.g., when presented by an MHC molecule). Virus-specific immune cells can express / contain such a receptor as a result of expression of endogenous nucleic acid encoding such an antigen receptor or as a result of being engineered to express such a receptor. Virus-specific immune cells preferably express / contain a TCR specific for a viral antigen peptide. Virus-specific T cells can display certain functional properties of T cells in response to the viral antigen for which the T cell is specific or in response to cells containing / expressing the virus / antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g., cytotoxic T cells.

[0138] In some embodiments, virus-specific T cells may exhibit one or more of the following properties: cytotoxicity towards cells containing / expressing the virus / viral antigen to which the T cell is specific; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression in response to stimulation with the virus / viral antigen to which the T cell is specific or in response to exposure to cells containing / expressing the virus / viral antigen to which the T cell is specific.

[0139] Virus-specific T cells express / contain a TCR that, when presented by the appropriate MHC molecule, is capable of recognizing a peptide of the viral antigen for which the T cell is specific. Virus-specific T cells may be CD4+ T cells and / or CD8+ T cells.

[0140] The virus that virus-specific immune cell is specific for can be any virus.For example, virus can be dsDNA virus (for example, adenovirus, herpesvirus, poxvirus), ssRNA virus (for example, parvovirus), dsRNA virus (for example, reovirus), (+)ssRNA virus (for example, picornavirus, togavirus), (-)ssRNA virus (for example, orthomyxovirus, rhabdovirus), ssRNA-RT virus (for example, retrovirus) or dsDNA-RT virus (for example, hepadnavirus). Specifically, the present disclosure contemplates viruses belonging to the families Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae. In some embodiments, the virus is selected from the group consisting of Epstein-Barr virus, adenovirus, herpes simplex 1 virus, herpes simplex 2 virus, varicella zoster virus, human cytomegalovirus, human herpesvirus 8, human papillomavirus, BK virus, JC virus, smallpox virus, hepatitis B virus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, The virus is selected from yellow fever virus, dengue virus, West Nile virus, TBE virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coltivirus, and bannavirus.

[0141] In some embodiments, the virus is selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).

[0142] In some embodiments, the virus-specific immune cells may be specific for a viral peptide / polypeptide selected from, for example, Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).

[0143] T cells specific for viral antigens are sometimes referred to herein as virus-specific T cells (VSTs). T cells specific for a particular viral antigen may also be described as specific for the related virus, for example, T cells specific for EBV antigens may also be referred to as EBV-specific T cells, or "EBVSTs."

[0144] Thus, in some embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVST), adenovirus-specific T cells (AdVST), cytomegalovirus-specific T cells (CMVST), human papillomavirus (HPVST), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B virus-specific T cells (HBVST), hepatitis C virus-specific T cells (HCVST), human immunodeficiency virus-specific T cells (HIVST), lymphocytic choriomeningitis virus-specific T cells (LCMVST), or herpes simplex virus-specific T cells (HSVST).

[0145] In some preferred embodiments, the virus-specific immune cells are specific for an EBV antigenic peptide / polypeptide. In preferred embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVST).

[0146] EBV virology is described, for example, in Stanfield and Luftiq, F1000 Res. (2017) 6:386 and Odumade et al., Clin Microbiol Rev (2011) 24(1):193-209, both of which are incorporated by reference in their entireties.

[0147] EBV infects epithelial cells through the binding of the viral protein BMFR2 to β1 integrin and the binding of the viral proteins gH / gL to integrins avβ6 and avβ8. EBV infects B cells through the interaction of the viral glycoprotein gp350 with CD21 and / or CD35, followed by the interaction of viral gp42 with MHC class II. These interactions initiate fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.

[0148] EBV has two replication modes: latent and lytic. The latent cycle does not result in the production of virions and can occur in niches, i.e., B cells and epithelial cells. The circular DNA of the EBV genome exists as an episome in the cell nucleus and is copied by the host cell's DNA polymerase. During latency, only a fraction of EBV's genes are expressed in one of three distinct patterns known as the latency program, which produces distinct sets of viral proteins and RNA. The latent cycle is described, for example, in Amon and Farrell, Reviews in Medical Virology (2004) 15(3):149-56, the entire contents of which are incorporated herein by reference.

[0149] The EBNA1 protein and the non-coding RNA EBER are expressed in each of the latency programs I to III. Latency programs II and III further include the expression of EBNALP, LMP1, LMP2A, and LMP2B proteins, while latency program III further includes the expression of EBNA2, EBNA3A, EBNA3B, and EBNA3C.

[0150] EBNA1 is multifunctional and plays a role in gene regulation, extrachromosomal replication, and genomic maintenance of EBV episomes through positive and negative regulation of viral promoters (Duellman et al., J Gen Virol. (2009); 90(Pt9):2251-2259). EBNA2 is involved in regulating latent viral transcription and contributes to the immortalization of EBV-infected cells (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391:35-59). EBNA-LP is required for the transformation of naive B cells and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog. (2018); 14(2):e1006890). EBNA3A, 3B, and 3C interact with RBPJ and influence gene expression, contributing to the survival and proliferation of infected cells (Wang et al., J Virol. (2016) 90(6):2906-2919). LMP1 regulates the expression of genes involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5):490-504). LMP2A and LMP2B inhibit normal B cell signaling by mimicking activated B cell receptors (Portis and Longnecker, Oncogene (2004) 23(53):8619-8628). EBERs form ribonucleoprotein complexes with host cell proteins and are proposed to play a role in cellular transformation.

[0151] The latent cycle can progress through either latency program I or III in B cells, usually progressing from III to II and from II to I. EBV enters latency program III when it infects resting naive B cells. Expression of latency III genes activates the B cell, which becomes a proliferating blast. EBV then typically progresses to latency II by restricting expression to a subset of genes, which causes the blast to differentiate into a memory B cell. Further restriction of gene expression causes EBV to enter latency I. When memory B cells divide, EBNA1 expression allows EBV to replicate. In epithelial cells, only latency II occurs.

[0152] During primary infection, EBV replicates in oropharyngeal epithelial cells and establishes latency III, II, and I infections in B lymphocytes. EBV latent infection of B lymphocytes is required for viral persistence and subsequent replication in epithelial cells and for the release of infectious virus into saliva. EBV latency III and II infections of B lymphocytes, latency II infections of oral epithelial cells, and latency II infections of NK or T cells can result in malignancies characterized by the presence and gene expression of the normal EBV genome.

[0153] Latent EBV in B cells can be reactivated and switch to lytic replication. The lytic cycle leads to the production of infectious virions and can occur in niches, i.e., B cells and epithelial cells, as reviewed, for example, by Kenney in Chapter 25 of Arvin et al., Human Herpesviruses: Biology, Therapy and Immunoprophylaxis; Cambridge University Press (2007), which is incorporated herein by reference in its entirety.

[0154] Lytic replication requires that the EBV genome be linear. The latent EBV genome is episomal and must be linearized for lytic reactivation. In B cells, lytic replication usually occurs only after reactivation from latency.

[0155] Immediate-early lytic gene products, such as BZFL1 and BRLF1, act as transactivators, enhancing their own expression and that of late lytic cycle genes. Early lytic gene products have roles in viral replication (e.g., EBV DNA polymerase catalytic component BALF5; DNA polymerase processivity factor BMRF1, DNA-binding protein BALF2, helicase BBLF4, primase BSLF1, and primase-related protein BBLF2 / 3) and deoxyribonucleotide metabolism (e.g., thymidine kinase BXLF1, dUTPase BORF2). Other early lytic gene products act as transcription factors (e.g., BMRF1, BRRF1), have roles in RNA stability and processing (e.g., BMLF1), or are involved in immune evasion (e.g., BHRF1, which inhibits apoptosis).

[0156] Late lytic gene products are traditionally classified as those expressed after the onset of viral replication. They generally encode structural components of the virion, such as nucleocapsid proteins, as well as glycoproteins (e.g., gp350 / 220, gp85, gp42, gp25) that mediate EBV binding and fusion. Other late lytic gene products have roles in immune evasion; BCLF1 encodes the viral homolog of IL-10, and BALF1 encodes a protein with homology to the anti-apoptotic protein Bcl2.

[0157] "EBV-specific immune cells," as used herein, refer to immune cells specific for Epstein-Barr virus (EBV). EBV-specific immune cells express / contain a receptor (preferably a T cell receptor) capable of recognizing an EBV antigenic peptide (e.g., when presented by an MHC molecule). EBV-specific immune cells preferably express / contain a TCR specific for an EBV antigenic peptide presented by MHC class I.

[0158] In some embodiments, the EBV-specific immune cells are T cells, e.g., CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (T H In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)).

[0159] EBV-specific T cells preferably express / contain a TCR that is capable of recognizing a peptide of an EBV antigen for which the T cell is specific when presented by an appropriate MHC molecule. EBV-specific T cells may be CD4+ and / or CD8+ T cells.

[0160] The EBV-specific immune cells may be specific for any EBV antigen, such as those described herein. A population of EBV-specific immune cells, or a composition comprising a plurality of EBV-specific immune cells, may comprise immune cells specific for one or more EBV antigens.

[0161] In some embodiments, the EBV antigen is an EBV latent antigen, such as a type III latent antigen (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latent antigen (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B or BARF1), or a type I latent antigen (e.g., EBNA1 or BARF1). In some embodiments, the EBV antigen is an EBV lytic antigen, such as an immediate early lytic antigen (e.g., BZLF1, BRLF1 or BMRF1), an early lytic antigen (e.g., BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU or EBNA1-FUK), or a late lytic antigen (e.g., BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3 or gp350).

[0162] In some embodiments according to various aspects of the present disclosure, the cells may contain / express more than one (e.g., two, three, four, etc.) CARs. In some embodiments, a cell may contain / express more than one non-identical CAR. A cell containing / expressing more than one non-identical CAR may contain / express CARs specific for non-identical target antigens. In some embodiments, each non-identical target antigen is independently a cancer cell antigen described herein. Functional properties of cells expressing the CAR of the present disclosure A cell (e.g., an immune cell, e.g., a T cell) expressing a CAR according to the present disclosure can exhibit certain functional properties in response to the target antigen for the CAR (e.g., CD30) or in response to a cell containing / expressing the target antigen for the CAR. In some embodiments, the properties are functional properties associated with effector T cells, e.g., cytotoxic T cells.

[0163] Cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure may exhibit one or more of the following properties: expression of one or more cytotoxic / effector factors (e.g., IFNγ, TNFα, GM-CSF), proliferation / population expansion, and / or growth factor (e.g., IL-2) expression in response to cells expressing a target antigen (e.g., CD30) for the CAR; cytotoxicity against cells expressing the target antigen for the CAR (e.g., CD30); no cytotoxicity (i.e., above baseline) against cells that do not express the target antigen for the CAR (e.g., CD30); Anti-cancer activity against cancers containing cells expressing the target antigen for the CAR (e.g., CD30) (e.g., cytotoxicity against cancer cells, inhibition of tumor growth, reduction of metastasis, etc.); Cytotoxicity against alloreactive immune cells, for example, alloreactive immune cells expressing the target antigen (e.g., CD30) for the CAR.

[0164] In some embodiments, a CAR-expressing T cell according to the present disclosure may exhibit one or more of the following properties: cytotoxicity against cells containing / expressing the target antigen for the CAR; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression in response to stimulation with or exposure to cells containing / expressing the target antigen for the CAR; anti-cancer activity against a cancer comprising cells expressing the target antigen for the CAR (e.g., cytotoxicity against cancer cells, tumor growth inhibition, reduced metastasis, etc.).

[0165] Cell proliferation / population expansion can be studied by analyzing cell division or cell number over a period of time. Cell division can be measured, for example, by: 3Proliferating cells may be analyzed by in vitro analysis of H-thymidine incorporation, or by the CFSE dilution assay described, for example, in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, which are incorporated herein by reference in their entireties. Proliferating cells can also be identified by analysis of 5-ethynyl-2'-deoxyuridine (EdU) incorporation by a suitable assay, for example, as described in Buck et al., Biotechniques. 2008 January;44(7):927-9, and Sali and Mitchison, PNAS USA 2008 February 19;105(7):2415-2420, both of which are incorporated herein by reference in their entireties.

[0166] "Expression" as used herein may refer to gene or protein expression. Gene expression encompasses the transcription of DNA into RNA and can be measured by various means known to those skilled in the art, for example, by measuring mRNA levels with quantitative real-time PCR (qRT-PCR) or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, for example, by antibody-based methods, such as Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0167] Cytotoxicity and cell killing can be investigated using, for example, any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include: 51Examples of suitable release assays include Cr release assays, lactate dehydrogenase (LDH) release assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assays, and calcein-acetoxymethyl (calcein-AM) release assays. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed, for example, by co-culturing the test cells with the effector immune cells and measuring the number / proportion of viable / dead (e.g., lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3): e0193498 (incorporated herein by reference in its entirety).

[0168] In some embodiments, cell killing of cells expressing the target antigen for the CAR by CAR-expressing cells can be assessed by xCELLigence assay, as described in Example 1.5 herein. Cell killing by CAR-expressing cells can also be assessed in vivo, for example, by assessing the number / proportion of cells expressing the target antigen for the CAR and inferring their killing / depletion by the CAR-expressing cells.

[0169] The cells may be evaluated for anti-cancer activity by suitable in vitro assays or analysis in an in vivo model of the relevant cancer. A cell comprising a CAR / CAR-encoding nucleic acid according to the present disclosure preferably possesses new and / or improved properties compared to a cell comprising a nucleic acid comprising / encoding a known CAR, such as a CAR comprising a known spacer domain.

[0170] In particular, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure preferably possess new and / or improved properties compared to cells comprising a nucleic acid encoding an identical CAR / equivalent CAR construct in all respects except that the spacer domain consists of the sequence set forth in SEQ ID NO: 96, 97, or 98. By way of example, the CAR construct of SEQ ID NO: 213 is identical to the CAR construct of SEQ ID NO: 119 except that the CAR of SEQ ID NO: 213 comprises a human IgG1 CH2-CH3 spacer domain set forth in SEQ ID NO: 96, whereas the CAR of SEQ ID NO: 119 comprises a 41BB spacer domain set forth in SEQ ID NO: 100. In preferred embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure possess new and / or improved properties compared to cells comprising a nucleic acid encoding an identical CAR / equivalent CAR construct in all respects except that the spacer domain consists of the sequence set forth in SEQ ID NO: 96.

[0171] For the sake of brevity, in the following paragraphs, such a reference CAR comprising a human IgG1 CH2-CH3 spacer domain may be simply referred to as an "equivalent CAR comprising a hIgG1 spacer."

[0172] In some aspects, a cell comprising a nucleic acid encoding a CAR / CAR according to the present disclosure comprises: expand and / or grow to an extent equal to or greater than the extent to which cells containing a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer expand / grow; expresses cytotoxic / effector factors (e.g., IFNγ, TNFα, GM-CSF) in response to stimulation by cells expressing the target antigen for the CAR to an extent comparable to or greater than the extent to which cells containing a nucleic acid encoding a CAR / equivalent CAR comprising a hIgG1 spacer express such factors; kills cells expressing the target antigen for the CAR with a potency / rate that is equal to or greater than the potency / rate of killing of such cells by cells containing a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer; exhibits off-target cytotoxicity (e.g., against NK cells) that is equivalent to or less than the off-target cytotoxicity exhibited by cells containing a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer; induces systemic inflammation in a recipient subject to an extent comparable to or less than that induced by administration of cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer; inhibits tumor growth of, for example, a cancer expressing the target antigen for the CAR with a potency / degree comparable to or greater than the tumor growth inhibition by cells containing a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer; increasing survival of a subject with cancer, e.g., a cancer that expresses a target antigen for a CAR, to an extent equal to or greater than the extent to which survival is increased by cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer; It exhibits in vivo persistence that is comparable to or greater than the in vivo persistence of cells containing nucleic acids encoding CARs / equivalent CARs comprising a hIgG1 spacer.

[0173] Cell proliferation and cell population expansion can be measured as described herein above. The rate of cell proliferation / population expansion for a given cell type can be determined over time, for example, by analyzing the number of such cells at various time points. Cell proliferation / population expansion can be measured in vitro or in vivo, for example, after administration to a subject (for example, a subject with cancer that expresses a target antigen against the cancer).

[0174] In some embodiments, the rate of cell proliferation or population expansion growth (e.g., in vitro or in vivo) of cells comprising a CAR / CAR-encoding nucleic acid according to the disclosure, as determined in a given assay, is one of: 0.5 times or more and 2 times or less, such as 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, or 0.95 times or more and 1.1 times or less, the rate of cell proliferation / population expansion growth of cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer. In some embodiments, the rate of cell proliferation / population expansion growth (e.g., in vitro or in vivo) of cells comprising a CAR / CAR-encoding nucleic acid according to the disclosure, as determined in a given assay, is more than 1-fold, such as one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the rate of cell proliferation / population expansion growth of cells comprising a CAR / nucleic acid encoding an equivalent CAR comprising an hIgG1 spacer.

[0175] In some embodiments, the level of expression of one or more cytotoxic / effector factors (e.g., IFNγ, TNFα, GM-CSF) by cells comprising a CAR / CAR-encoding nucleic acid according to the present disclosure in response to stimulation by cells expressing the target antigen for the CAR, as determined in a given assay, is one of: 0.5-fold or more and 2-fold or less, such as 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold the level of expression of such factor(s) by cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer in response to stimulation by cells expressing the target antigen for the CAR. In some embodiments, the level of expression of one or more cytotoxic / effector factors (e.g., IFNγ, TNFα, GM-CSF) by cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure in response to stimulation by cells expressing the target antigen for the CAR, as determined in a given assay, is more than 1-fold, such as one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the level of expression of such factor(s) by cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer in response to stimulation by cells expressing the target antigen for the CAR.

[0176] In some embodiments, the efficacy or rate of cell killing (e.g., in vitro or in vivo) of cells expressing a target antigen for a CAR by cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure is one of: 0.5-fold or more and 2-fold or less, such as 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold, of the efficacy or rate of cell killing of such cells by cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer, as determined in a given assay. In some embodiments, the efficacy or rate of cell killing (e.g., in vitro or in vivo) of cells expressing a target antigen for a CAR by cells comprising a nucleic acid encoding a CAR / CAR according to the disclosure, as determined in a given assay, is more than 1-fold, such as one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the efficacy or rate of cell killing of such cells by cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer.

[0177] In some aspects, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure induce cell killing of cells that do not express the target antigen for the CAR to an extent that is similar to or less than the extent to which cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer induce cell killing of cells that do not express the target antigen for the CAR.

[0178] In some embodiments, cells comprising a CAR / CAR-encoding nucleic acid according to the present disclosure induce cell killing (e.g., in vitro or in vivo) of cells that do not express the target antigen for the CAR, as determined in a given assay, to a level that is one of: 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold the level of cell killing of such cells by cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer. In some embodiments, cells comprising a CAR / CAR-encoding nucleic acid according to the present disclosure induce cell killing (e.g., in vitro or in vivo) of cells that do not express the target antigen for the CAR to a level that is less than 1 fold, e.g., 0.99 fold or less, 0.95 fold or less, 0.9 fold or less, 0.85 fold or less, 0.8 fold or less, 0.75 fold or less, 0.7 fold or less, 0.65 fold or less, 0.6 fold or less, 0.55 fold or less, 0.5 fold or less, 0.45 fold or less, 0.4 fold or less, 0.35 fold or less, 0.3 fold or less, 0.25 fold or less, 0.2 fold or less, 0.15 fold or less, 0.1 fold or less, 0.05 fold or less, or 0.01 fold or less, the level of cell killing of such cells by cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer, as determined in a given assay.

[0179] In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure induce systemic inflammation (e.g., cytokine release syndrome) in a recipient subject of such cells to an extent comparable to or less than that of cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer. The induction of systemic inflammation by CAR-expressing cells in a recipient subject can be assessed by measuring one or more markers of systemic inflammation in the recipient subject after administration of such cells. Markers of systemic inflammation include, for example, levels of inflammatory cytokines (e.g., IL-6, IL-8, TNFα, and GM-CSF) in peripheral blood. In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure induce expression of one or more inflammatory cytokines (e.g., selected from IL-6, IL-8, TNFα, and GM-CSF) to an extent comparable to or less than that of cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer.

[0180] In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the present disclosure induce expression of one or more inflammatory cytokines (e.g., selected from IL-6, IL-8, and TNFα and GM-CSF) to a level that is one of: 0.5-fold or more and 2-fold or less, such as 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold, the level induced by cells comprising a nucleic acid encoding a CAR / equivalent CAR whose expression comprises an hIgG1 spacer, as determined in a given assay. In some embodiments, cells comprising a nucleic acid encoding a CAR / CAR according to the disclosure induce expression of one or more inflammatory cytokines (e.g., selected from IL-6, IL-8 and TNFα and GM-CSF) to a level that is less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less, of the level induced by cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer, as determined in a given assay.

[0181] In some embodiments, the level of inhibition of tumor growth (e.g., of a tumor expressing the target antigen for a CAR) achieved by a CAR / cells comprising a nucleic acid encoding a CAR according to the present disclosure is one of: 0.5-fold or more and 2-fold or less, such as 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold, of the level achieved by administration of a comparable amount of cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer, as determined in a given assay. In some embodiments, the level of inhibition of tumor growth (e.g., of a tumor expressing the target antigen for a CAR) achieved by cells comprising a CAR / CAR-encoding nucleic acid according to the present disclosure is more than 1-fold, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, of the level achieved by administration of a comparable amount of cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer, as determined in a given assay.

[0182] In some embodiments, administration of a CAR / cells comprising a nucleic acid encoding a CAR according to the present disclosure increases the survival rate of a recipient subject having a cancer that expresses the target antigen for the CAR by one of the following: 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold the level of survival achieved by administration of a comparable amount of cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer, as determined in a given assay. In some embodiments, administration of a CAR / cells comprising a nucleic acid encoding a CAR according to the present disclosure increases the survival rate of a recipient subject having a cancer expressing the target antigen for the CAR by more than 1-fold, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the level of survival achieved by administration of a comparable amount of cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer, as determined in a given assay.

[0183] In some embodiments, the in vivo persistence of a CAR / cells comprising a nucleic acid encoding a CAR according to the present disclosure following administration to a subject (e.g., a subject with cancer, e.g., a cancer that expresses a target antigen for the CAR), as determined in a given assay, is one of: 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold, 0.6-fold or more and 1.8-fold, 0.65-fold or more and 1.7-fold, 0.7-fold or more and 1.6-fold, 0.75-fold or more and 1.5-fold, 0.8-fold or more and 1.4-fold, 0.85-fold or more and 1.3-fold, 0.9-fold or more and 1.2-fold, or 0.95-fold or more and 1.1-fold the in vivo persistence observed for cells comprising a nucleic acid encoding a CAR / equivalent CAR comprising an hIgG1 spacer. In some embodiments, the in vivo persistence following administration of a CAR / cells comprising a nucleic acid encoding a CAR according to the present disclosure to a subject (e.g., a subject having cancer, e.g., a cancer that expresses a target antigen for a CAR), as determined in a given assay, is more than 1-fold the in vivo persistence observed for cells comprising a nucleic acid encoding a CAR / equivalent CAR that comprises an hIgG1 spacer, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more. Producing cells expressing the CAR of the present disclosure The present disclosure provides a method of producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid or vector according to the present disclosure into a cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0184] The present disclosure also provides a method for producing a cell that expresses / contains a CAR according to the present disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present disclosure into the cell. In some embodiments, the method additionally comprises culturing the cell under conditions suitable for expression of the nucleic acid or vector by the cell. In some embodiments, the method is carried out in vitro.

[0185] Methods for producing cells that express a CAR are well known to those of skill in the art. They generally involve modifying a cell (e.g., an immune cell, such as a T cell or an NK cell) to express / contain a CAR, e.g., introducing a nucleic acid encoding a CAR into the immune cell.

[0186] Immune cells may be modified to contain / express a CAR or a nucleic acid encoding a CAR described herein by methods well known to those of skill in the art. The methods generally involve transfer of a nucleic acid for permanent (stable) or transient expression of the transferred nucleic acid.

[0187] Any suitable genetic engineering platform can be used to modify cells according to the present disclosure.Suitable methods for modifying cells include using genetic engineering platforms, such as gammaretroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery and RNA transfection, as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, the entire contents of which are incorporated herein by reference.

[0188] Methods also include, for example, those described in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is incorporated herein by reference in its entirety. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0189] Methods for generating / expanding a population of immune cells expressing a CAR in vitro / ex vivo are well known to those skilled in the art. Suitable culture conditions (i.e., cell culture medium, additives, stimuli, temperature, gas atmosphere), cell number, culture period, and method for introducing a nucleic acid encoding a CAR into cells can be determined by reference to, for example, Hombach et al., J Immunol (2001) 167:6123-6131, Ramos et al., J. Clin. Invest. (2017) 127(9):3462-3471, and WO2015 / 028444A1, all of which are incorporated herein by reference in their entirety.

[0190] Conveniently, cultures of cells according to the present disclosure may be maintained in a humidified atmosphere containing 5% CO at 37° C. Cells in cell culture may be established and / or maintained at any suitable density, as can be readily determined by one of skill in the art.

[0191] Culturing can be carried out in any vessel suitable for the volume of culture, such as a well of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, the cells are cultured in a bioreactor, such as those described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, which is incorporated herein by reference in its entirety. In some embodiments, the cells are cultured in a GRex cell culture vessel, such as a GRex flask or a GRex100 bioreactor.

[0192] Immune cells (e.g., T cells) may be activated prior to introduction of a nucleic acid encoding a CAR. For example, T cells within a population of PBMCs may be nonspecifically activated in vitro by stimulation with agonistic anti-CD3 and anti-CD28 antibodies in the presence of IL-2.

[0193] Introducing nucleic acid / vector into cell can include transduction, for example, retroviral transduction.Therefore, in some embodiments, nucleic acid is contained in a viral vector, or vector is a viral vector.The transduction of immune cells with viral vector is described, for example, in Simmons and Alberola-Ila, Methods Mol Biol.(2016)1323:99-108, which is incorporated herein by reference in its entirety.

[0194] Drugs may be employed to enhance transduction efficiency. Hexadimethrine bromide (polybrene) is a cationic polymer commonly used to improve transduction by neutralizing charge repulsion between virions and sialic acid residues expressed on the cell surface. Other drugs commonly used to enhance transduction include, for example, poloxamer-based drugs such as LentiBOOST (Sirion Biotech), Retronectin (Takara), and Vectofusin (Miltenyi Biotech), as well as SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0195] In some embodiments, the method comprises centrifuging cells into which it is desired to introduce a nucleic acid encoding a CAR in the presence of cell culture medium containing a viral vector comprising the nucleic acid (referred to in the art as "spinfection").

[0196] In some embodiments, the method comprises introducing a nucleic acid or vector according to the present disclosure into an immune cell by electroporation, e.g., as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is incorporated herein by reference in its entirety.

[0197] The methods generally include introducing a nucleic acid encoding a CAR into a cell and culturing the cell under conditions suitable for expression of the nucleic acid / CAR by the cell. In some embodiments, the methods include culturing the immune cells into which the nucleic acid encoding the CAR has been introduced to expand their numbers. In some embodiments, the methods include culturing the immune cells into which the nucleic acid encoding the CAR has been introduced in the presence of IL-7 and / or IL-15 (e.g., recombinant IL-7 and / or IL-15).

[0198] In some embodiments, the method further comprises purifying / isolating cells that express a CAR, e.g., from other cells (e.g., cells that do not express a CAR). Methods for purifying / isolating immune cells from a heterogeneous population of cells are well known in the art, and for example, FACS- or MACS-based methods can be employed to sort a population of cells based on the expression of immune cell markers. In some embodiments, the method purifies / isolates a specific type of cell, e.g., CD8+ T cells that express a CAR, CTLs that express a CAR.

[0199] In a preferred embodiment, T cells expressing a CAR may be generated from T cells within a population of PBMCs by a process comprising stimulating the PBMCs with antagonist anti-CD3 and anti-CD28 antibodies, transducing the cells with a viral vector (e.g., a gamma-retroviral vector) encoding the CAR, and then culturing the cells in the presence of IL-7 and IL-15.

[0200] The present disclosure also provides a CAR-expressing cell obtained or obtainable by a method according to the present disclosure. composition The present disclosure also provides compositions comprising the CARs, nucleic acids, expression vectors, and cells described herein.

[0201] The cells, CARs, nucleic acids, and expression vectors described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may include a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0202] The compositions of the present disclosure may be formulated with one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants, or the like. The formulation may contain an agent (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), a lubricant (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), a binder (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), a stabilizer, a solubilizer, a surfactant (e.g., a wetting agent), a masking agent, or a colorant (e.g., titanium dioxide).

[0203] The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of a subject of interest (e.g., a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of a composition according to the present disclosure must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings or sweeteners can be found in standard pharmaceutical textbooks, for example, Remington's "The Science and Practice of Pharmacy" (A. Adejare, ed.), 23rd Edition (2020), Academic Press.

[0204] The composition may be formulated for local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration. In some embodiments, the pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or by ingestion.

[0205] Suitable formulations may include the relevant items in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid form, such as gels. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to a selected region of the human or animal body.

[0206] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel, a tissue / organ of interest, or a tumor. The present disclosure also provides methods for producing a pharmaceutically useful composition, which may include one or more steps selected from the following: producing a CAR or a cell comprising / expressing a CAR according to the present disclosure; isolating a CAR or a cell comprising / expressing a CAR according to the present disclosure; and / or mixing a CAR or a cell comprising / expressing a CAR according to the present disclosure with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0207] For example, a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., cancer), the method comprising formulating the pharmaceutical composition or medicament by mixing a CAR or a cell comprising / expressing a CAR according to the present disclosure with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent. Therapeutic and prophylactic uses The CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.

[0208] The present disclosure provides a CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein for use in a method of medical therapy or prevention. Also provided is a CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of a CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of a CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein.

[0209] The method may be effective in reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. The method may be effective in preventing the progression of a disease / condition, for example, preventing the worsening of a disease / condition or slowing the rate of its onset. In some embodiments, the method may result in an improvement in a disease / condition, for example, a reduction in the symptoms of a disease / condition or a reduction in some other correlates of the severity / activity of a disease / condition. In some embodiments, the method may prevent the disease / condition from progressing to a later stage (e.g., a chronic stage or metastasis).

[0210] It will be understood that the articles of the present disclosure can be used to treat / prevent any disease / condition that derives a therapeutic or prophylactic benefit from reducing the level / activity of a target antigen for a CAR (e.g., CD30), or reducing the number or activity of cells containing / expressing a target antigen for a CAR.

[0211] For example, the disease / condition may be one in which the target antigen for the CAR, or cells containing / expressing the target antigen for the CAR, is pathologically involved, e.g., a disease / condition in which an increase in the level / activity of the target antigen for the CAR, or an increase in the number / proportion of cells containing / expressing the target antigen for the CAR, is positively associated with the onset, development, or progression of the disease / condition, and / or the severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of the target antigen for the CAR, or an increase in the number / proportion of cells containing / expressing the target antigen for the CAR, may be a risk factor for the onset, development, or progression of the disease / condition.

[0212] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by an increased level of expression or activity of the target antigen for the CAR (e.g., CD30), for example, compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterized by an increased number / proportion / activity of cells expressing the target antigen for the CAR, for example, compared to the level / number / proportion / activity in the absence of the disease / condition (e.g., in a healthy subject or in an equivalent non-diseased tissue). If the disease / condition is cancer, the level of expression or activity of the target antigen for the CAR may be greater than the level of expression or activity of the target antigen for the CAR in an equivalent non-cancerous cell / non-tumor tissue. The cancer / its cells may contain one or more mutations that result in upregulation of the expression or activity of the target antigen for the CAR (e.g., compared to an equivalent non-cancerous cell / non-tumor tissue).

[0213] Treatment with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or a subject treated with an appropriate control): a reduction in the level of the target antigen for the CAR; a reduction in the activity of the target antigen for the CAR; and / or a reduction in the number / proportion of cells containing / expressing the target antigen for the CAR.

[0214] In aspects and embodiments according to the present disclosure, cells (particularly immune cells, more particularly T cells) comprising / expressing a CAR according to the present disclosure are provided for therapeutic and prophylactic use. It will be understood that the methods generally include administering to a subject a population of immune cells expressing a CAR according to the present disclosure. In some embodiments, immune cells expressing a CAR according to the present disclosure may be administered in the form of a pharmaceutical composition comprising such cells.

[0215] In particular, the use of immune cells expressing a CAR according to the present disclosure in methods for treating / preventing diseases / conditions by adoptive cell transfer (ACT) is envisioned. Adoptive cell transfer generally refers to the process of obtaining cells (e.g., immune cells) from a subject, typically by taking a blood sample and isolating the cells therefrom. The cells are then typically modified and / or expanded, and then administered to either the same subject (in the case of adoptive transfer of autologous / autologous cells) or a different subject (in the case of adoptive transfer of allogeneic cells). The treatment typically aims to provide a population of cells with certain desired characteristics to the subject, or to increase the frequency of such cells with such characteristics in the subject. Adoptive transfer may be performed with the aim of introducing a cell or a population of cells into the subject and / or increasing the frequency of a cell or a population of cells in the subject.

[0216] Adoptive transfer of immune cells is described, for example, in Kalos and June (2013), Immunity 39(1):49-60, and Davis et al. (2015), Cancer J. 21(6):486-491, both of which are incorporated by reference in their entireties. One of skill in the art can determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure by referring, for example, to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, which is incorporated by reference in its entirety.

[0217] In some embodiments, the target antigen for the CAR is CD30. The biology of CD30 and CD30-targeted interventions for disease treatment and prevention are reviewed, for example, in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1-3):151-8, both of which are incorporated herein by reference in their entireties.

[0218] The utility of immune cells expressing a CD30-specific CAR in the treatment / prevention of cancer is described, for example, in Hombach et al., Cancer Res. (1998) 58(6):1116-9; Hombach et al., Gene Therapy (2000) 7:1067-1075; Hombach et al., J Immunother. (1999) 22(6):473-80; Hombach et al., Cancer Res. (2001) 61:1976-1982; Hombach et al., J Immunol (2001) 167:6123-6131; Savoldo et al., Blood (2007) 110(7):2620-30; Koehler et al., Cancer Res. (2007) 67(5):2265-2273; Di Stasi et al., Blood (2009) 113(25):6392-402; Hombach et al., Gene Therapy (2010) 17:1206-1213; Chmielewski et al., Gene Therapy (2011) 18:62-72; Kofler et al., Mol. Ther. (2011) 19(4):760-767; Gilham, Abken and Pule. Trends in Mol. Med. (2012) 18(7):377-384; Chmielewski et al., Gene Therapy (2013) 20:177-186, Hombach et al., Mol. Ther. (2016) 24(8):1423-1434, Ramos et al., J. Clin. Invest. (2017) 127(9):3462-3471, WO2015 / 028444 A1, WO2016 / 008973 A1, WO2021 / 222927 A1 and WO2021 / 222928 A1.

[0219] Immune cells expressing a CAR according to the present disclosure can be employed in the treatment / prevention of diseases / conditions by allogeneic or autologous transplantation. "Allogeneic transplantation," as used herein, refers to the transplantation of cells, tissues, or organs into a recipient subject that are genetically non-identical to the recipient subject. The cells, tissues, or organs may be of or derived from the cells, tissues, or organs of a donor subject that is genetically non-identical to the recipient subject. Allogeneic transplantation differs from autologous transplantation in that it refers to the transplantation of cells, tissues, or organs that are / are from a donor subject that is genetically identical to the recipient subject (i.e., autologous material). It will be understood that adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation, and adoptive transfer of autologous immune cells is a form of autologous transplantation.

[0220] The present disclosure provides methods that include administering to a subject immune cells that contain / express a CAR according to the disclosure, or immune cells that contain / express a nucleic acid encoding a CAR according to the disclosure.

[0221] In some embodiments, the method comprises modifying an immune cell to contain / express a CAR according to the present disclosure. In some embodiments, the method comprises modifying a virus-specific immune cell to contain / express a nucleic acid encoding a CAR according to the present disclosure.

[0222] In some embodiments, the method comprises: (a) modifying an immune cell to express or contain a CAR according to the present disclosure, or to express or contain a nucleic acid encoding a CAR according to the present disclosure, and (b) administering to the subject virus-specific immune cells that have been modified to express or contain a CAR according to the disclosure, or that have been modified to express or contain a nucleic acid encoding a CAR according to the disclosure. Includes:

[0223] In some embodiments, the method comprises: (a) isolating or obtaining immune cells; (b) modifying an immune cell to express or contain a CAR according to the disclosure, or to express or contain a nucleic acid encoding a CAR according to the disclosure, and (c) administering to the subject immune cells that have been modified to express or contain a CAR according to the disclosure, or that have been modified to express or contain a nucleic acid encoding a CAR according to the disclosure. Includes:

[0224] In some embodiments, the method comprises: (a) isolating immune cells (e.g., PBMCs) from a subject; (b) generating / expanding a population of virus-specific immune cells; (c) modifying virus-specific immune cells to express or contain a CAR according to the present disclosure, or to express or contain a nucleic acid encoding a CAR according to the present disclosure; and (d) administering to the subject virus-specific immune cells that have been modified to express or contain a CAR according to the disclosure, or that have been modified to express or contain a nucleic acid encoding a CAR according to the disclosure. Includes:

[0225] In some embodiments, the method includes administering to a subject EBV-specific immune cells that have been modified to express or contain a CD30-specific CAR according to the present disclosure, or that have been modified to express or contain a nucleic acid encoding a CD30-specific CAR according to the present disclosure.

[0226] In some embodiments, the subject from which the immune cells (e.g., PBMCs) are isolated is the same subject to which the cells are administered (i.e., adoptive transfer can be transfer of autologous / autologous cells). In some embodiments, the subject from which the immune cells (e.g., PBMCs) are isolated is a different subject from the subject to which the cells are administered (i.e., adoptive transfer can be transfer of allogeneic cells).

[0227] In some embodiments, the method may include one or more of the following: obtaining a blood sample from the subject; isolating immune cells (e.g., PBMCs) from a blood sample obtained from the subject; generating / expanding a population of immune cells; Culturing immune cells in in vitro or ex vivo cell culture; modifying immune cells to express or contain a CAR according to the disclosure, or to express or contain a nucleic acid encoding a CAR according to the disclosure (e.g., by transduction with a viral vector encoding such a CAR or containing such a nucleic acid); culturing immune cells that express / comprise a CAR according to the present disclosure or that express / comprise a nucleic acid encoding a CAR according to the present disclosure in an in vitro or ex vivo cell culture; collecting / isolating immune cells that express / comprise a CAR according to the present disclosure or that express / comprise a nucleic acid encoding a CAR according to the present disclosure; formulating the immune cells expressing / comprising a CAR according to the disclosure, or a nucleic acid encoding a CAR according to the disclosure, into a pharmaceutical composition, e.g., by mixing the cells with a pharmaceutically acceptable adjuvant, diluent, or carrier; Administering immune cells that express / comprise a CAR according to the disclosure, or that express / comprise a nucleic acid encoding a CAR according to the disclosure, or a pharmaceutical composition comprising such cells to the subject.

[0228] In some embodiments, the method may additionally include treating a cell or subject to induce / enhance expression of a CAR and / or to induce / enhance proliferation or survival of virus-specific immune cells that contain / express a CAR.

[0229] cancer In some embodiments, the disease to be treated / prevented in accordance with the present disclosure is cancer. Cancer can also refer to any unwanted cell proliferation (or any disease that manifests itself as unwanted cell proliferation), neoplasm, or tumor. Cancer can be benign or malignant, primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. The cancer may be, for example, a cancer of the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, and / or tissue / cells derived from white blood cells.

[0230] The tumor may be a nervous system tumor or a non-nervous system tumor. Nervous system tumors may originate from either the central or peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-nervous system cancers / tumors may originate from any other non-nervous tissue, such as melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0231] In some embodiments, the cancer is a solid cancer, a hematological cancer, gastric cancer (e.g., gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), liver cancer (hepatocellular carcinoma, cholangiocarcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), oral cancer (e.g., oropharyngeal cancer (e.g., oropharyngeal carcinoma), oral cancer, laryngeal cancer, nasopharyngeal carcinoma, esophageal cancer), colorectal cancer (e.g., colorectal carcinoma), colon cancer, colon carcinoma, cervical cancer, prostate cancer, lung cancer (e.g., NSCLC, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), bladder cancer, urothelial cancer, skin cancer (e.g., For example, the cancer is selected from the group consisting of melanoma, advanced melanoma), renal cell cancer (e.g., renal cell carcinoma), ovarian cancer (e.g., ovarian carcinoma), mesothelioma, breast cancer, brain cancer (e.g., glioblastoma), prostate cancer, pancreatic cancer, myeloid hematologic malignancies, lymphoblastic hematologic malignancies, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, non-Hodgkin's lymphoma (NHL), thymoma, or multiple myeloma (MM).

[0232] In some embodiments, the cancer is a cancer in which the target antigen for the CAR (e.g., CD30) is pathologically involved. That is, in some embodiments, the cancer is a cancer caused or exacerbated by expression of the target antigen for the CAR, a cancer in which expression of the target antigen for the CAR is a risk factor, and / or a cancer in which expression of the target antigen for the CAR is positively associated with the onset, development, progression, severity, or metastasis of the cancer. A cancer may be characterized by expression of the target antigen for the CAR, for example, the cancer may contain cells that express the target antigen for the CAR. Such a cancer may be referred to as being positive for the target antigen for the CAR. A cancer that is "positive" for the target antigen for the CAR may be a cancer that contains cells that express (e.g., on the cell surface) the target antigen for the CAR. A cancer that is "positive" for the target antigen for the CAR may overexpress the target antigen for the CAR.

[0233] CD30-positive cancers have been described, for example, in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1-3):151-8. CD30 is expressed on a small subset of activated T and B lymphocytes and by various lymphoid neoplasms, including classical Hodgkin's lymphoma and anaplastic large cell lymphoma. Variable expression of CD30 has also been shown in peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), adult T-cell leukemia / lymphoma, cutaneous T-cell lymphoma (CTCL), extranodal NK-T cell lymphoma, various B-cell non-Hodgkin's lymphomas (including diffuse large B-cell lymphoma, particularly EBV-positive diffuse large B-cell lymphoma), and advanced systemic mastocytosis. CD30 expression has also been observed in several non-hematopoietic malignancies, including germ cell tumors and testicular embryonal carcinoma.

[0234] The transmembrane glycoprotein CD30 is a member of the tumor necrosis factor receptor superfamily (Falini et al., Blood (1995) 85(1):1-14). Members of the TNF / TNF receptor (TNF-R) superfamily orchestrate immune responses at multiple levels, and CD30 plays a role in regulating the function or proliferation of normal lymphoid cells. CD30 was originally described as an antigen recognized by the monoclonal antibody Ki-1, which was generated by immunizing mice with the HL-derived cell line L428 (Muta and Podack, Immunol Res (2013) 57:151-158). Expression of the CD30 antigen has been used to identify ALCL and Reed-Sternberg cells in Hodgkin's disease (Falini et al., Blood (1995) 85(1):1-14). Therefore, due to its widespread expression in lymphoma malignant cells, CD30 is a promising target for developing both antibody-based immunotherapy and cell-based therapy. Importantly, CD30 is not normally expressed on normal tissues under physiological conditions, and is therefore not present on resting mature or precursor B cells or T cells in particular (Younes and Ansell, Semin Hematol (2016) 53:186-189). Brentuximab vedotin, an antibody-drug conjugate targeting CD30, was initially approved for the treatment of CD30-positive HL (Adcetris® US Package Insert 2018). Clinical trial data for brentuximab vedotin support CD30 as a therapeutic target for the treatment of CD30-positive lymphoma.

[0235] Hodgkin lymphoma (HL) is a rare malignant tumor involving the lymph nodes and lymphatic system. The incidence of HL is bimodal, with most patients diagnosed between the ages of 15 and 30, followed by another peak in adults aged 55 or older. In 2019, an estimated 8,110 new cases (3,540 women and 4,570 men) were reported in the United States, and 1,000 people (410 women and 590 men) died from the disease (American Cancer Society 2019). Based on cases from 2012 to 2016 in the National Cancer Institute's SEER database, the incidence rates for pediatric HL patients in the United States are as follows: 1-4 years: 0.1 per 100,000; 5-9 years: 0.3; 10-14 years: 1.3; and 15-19 years: 3.3 per 100,000 (SEER Cancer Statistics Review, 1975-2016). The World Health Organization (WHO) classification divides HL into two major types: classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). In Western countries, cHL accounts for 95% of all HL cases, and NLPHL accounts for 5% (National Comprehensive Cancer Network Guidelines 2019).

[0236] First-line chemotherapy for cHL patients with advanced disease is associated with a cure rate of 70%–75% (Karantanos et al., Blood Lymphat Cancer (2017) 7:37–52). For patients who relapse after first-line therapy, salvage chemotherapy followed by autologous stem cell transplantation (ASCT) is commonly used. Unfortunately, up to 50% of cHL patients experience disease recurrence after ASCT. The median overall survival for patients who relapse after ASCT is approximately 2 years (Alinari Blood (2016) 127:287–295). Despite aggressive combination chemotherapy, 10%–40% of patients do not respond to salvage chemotherapy, and no randomized clinical trial data support ASCT in non-responders. For patients who do not respond to salvage chemotherapy, who relapse after ASCT, or who are not candidates for this approach, the prognosis remains grim, and new treatment approaches are urgently needed (Keudell British Journal of Haematology (2019) 184:105-112).

[0237] Although the majority of pediatric patients (children, adolescents, and young adults) are cured with currently available therapies, a small proportion of patients have refractory or recurrent disease and may require novel therapies with acceptable safety profiles combined with improved efficacy benefits (Flerlage et al., Blood (2018) 132:376-384; Kelly, Blood (2015) 126:2452-2458; McClain and Kamdar, in UpToDate 2019; Moskowitz, ASCO Educational Book (2019) 477-486). HL patients treated with high-dose chemotherapy during childhood commonly experience treatment-related long-term sequelae, including cardiac, pulmonary, gonadal, and endocrine toxicity, as well as secondary malignant neoplasms (Castellino et al., Blood (2011) 117(6):1806-1816).

[0238] In some embodiments, the cancer to be treated / prevented is an EBV-associated cancer. EBV infection is involved in several cancers, for example, as reviewed in Jha et al., Front Microbiol. (2016) 7:1602, the entire contents of which are incorporated herein by reference. In some embodiments, the cancer is a cancer caused or exacerbated by EBV infection, a cancer for which EBV infection is a risk factor, and / or a cancer for which EBV infection is positively associated with the onset, development, progression, severity, or metastasis of cancer. The cancer may be characterized by EBV infection, for example, the cancer may contain cells infected with EBV. Such cancer may be referred to as an EBV-positive cancer.

[0239] EBV-associated cancers that may be treated / prevented according to the present disclosure include B-cell-associated cancers such as Burkitt's lymphoma, post-transplant lymphoproliferative disorder (PTLD), central nervous system lymphoma (CNS lymphoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and EBV-associated lymphomas associated with immunodeficiency (including, for example, EBV-positive lymphomas associated with X-linked lymphoproliferative disorder, EBV-positive lymphomas associated with HIV infection / AIDS, and oral hairy leukoplakia), as well as epithelial cell-associated cancers such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC). In some embodiments, the cancer is selected from lymphoma (e.g., EBV-positive lymphoma), head and neck squamous cell carcinoma (HNSCC; e.g., EBV-positive HNSCC), nasopharyngeal carcinoma (NPC; e.g., EBV-positive NPC), and gastric cancer (GC; e.g., EBV-positive GC).

[0240] In some embodiments, the CD30-positive cancer may be chosen from a solid tumor, a blood cancer, a hematopoietic malignancy, Hodgkin's lymphoma (HL), anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma (e.g., PTCL-NOS), T-cell leukemia, T-cell lymphoma, cutaneous T-cell lymphoma (CTCL), NK-T-cell lymphoma (e.g., extranodal NK-T-cell lymphoma), non-Hodgkin's lymphoma (NHL), B-cell non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (e.g., diffuse large B-cell lymphoma-NOS), primary mediastinal B-cell lymphoma, EBV-positive B-cell lymphoma, EBV-positive diffuse large B-cell lymphoma, advanced systemic mastocytosis, germ cell tumor, or testicular embryonal carcinoma.

[0241] In some embodiments, the cancer is selected from the group consisting of CD30-positive cancer, EBV-associated cancer, hematological cancer, myeloid hematological malignancies, hematopoietic malignancies, lymphoblastic hematological malignancies, myelodysplastic syndrome, leukemia, T-cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, Lymphoma, EBV-associated lymphoma, EBV-positive B-cell lymphoma, EBV-positive diffuse large B-cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection / AIDS, oral hairy leukoplakia, Burkitt lymphoma, post-transplant lymphoproliferative disorder, central nervous system lymphoma, anaplastic large cell lymphoma, T-cell lymphoma, ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, NK-T-cell lymphoma, extranodal NK-T-cell lymphoma, thymoma, multiple myeloma, solid tumors, epithelial cell carcinoma, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, oropharyngeal cancer, oropharyngeal cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, colon Selected from rectal cancer, colorectal cancer, colon cancer, cervical cancer, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer, ovarian cancer, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor, or testicular embryonal carcinoma.

[0242] In some embodiments, cancer may be a recurrent cancer. As used herein, "recurrent" cancer refers to a cancer that has responded to treatment (e.g., a first-line therapy for cancer), but then reappears / progresses, for example, after a period of remission. For example, a recurrent cancer may be a cancer whose growth / progression has been inhibited by treatment (e.g., a first-line therapy for cancer), and then grows / progresses.

[0243] In some embodiments, the cancer may be a refractory cancer. As used herein, "refractory" cancer refers to a cancer that has not responded to treatment (e.g., a first-line therapy for cancer). For example, a refractory cancer may be a cancer whose growth / progression has not been inhibited by treatment (e.g., a first-line therapy for cancer). In some embodiments, a refractory cancer may be a cancer in which a subject receiving treatment for cancer has not shown a partial or complete response to the treatment.

[0244] In embodiments where the cancer is anaplastic large cell lymphoma, the cancer may be relapsed or refractory to treatment with chemotherapy, brentuximab vedotin, or crizotinib. In embodiments where the cancer is peripheral T-cell lymphoma, the cancer may be relapsed or refractory to treatment with chemotherapy or brentuximab vedotin. In embodiments where the cancer is extranodal NK-T cell lymphoma, the cancer may be relapsed or refractory to treatment with chemotherapy (with or without asparaginase) or brentuximab vedotin. In embodiments where the cancer is diffuse large B-cell lymphoma, the cancer may be relapsed or refractory to treatment with chemotherapy (with or without rituximab) or CD19 CAR-T therapy. In embodiments where the cancer is primary mediastinal B-cell lymphoma, the cancer may be relapsed or refractory to treatment with chemotherapy, immune checkpoint inhibitors (e.g., PD-1 inhibitors), or CD19 CAR-T therapy.

[0245] Treating cancer with the methods of the present disclosure achieves one or more of the following effects of treatment: reducing the number of cancer cells in a subject; reducing the size of a cancerous tumor / lesion in a subject; inhibiting (e.g., preventing or slowing) the growth of cancer cells in a subject; inhibiting (e.g., preventing or slowing) the growth of a cancerous tumor / lesion in a subject; inhibiting (e.g., preventing or slowing) the onset / progression of cancer (e.g., to a later stage or to metastasis); reducing the severity of cancer symptoms in a subject; prolonging the survival (e.g., progression-free survival or overall survival) of a subject; reducing the number or activity correlates of cancer cells in a subject; and / or reducing the cancer burden in a subject.

[0246] The subject can be evaluated according to the Revised Criteria for Response Assessment: The Lugano Classification (e.g., as described in Cheson et al., J Clin Oncol (2014) 32:3059-3068, incorporated by reference above) to determine their response to treatment. In some embodiments, treatment of a subject with the methods of the present disclosure achieves one of the following: a complete response, a partial response, or stable disease. Applications for treating / preventing alloreactive immune responses The CAR-expressing immune cells and compositions of the present disclosure can be used, for example, in methods including allogeneic transplantation to treat / prevent a disease / condition in a subject. The CAR-expressing immune cells and compositions of the present disclosure are useful in methods to reduce / prevent alloreactive immune responses (particularly T cell-mediated alloreactive immune responses) and their adverse consequences.

[0247] Alloreactive T cells express CD30. Chan et al., J Immunol (2002) 169(4):1784-91, identified CD30-expressing T cells as a subset of activated T cells (which also express CD25 and CD45RO) that play an important role in the alloimmune response. CD30 expression and proliferation of CD30-expressing T cells increase in response to alloantigens. Chen et al., Blood (2012) 120(3):691-6, identified CD30 expression on the CD8+ T cell subset as a potential biomarker for GVHD and proposed CD30 as a therapeutic target for GVHD.

[0248] The utility of immune cells expressing a CD30-specific CAR in methods for reducing / preventing alloreactive immune responses is described, for example, in WO2021 / 222929 A1, the entire contents of which are incorporated herein by reference.

[0249] The CAR-expressing immune cells and compositions of the present disclosure are particularly useful in methods involving allogeneic transplantation, as well as in the treatment / production of allografts. In particular, CAR-expressing immune cells and compositions are being considered for use in the production and administration of "off-the-shelf" materials for use in therapeutic and prophylactic methods, including the administration of allogeneic materials.

[0250] As explained above, the CAR-expressing immune cells of the present disclosure are useful for treating / preventing diseases / conditions by adoptive cell transfer. The CAR-expressing immune cells of the present disclosure are less susceptible to T cell-mediated alloreactive immune responses in the recipient after adoptive transfer, and therefore exhibit enhanced proliferation / survival rates and superior therapeutic / prophylactic effects in the recipient after transfer.

[0251] The CAR-expressing immune cells and compositions of the present disclosure are also useful in methods involving allogeneic transplantation of allogeneic cells other than the CAR-expressing immune cells of the present disclosure. In particular, the CAR-expressing immune cells and compositions of the present disclosure are useful for depleting alloreactive immune cells (e.g., alloreactive T cells) from allografts (collections of cells, tissues, and organs) and subjects.

[0252] In such methods, the CAR-expressing immune cells and compositions are useful for conditioning the donor and / or recipient subject and / or treating the allograft to reduce / prevent an alloreactive immune response following allotransplantation.

[0253] Cells, tissues, and organs to be allogeneically transplanted include, for example, immune cells (e.g., adoptive cell transfer), heart, lung, kidney, liver, pancreas, intestine, face, cornea, skin, hematopoietic stem cells (bone marrow), blood, hand, leg, penis, bone, uterus, thymus, islets of Langerhans, heart valves, and ovaries. A collection of cells, tissues, and organs to be allogeneically transplanted may be referred to as an "allograft."

[0254] The disease / condition to be treated / prevented by allogeneic transplantation can be any disease / condition that derives therapeutic or prophylactic benefit from allogeneic transplantation. In some embodiments, the disease / condition to be treated / prevented by allogeneic transplantation can be, for example, T cell dysfunction, cancer, infectious disease, or autoimmune disease.

[0255] T cell dysfunction can be a disease / condition in which normal T cell function is impaired, resulting in a downregulation of a subject's immune response to pathogenic antigens produced by infection with exogenous factors such as microorganisms, bacteria, and viruses, or to pathogenic antigens produced by the host (e.g., in the form of tumor-associated antigens) in some disease states, such as some forms of cancer. T cell dysfunction can include T cell exhaustion or T cell anergy. T cell exhaustion includes a condition in which CD8+ T cells are unable to proliferate or exert T cell effector functions, such as cytotoxicity and cytokine (e.g., IFNγ) secretion, in response to antigenic stimulation. Exhausted T cells can also be characterized by the persistent expression of one or more markers of T cell exhaustion, such as PD-1, CTLA-4, LAG-3, and TIM-3. T cell dysfunction can manifest as infection or an inability to mount an effective immune response to infection. Infection can be chronic, persistent, latent, or delayed, and can be the result of bacterial, viral, fungal, or parasitic infection. Thus, treatment can be provided to patients with bacterial, viral, or fungal infections. Examples of bacterial infections include infection with Helicobacter pylori. Examples of viral infections include infection with HIV, hepatitis B, or hepatitis C. T cell dysfunction, such as tumor immune escape, can be associated with cancer. Many human tumors express tumor-associated antigens that can be recognized by T cells and induce an immune response.

[0256] Infectious disease can be, for example, bacterial, viral, fungal or parasitic infection.In some embodiments, it may be particularly desirable to treat chronic / persistent infection, for example, when this infection is associated with T cell dysfunction or T cell exhaustion.It has been well established that T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections (including viral, bacterial and parasitic) and in cancer (Wherry Nature Immunology Vol.12, No.6, p492-499, June 2011). Examples of bacterial infections that may be treated include Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp., Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria spp., Helicobacter pylori, mycobacteria (e.g., Mycobacterium tuberculosis), and the like. Examples of bacterial infections that can be treated include infections caused by Bacillus subtilis (B. tuberculosis) and Pseudomonas aeruginosa. For example, the bacterial infection can be sepsis or tuberculosis. Examples of viral infections that can be treated include infections caused by influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus, and human papillomavirus (HPV). Examples of fungal infections that can be treated include infections caused by Alternaria sp., Aspergillus sp., Candida sp., and Histoplasma sp.The fungal infection may be fungal septicemia or histoplasmosis. Examples of parasitic infections that can be treated include infections with Plasmodium species (e.g., Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi). The parasitic infection may be a disease such as malaria, leishmaniasis, or toxoplasmosis.

[0257] In some embodiments, the disease / condition is an autoimmune disease.In such embodiments, treatment can be aimed at reducing the number of autoimmune effector cells.In some embodiments, the autoimmune disease is selected from type 1 diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus.

[0258] The CAR-expressing immune cells and compositions of the present disclosure are also useful for treating / preventing alloreactive immune responses and diseases / conditions characterized by alloreactive immune responses. Diseases and conditions characterized by alloreactive immune responses include diseases / conditions caused or exacerbated by alloreactive immune responses associated with allogeneic transplants. Such diseases / conditions include graft-versus-host disease (GVHD) and graft rejection, as described in detail in Perky and Maillard Annu Rev Pathol. (2018) 13:219-245, the entire contents of which are incorporated herein by reference.

[0259] Graft-versus-host disease (GVHD) can occur after allogeneic transplantation of large numbers of donor immune cells and involves the reactivity of donor-derived immune cells against allogeneic recipient cells / tissues / organs. Graft rejection refers to the destruction of transplanted cells / tissues / organs by the recipient's immune system after transplantation. When the graft rejection is an allograft, the graft rejection can be referred to as allograft rejection.

[0260] The CAR-expressing immune cells and compositions of the present disclosure can be used to deplete alloreactive T cells in allografts that may otherwise cause graft-versus-host disease (GVHD) in the recipient upon allogeneic transplantation.

[0261] The CAR-expressing immune cells and compositions of the present disclosure can be used to deplete alloreactive T cells in donors for allografts (e.g., prior to retrieving / harvesting the allograft) that may otherwise cause GVHD in recipients upon allograft transplantation.

[0262] The CAR-expressing immune cells and compositions of the present disclosure can be used to deplete alloreactive T cells in recipients of allografts that may otherwise cause / promote graft rejection.

[0263] The present disclosure provides methods for treating / preventing graft-versus-host disease (GVHD) after allogeneic transplantation, the methods comprising administering a CAR-expressing immune cell or composition according to the present disclosure to a donor subject associated with the allograft. The present disclosure also provides methods for treating / preventing graft-versus-host disease (GVHD) after allogeneic transplantation, the methods comprising contacting the allograft with a CAR-expressing immune cell or composition according to the present disclosure. The purpose of such methods is to reduce / eliminate the ability of alloreactive immune cells in the allograft to mount an alloreactive immune response against the recipient's cells, tissues, and / or organs associated with the allograft.

[0264] The present disclosure provides a method for treating / preventing graft rejection after allogeneic transplantation, comprising administering a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject of the allograft. The purpose of such a method is to reduce / eliminate the recipient subject's ability to mount an alloreactive immune response against the allograft. The CAR-expressing immune cell is useful for eliminating immune cells in the recipient that would otherwise cause an alloreactive immune response against donor cells, tissues, and / or organs.

[0265] The present disclosure provides methods comprising depleting alloreactive immune cells (e.g., alloreactive T cells) from an allograft (e.g., a population of cells, tissues, or organs to be transplanted) comprising contacting the allograft with a CAR-expressing immune cell or composition of the present disclosure. The method may comprise administering the CAR-expressing immune cell or composition of the present disclosure to a donor subject for the allograft. The purpose of such methods is to reduce / eliminate the ability of alloreactive immune cells in the allograft to mount an alloreactive immune response against the recipient's cells, tissues, and / or organs for the allograft.

[0266] In some embodiments, the method includes one or more of the following: Obtaining / collecting a population of cells, tissues or organs from a subject; contacting the population of cells, tissues, or organs with a CAR-expressing immune cell or composition according to the present disclosure; Culturing the population of cells, tissues, or organs in vitro or ex vivo in the presence of CAR-expressing immune cells according to the present disclosure; harvesting / collecting the population of cells, tissues or organs depleted of alloreactive immune cells; and Transplanting / administering the population of cells, tissues or organs depleted of alloreactive immune cells into the subject.

[0267] The present disclosure also provides methods comprising a step of depleting alloreactive immune cells (e.g., alloreactive T cells) from a subject, comprising administering to the subject a CAR-expressing immune cell or composition of the present disclosure. The subject may be a donor subject for an allograft or may be an intended recipient subject for an allograft.

[0268] In some embodiments, the method includes one or more of the following: administering to the subject a CAR-expressing immune cell or composition according to the present disclosure so as to deplete alloreactive immune cells in the subject; Obtaining / collecting a population of cells, tissues, or organs from a subject to which a CAR-expressing immune cell or composition according to the present disclosure has been administered; and Transplanting / administering the population of cells, tissues or organs depleted of alloreactive immune cells into the subject.

[0269] In some embodiments, the method includes one or more of the following: administering to the subject a CAR-expressing immune cell or composition according to the present disclosure to deplete alloreactive immune cells in the subject; and Transplanting / administering the population of cells, tissues or organs into a subject that has previously been administered a CAR-expressing immune cell or composition according to the present disclosure.

[0270] Depletion of alloreactive immune cells can result in, for example, a 2-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or greater reduction in the amount of alloreactive immune cells in the allograft or subject.

[0271] The method may be performed in vitro or ex vivo, or in vivo in a subject. Method steps performed in vitro or ex vivo may include in vitro or ex vivo cell culture.

[0272] The method may further comprise method steps for the production of CAR-expressing immune cells and compositions according to the present disclosure. In some embodiments, administration of a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for allogeneic transplantation and the allogeneic transplantation are performed simultaneously (i.e., at the same time or, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, or 48 hours).

[0273] In some embodiments, the administration of CAR-expressing immune cells or compositions according to the present disclosure to a recipient subject for allogeneic transplantation and allogeneic transplantation are carried out sequentially.The time interval between the administration of CAR-expressing immune cells or compositions and allogeneic transplantation can be any time interval, including several hours, several days, several weeks, several months, or several years.The CAR-expressing immune cells or compositions can be administered to the recipient subject before or after allogeneic transplantation.The CAR-expressing immune cells or compositions are preferably administered to the recipient subject prior to allogeneic transplantation.

[0274] In some embodiments, the administration of CAR-expressing immune cells or compositions according to the present disclosure to a donor subject for allogeneic transplantation and the collection of the allograft (i.e., collection of cells, tissues, and / or organs) from the subject are performed simultaneously (i.e., at the same time, or within, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, or 48 hours). In some embodiments, the administration of CAR-expressing immune cells or compositions according to the present disclosure to a donor subject for allogeneic transplantation and the collection of the allograft (i.e., collection of cells, tissues, and / or organs) from the subject are performed sequentially. The time interval between the administration of the CAR-expressing immune cells or compositions and the collection of the allograft can be any time interval, including hours, days, weeks, months, or years. The CAR-expressing immune cells or compositions can be administered to the donor subject before or after the collection of the allograft. The CAR-expressing immune cells or compositions are preferably administered to the donor subject prior to the collection of the allograft.

[0275] In some embodiments, the methods include further interventions to treat / prevent an alloreactive immune response, graft rejection and / or GVHD. In some embodiments, methods of treating / preventing alloreactivity, graft rejection and / or GVHD include administration of immunosuppressive and / or lymphadenectomy therapy, such as treatment with corticosteroids (e.g., prednisolone, hydrocortisone), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), antiproliferative agents (e.g., azathioprinem, mycophenolic acid) and / or mTOR inhibitors (e.g., sirolimus, everolimus).

[0276] In some embodiments, methods for treating / preventing alloreactive reactions and / or graft rejection include antibody therapy, such as treatment with monoclonal anti-IL-2Rα receptor antibodies (e.g., basiliximab, daclizumab), anti-T cell antibodies (e.g., anti-thymocyte globulin, anti-lymphocyte globulin), and / or anti-CD20 antibodies (e.g., rituximab).

[0277] In some embodiments, methods of treating / preventing alloreactivity and / or graft rejection include blood transfusion and / or bone marrow transplantation. Where a method is disclosed herein, the disclosure also provides the CAR-expressing immune cells and compositions of the disclosure for use in such a method. Also provided is the use of the CAR-expressing immune cells or compositions of the disclosure in the manufacture of a product (e.g., a medicament) for use in such a method.

[0278] In some embodiments, the methods of various aspects of the present disclosure cause less depletion and / or increased survival of non-allo-reactive immune cells when compared to methods using immunosuppressant(s). For example, the methods are useful for preserving / maintaining the non-allo-reactive immune cell compartment in a recipient subject for an allograft or in an allograft.

[0279] In some embodiments of the disclosed methods involving allogeneic transplantation, the methods are associated with an increased number / proportion of non-allo-reactive immune cells in the recipient subject with respect to the allograft when compared to methods involving treatment with an immunosuppressant. In some embodiments of the disclosed methods involving adoptive transfer of allogeneic immune cells, the methods are associated with an increased number / proportion of non-allo-reactive immune cells in the recipient subject with respect to the allogeneic immune cells when compared to methods involving treatment with an immunosuppressant.

[0280] In some aspects of the disclosed methods involving allogeneic transplantation, the methods are associated with an increase in the number / proportion of non-allo-reactive immune cells in the allograft when compared to methods involving treatment with immunosuppressants.

[0281] The present disclosure also provides a CAR-expressing immune cell or composition of the present disclosure for use in the following methods: Killing cells that express the target antigen for which the CAR is specific (e.g., cells that express CD30); and / or Killing alloreactive immune cells (e.g., T cells expressing CD30).

[0282] The present disclosure also provides the use of such CAR-expressing immune cells and compositions in such methods, as well as methods of using the CAR-expressing immune cells and compositions for such purposes. Administration Administration of the articles of the present disclosure is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is an amount sufficient to provide a therapeutic or prophylactic benefit to the subject. The actual amount administered, and the rate and time course of administration, will depend on the nature and severity of the disease / condition and the particular article being administered. Prescribing treatment, such as determining dosage, is within the responsibility of a general practitioner or other physician, and typically takes into account the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and protocols described above can be found in Remington's "The Science and Practice of Pharmacy" (A. Adejare, ed.), 23rd Edition (2020), Academic Press.

[0283] Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, intranasal, or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral.

[0284] In some aspects and embodiments of the present disclosure, the article of the present disclosure is delivered in a targeted manner, i.e., the concentration of the relevant drug in a subject is increased in some parts of the body compared to other parts of the body. In some embodiments, the method includes intravenous, intraarterial, intramuscular, or subcutaneous administration, in which case the relevant article is formulated in the form of a targeted drug delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes, or micro-sized silica rods. Such systems may also include magnetic elements that direct the drug to the desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to those skilled in the art.

[0285] In some cases, the articles of the present disclosure are formulated for targeted delivery to specific cells, tissues, organs and / or tumors. Further interventions Administration can be alone or in combination with other treatments, either simultaneously or sequentially, depending on the disease / condition to be treated. The antigen-binding molecules, CARs, cells or compositions described herein and another prophylactic / therapeutic agent can be administered simultaneously or sequentially.

[0286] In some embodiments, the method includes additional therapeutic or preventive intervention, for example, for the treatment / prevention of cancer. In some embodiments, the therapeutic or preventive intervention is selected from chemotherapeutic agents, immunotherapy, radiation therapy, surgery, vaccination, and / or hormone therapy. In some embodiments, the therapeutic or preventive intervention includes leukapheresis. In some embodiments, the therapeutic or preventive intervention includes stem cell transplantation.

[0287] Simultaneous administration refers to the administration of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), cell, or composition and a therapeutic agent together, for example, as a pharmaceutical composition (combined preparation) containing both agents, or administered immediately after each other, optionally via the same administration route, for example, into the same artery, vein, or other blood vessel. Sequential administration refers to the administration of one of the antigen-binding molecule / composition or therapeutic agent, followed by separate administration of the other agent after a given time interval. The two agents do not necessarily have to be administered by the same route, although in some embodiments they are. The time interval may be any time interval.

[0288] In some embodiments, the cancer treatment further includes chemotherapy and / or radiation therapy. Chemotherapy and radiation therapy refer to the treatment of cancer with drugs or ionizing radiation (e.g., radiation therapy using X-rays or gamma rays), respectively. The drug may be a chemical entity, such as a small molecule drug, an antibiotic, a DNA intercalator, a protein inhibitor (e.g., a kinase inhibitor), or a biological substance, such as an antibody, an antibody fragment, an aptamer, a nucleic acid (e.g., DNA, RNA), a peptide, a polypeptide, or a protein. The drug may be formulated as a pharmaceutical composition or medicament. The formulation may include one or more drugs (e.g., one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0289] Chemotherapeutic agents may involve the administration of more than one drug, which may be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated.

[0290] The chemotherapeutic agent may be administered by one or more routes of administration, for example, parenterally, intravenously, orally, subcutaneously, intradermally, or intratumorally. Chemotherapeutic agents may be administered according to a treatment plan. A treatment plan may be a predetermined timetable, plan, scheme, or schedule for administering chemotherapy, which can be created by a doctor or medical professional and can be tailored to the patient who needs treatment. A treatment plan may indicate one or more of the following: the type of chemotherapy agent to be administered to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; and, if there is a treatment break, the number and nature of any treatment breaks. In the case of simultaneous therapy, a single treatment plan may be provided that indicates how each drug should be administered.

[0291] Chemotherapy drugs include abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (albumin-stabilized nanoparticle formulation of paclitaxel), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta ( Pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran (melphalan hydrochloride) for injection, Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), Arsenic trioxide, Arzera (ofatumumab), Asparaginase from Erwinia blackleg chrysanthemi), atezolizumab, Avastin (bevacizumab), avelumab, axiconib-ciloreucel, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodak (belinostat), belinamustine hydrochloride, BEP, Besponsa (inotuzumab ozolomide) gamycin), bevacizumab, bexarotene, Bexar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Bilincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan),Cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Calquence (acalabrutinib), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (topical fluorouracil), carboplatin, carboplatin-taxol, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide) ), CEM, ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), chloral (clofarabine), CMF, cobimetinib, Cometrik (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-AB V, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride Salt and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, DepoCyt (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab,Efudex (topical fluorouracil), Elitek (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, Epoch, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase derived from blackleg disease bacteria), Ethio (amifostine), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil topical), Fairston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), Filgra Stim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (topical fluorouracil), fluorouracil injection, topical fluorouracil, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, Folfox, Folotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil Lu9 (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Giotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemandiol (propranolol hydrochloride),Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nonavalent vaccine, recombinant HPV quadrivalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), Ifo Sufamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene laherparepvec), Inlyta (Axitinib), Inotuzumab ozogamicin, Interferon alfa-2b, Recombinant Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon alfa-2b), Iodine I131 Tositumomab and Tositumomab Mab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah ( Tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukelan (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levran (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposomal),Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marquibo (vincristine sulfate liposomal), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), metazolastone (Methazolamide) olastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), mutamycin (mitomycin C), Myleran (busulfan), Mirosar Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (albumin-stabilized nanoparticle formulation of paclitaxel), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexa Bar (sorafenib tosylate), Nilandrone (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal),Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, albumin-stabilized nanoparticle formulation of paclitaxel, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate dihydrate, Tritium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst ( pomalidomide), ponatinib hydrochloride, Portraza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human Human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, Relistol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycera (rituximab and human-derived hyaluronidase), rituximab, rituximab and human-derived hyaluronan Nidase, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparibe camsylate), rucaparibe camsylate, ruxolitinib phosphate, Rydapt (midostaurin), Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc), Steritalc (talc),Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxine mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (venom) Xarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (topical fluorouracil), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I-131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, tra Bectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, valrubicin, Valstar (valrubicin), vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib) vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate),Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xelori, Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicabtagene cilolucel), Yondelis (trabectedin), Zaltrap (Z iv-aflibercept), Zarxio (filgrastim), Zejula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zynecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0292] In some embodiments, treatment may include administration of corticosteroids, such as dexamethasone and / or prednisone. In some embodiments, the subject is administered lymphodepleting chemotherapy prior to administration of immune cells expressing / comprising a CAR described herein (or expressing / comprising a nucleic acid encoding such a CAR).

[0293] That is, in some embodiments, a method of treating / preventing a disease / condition in accordance with the present disclosure comprises (i) administering lymphodepleting chemotherapy to a subject, and (ii) thereafter administering immune cells that express / comprise a CAR according to the present disclosure or that express / comprise a nucleic acid encoding a CAR according to the present disclosure.

[0294] "Lymphodepleting chemotherapy," as used herein, refers to treatment with a chemotherapeutic agent that results in the depletion of lymphocytes (e.g., T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors) in the subject to whom the treatment is administered. "Lymphodepleting chemotherapeutic agent" refers to a chemotherapeutic agent that results in lymphocyte depletion.

[0295] Lymphodepleting chemotherapy and its use in methods of treatment by adoptive cell transfer are described, for example, in Klebanoff et al., Trends Immunol. (2005) 26(2):111-7 and Muranski et al., Nat Clin Pract Oncol. (2006)(12):668-81, both of which are incorporated herein by reference in their entireties. The goal of lymphodepleting chemotherapy is to deplete the recipient subject's endogenous lymphocyte populations.

[0296] In the context of disease treatment by adoptive transfer of immune cells, lymphodepleting chemotherapy is typically administered before adoptive cell transfer to prepare recipient subject to receive adoptively transferred cells.Lymphodepleting chemotherapy is thought to promote the persistence and activity of adoptively transferred cells by, for example, creating a permissive environment through the elimination of cells that express immunosuppressive cytokines, and creating the "lymphocyte space" that is necessary for the expansion and activity of adoptively transferred lymphoid cells.

[0297] Chemotherapeutic agents commonly used in lymphodepleting chemotherapy include, for example, fludarabine, cyclophosphamide, bedamustine, and pentostatin.

[0298] Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or nucleic acids), expression vector (or expression vectors), cell, or composition may be provided, and one or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0299] The multiple doses may be separated by predetermined time intervals, which may be selected to be one 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, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. As an example, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0300] According to various aspects of the present disclosure, methods of treating and / or preventing a disease / condition may include one or more of the following: reducing the number / ratio of cells expressing a target antigen for a CAR (e.g., CD30); inhibiting tumor growth (e.g., of a tumor expressing a target antigen for a CAR); reducing metastasis of a cancer (e.g., a cancer expressing a target antigen for a CAR); or increasing the survival rate of a subject with a cancer (e.g., a cancer expressing a target antigen for a CAR). subject The subject according to various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in humans or animals (veterinary use).

[0301] The subject to which the article of the present disclosure is to be administered (e.g., following a therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient.

[0302] The subject may have (e.g., been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / obtaining such a disease / condition. In embodiments according to the present disclosure, subjects may be selected for treatment by the present methods based on their characterization with respect to one or more markers for such a disease / condition.

[0303] In some embodiments, a subject can be selected for a therapeutic or prophylactic intervention as described herein based on, for example, detection of cells / tissues that express a target antigen for a CAR (e.g., CD30) or that overexpress a target antigen for a CAR in a sample obtained from the subject.

[0304] The subject may be an allogeneic subject for intervention according to the present disclosure. The subject to be treated / prevented according to the present disclosure may be genetically non-identical to the subject from which the CAR-expressing immune cells are derived. The subject to be treated / prevented according to the present disclosure may be HLA-mismatched to the subject from which the CAR-expressing immune cells are derived. The subject to be treated / prevented according to the present disclosure may be HLA-matched to the subject from which the CAR-expressing immune cells are derived.

[0305] The subject to whom cells are administered in accordance with the present disclosure may be allogeneic / non-autologous with respect to the source from which the cells are derived. The subject to whom cells are administered may be a different subject from the subject from which the cells were obtained / derived for the production of the cells to be administered. The subject to whom cells are administered may be genetically non-identical to the subject from which the cells were obtained / derived for the production of the cells to be administered.

[0306] The subject to whom the cells are administered may contain MHC / HLA genes encoding MHC / HLA molecules that are not identical to the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from which the cells were / are obtained for the production of the cells to be administered.The subject to whom the cells are administered may contain MHC / HLA genes encoding MHC / HLA molecules that are identical to the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from which the cells were / are obtained for the production of the cells to be administered.

[0307] In some embodiments, the subject to whom the cells are administered is HLA-matched to the subject from whom the cells were obtained / derived for the production of the cells to be administered. In some embodiments, the subject to whom the cells are administered is nearly or perfectly HLA-matched to the subject from whom the cells were obtained / derived for the production of the cells to be administered.

[0308] In some embodiments, the subject is ≧4 / 8 (i.e., 4 / 8, 5 / 8, 6 / 8, 7 / 8, or 8 / 8) matched across HLA-A, -B, -C, and -DRB1. In some embodiments, the subject is ≧5 / 10 (i.e., 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10, or 10 / 10) matched across HLA-A, -B, -C, -DRB1, and -DQB1. In some embodiments, the subject is ≧6 / 12 (i.e., 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, 11 / 12, or 12 / 12) matched across HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1. In some embodiments, the subject is 8 / 8 matched across HLA-A, -B, -C, and -DRB1. In some embodiments, the subject is 10 / 10 matched across HLA-A, -B, -C, -DRB1 and -DQB1, hi some embodiments, the subject is 12 / 12 matched across HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1. kit In some aspects of the present disclosure, a kit of parts is provided. In some embodiments, the kit may have at least one container with a predetermined amount of the CAR, nucleic acid (or multiple nucleic acids), expression vector (or multiple expression vectors), cell or composition described herein.

[0309] In some embodiments, the kit may include materials for producing a CAR, nucleic acid(s), expression vector(s), cell(s), or composition described herein.

[0310] The kit can provide a CAR, nucleic acid(s), expression vector(s), cells or compositions to treat a specified disease / condition, along with instructions for administration to a patient.

[0311] In some embodiments, the kit may further include at least one container with a predetermined amount of another therapeutic agent (e.g., as described herein). In such embodiments, the kit may also include a second medicament or pharmaceutical composition, such that the two medicaments or pharmaceutical compositions can be administered simultaneously or separately to provide a combined treatment for a particular disease or condition.

[0312] Kits according to the present disclosure may include instructions, for example in the form of an instruction booklet or leaflet, which may include protocols for carrying out any one or more of the methods described herein. Sequence identity "Sequence identity," as used herein, refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780). When using such software, default parameters are preferably used, for example, with respect to gap penalties and extension penalties. array

[0313] [Table 1-1]

[0314] [Table 1-2]

[0315] [Table 1-3]

[0316] [Table 1-4]

[0317]

Table 1-5

[0318]

Table 1-6

[0319]

Table 1-7

[0320]

Table 1-8

[0321]

Table 1-9

[0322]

Table 1-10

[0323]

Table 1-11

[0324]

Table 1-12

[0325]

Table 1-13

[0326]

Table 1-14

[0327]

Table 1-15

[0328]

Table 1-16

[0329]

Table 1-17

[0330]

Table 1-18

[0331]

Table 1-19

[0332]

Table 1-20

[0333]

Table 1-21

[0334]

Table 1-22

[0335]

Table 1-23

[0336]

Table 1-24

[0337]

Table 1-25

[0338] [Table 1-26]

[0339] [Table 2-1]

[0340] [Table 2-2]

[0341] [Table 3-1]

[0342] [Table 3-2]

[0343] [Table 4-1]

[0344] [Table 4-2]

[0345] The present disclosure includes combinations of the described aspects and preferred features except where such combinations are clearly unacceptable or explicitly avoided. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0346] Aspects and embodiments of the present disclosure will now be illustrated by way of example with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this document are incorporated herein by reference.

[0347] Throughout this specification, including the claims set out below, and unless the context requires otherwise, the terms "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0348] As used herein, an amino acid sequence or region of a polypeptide that is "corresponding" to a specified reference amino acid sequence or region of a polypeptide has at least 60%, for example at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence that is "corresponding" to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, for example using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960).

[0349] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0350] When a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated. The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed with cultured cells, whereas the term "in vivo" is intended to encompass procedures with / on intact multicellular organisms.

[0351] Aspects and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings. [Brief explanation of the drawings]

[0352] [Figure 1A] Expression and functionality evaluation of novel alternative spacers in CD30 CARs. (1A) Expression of CD30 CARs with various spacers on the surface of transduced primary T cells. (1B) Cytotoxic efficacy of CAR T cells expressing CARs with various spacers when used with CD30+KM-H2 cells. (1C) Basal GM-CSF, interferon-γ, and TNF-α cytokine secretion of CD30 CAR T cells with various spacers for 24 hours with or without stimulation with KM-H2 cells. [Figure 1B]Expression and functionality evaluation of novel alternative spacers in CD30 CARs. (1A) Expression of CD30 CARs with various spacers on the surface of transduced primary T cells. (1B) Cytotoxic efficacy of CAR T cells expressing CARs with various spacers when used with CD30+KM-H2 cells. (1C) Basal GM-CSF, interferon-γ, and TNF-α cytokine secretion of CD30 CAR T cells with various spacers for 24 hours with or without stimulation with KM-H2 cells. [Figure 1C] Expression and functionality evaluation of novel alternative spacers in CD30 CARs. (1A) Expression of CD30 CARs with various spacers on the surface of transduced primary T cells. (1B) Cytotoxic efficacy of CAR T cells expressing CARs with various spacers when used with CD30+KM-H2 cells. (1C) Basal GM-CSF, interferon-γ, and TNF-α cytokine secretion of CD30 CAR T cells with various spacers for 24 hours with or without stimulation with KM-H2 cells. [Figure 2A] Functional evaluation of novel alternative spacers in CD30 CARs using Raji expressing various CD30 densities. (2A) Flow cytometry analysis of transduced Raji clones 1B1, 1C4, and 2C5 expressing various levels of CD30 compared to CD30-expressing KM-H2 cells. (2B) Cytotoxicity assay of Raji clones expressing various levels of CD30 using CD30 CAR T cells expressing IgG1, IgG2, OX40, 4-1BB, CD96, and CD33 spacers at an effector-target ratio of 1:1. (2C) Interferon-γ, GM-CSF, and TNF-α secretion by CD30 CAR T cells expressing various spacers upon activation with various CD30+ cell lines. Data are representative of three independent donors tested. [Figure 2B]Functional evaluation of novel alternative spacers in CD30 CARs using Raji expressing various CD30 densities. (2A) Flow cytometry analysis of transduced Raji clones 1B1, 1C4, and 2C5 expressing various levels of CD30 compared to CD30-expressing KM-H2 cells. (2B) Cytotoxicity assay of Raji clones expressing various levels of CD30 using CD30 CAR T cells expressing IgG1, IgG2, OX40, 4-1BB, CD96, and CD33 spacers at an effector-target ratio of 1:1. (2C) Interferon-γ, GM-CSF, and TNF-α secretion by CD30 CAR T cells expressing various spacers upon activation with various CD30+ cell lines. Data are representative of three independent donors tested. [Figure 2C] Functional evaluation of novel alternative spacers in CD30 CARs using Raji expressing various CD30 densities. (2A) Flow cytometry analysis of transduced Raji clones 1B1, 1C4, and 2C5 expressing various levels of CD30 compared to CD30-expressing KM-H2 cells. (2B) Cytotoxicity assay of Raji clones expressing various levels of CD30 using CD30 CAR T cells expressing IgG1, IgG2, OX40, 4-1BB, CD96, and CD33 spacers at an effector-target ratio of 1:1. (2C) Interferon-γ, GM-CSF, and TNF-α secretion by CD30 CAR T cells expressing various spacers upon activation with various CD30+ cell lines. Data are representative of three independent donors tested. [Figure 3A] Expression and functionality assessment of CARs expressing the novel 4-1BB spacer with a humanized CD30 scFv. (3A) Expression of a humanized CD30 CAR with a 4-1BB spacer on the surface of transduced primary T cells. (3B) Cytotoxic efficacy of CAR T cells expressing a CAR with a 4-1BB spacer. (3C) Levels of GM-CSF and TNF-α secretion by resting CAR T cells expressing a humanized CAR with 4-1BB. [Figure 3B]Expression and functionality assessment of CARs expressing the novel 4-1BB spacer with a humanized CD30 scFv. (3A) Expression of a humanized CD30 CAR with a 4-1BB spacer on the surface of transduced primary T cells. (3B) Cytotoxic efficacy of CAR T cells expressing a CAR with a 4-1BB spacer. (3C) Levels of GM-CSF and TNF-α secretion by resting CAR T cells expressing a humanized CAR with 4-1BB. [Figure 3C] Expression and functionality assessment of CARs expressing the novel 4-1BB spacer with a humanized CD30 scFv. (3A) Expression of a humanized CD30 CAR with a 4-1BB spacer on the surface of transduced primary T cells. (3B) Cytotoxic efficacy of CAR T cells expressing a CAR with a 4-1BB spacer. (3C) Levels of GM-CSF and TNF-α secretion by resting CAR T cells expressing a humanized CAR with 4-1BB. [Figure 4A] Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 4B]Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 4C] Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 4D]Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 4E] Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 4F]Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 4G] Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experimental layout of the assay evaluating monocyte interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with various spacers after co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with various spacers. (4E) Experimental layout of the assay evaluating NK cell interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and the number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells carrying various spacers. [Figure 5A]In vivo persistence and efficacy of CD30 CAR ATC with a novel spacer against NK-T lymphoma cell lines. (5A) Experimental scheme for the SNK6 model. (5B) Twice-weekly IVIS monitoring to track T cell biodistribution. (5C) Mean radiance of transferred T cells in tumors after treatment. (5D) Endpoint mean radiance measured by IVIS and flow cytometry quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry. [Figure 5B] In vivo persistence and efficacy of CD30 CAR ATC with a novel spacer against NK-T lymphoma cell lines. (5A) Experimental scheme for the SNK6 model. (5B) Twice-weekly IVIS monitoring to track T cell biodistribution. (5C) Mean radiance of transferred T cells in tumors after treatment. (5D) Endpoint mean radiance measured by IVIS and flow cytometry quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry. [Figure 5C] In vivo persistence and efficacy of CD30 CAR ATC with a novel spacer against NK-T lymphoma cell lines. (5A) Experimental scheme for the SNK6 model. (5B) Twice-weekly IVIS monitoring to track T cell biodistribution. (5C) Mean radiance of transferred T cells in tumors after treatment. (5D) Endpoint mean radiance measured by IVIS and flow cytometry quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry. [Figure 5D]In vivo persistence and efficacy of CD30 CAR ATC with a novel spacer against NK-T lymphoma cell lines. (5A) Experimental scheme for the SNK6 model. (5B) Twice-weekly IVIS monitoring to track T cell biodistribution. (5C) Mean radiance of transferred T cells in tumors after treatment. (5D) Endpoint mean radiance measured by IVIS and flow cytometry quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry. [Figure 5E] In vivo persistence and efficacy of CD30 CAR ATC with a novel spacer against NK-T lymphoma cell lines. (5A) Experimental scheme for the SNK6 model. (5B) Twice-weekly IVIS monitoring to track T cell biodistribution. (5C) Mean radiance of transferred T cells in tumors after treatment. (5D) Endpoint mean radiance measured by IVIS and flow cytometry quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry. [Figure 5F] In vivo persistence and efficacy of CD30 CAR ATC with a novel spacer against NK-T lymphoma cell lines. (5A) Experimental scheme for the SNK6 model. (5B) Twice-weekly IVIS monitoring to track T cell biodistribution. (5C) Mean radiance of transferred T cells in tumors after treatment. (5D) Endpoint mean radiance measured by IVIS and flow cytometry quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry. [Figure 6A]In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice xenografted with peripheral T-cell lymphoma. (6A) Experimental setup of the human peripheral T-cell lymphoma model. (6B) Twice-weekly IVIS monitoring to track T-cell biodistribution and (6C) endpoint mean radiance measured by IVIS. (6D) Absolute tumor counts quantified by flow cytometry. (6E) Cytokine levels in mouse plasma 6 days after treatment. [Figure 6B] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice xenografted with peripheral T-cell lymphoma. (6A) Experimental setup of the human peripheral T-cell lymphoma model. (6B) Twice-weekly IVIS monitoring to track T-cell biodistribution and (6C) endpoint mean radiance measured by IVIS. (6D) Absolute tumor counts quantified by flow cytometry. (6E) Cytokine levels in mouse plasma 6 days after treatment. [Figure 6C] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice xenografted with peripheral T-cell lymphoma. (6A) Experimental setup of the human peripheral T-cell lymphoma model. (6B) Twice-weekly IVIS monitoring to track T-cell biodistribution and (6C) endpoint mean radiance measured by IVIS. (6D) Absolute tumor counts quantified by flow cytometry. (6E) Cytokine levels in mouse plasma 6 days after treatment. [Figure 6D] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice xenografted with peripheral T-cell lymphoma. (6A) Experimental setup of the human peripheral T-cell lymphoma model. (6B) Twice-weekly IVIS monitoring to track T-cell biodistribution and (6C) endpoint mean radiance measured by IVIS. (6D) Absolute tumor counts quantified by flow cytometry. (6E) Cytokine levels in mouse plasma 6 days after treatment. [Figure 6E]In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice xenografted with peripheral T-cell lymphoma. (6A) Experimental setup of the human peripheral T-cell lymphoma model. (6B) Twice-weekly IVIS monitoring to track T-cell biodistribution and (6C) endpoint mean radiance measured by IVIS. (6D) Absolute tumor counts quantified by flow cytometry. (6E) Cytokine levels in mouse plasma 6 days after treatment. [Figure 7A] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 7B] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 7C]In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 7D] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 7E] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 7F]In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 7G] In vivo persistence, efficacy, and toxicity of CD30 CAR ATC in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experimental setup of the CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days after IVIG administration. (7C) Body weight change after T cell treatment. (7D) IVIS images taken twice a week to follow NALM-6 disease. (7E) CD30 CAR T cells present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 8A] Generation and characterization of T cells bearing a humanized CD30 CAR with a 4-1BB spacer. (8A) Fold expansion in cultures of untransduced T cells and T cells expressing HRS3-41BB, VH3Vk3-Cys-41BB, or VH5Vk3-Cys-41BB. (8B) Transduction efficiency of CD30CAR variants at day 11. (8C) Ratio of CD4 T cells to CD8 T cells in T cells transduced with CD30CAR variants at day 11. Each donor represents a unique donor consistent across 8A to 8C. (8D) CD30 expression detected by BerH8 and BY88 clones on total T cells over the culture period to demonstrate CD30 masking in cis by the CAR. (8E) Ratio of Tim3-, PD-1-, LAG3-expressing cells, and their combinations, among CD4 and CD8 T cells in the final product at day 11. [Figure 8B]Generation and characterization of T cells bearing a humanized CD30 CAR with a 4-1BB spacer. (8A) Fold expansion in cultures of untransduced T cells and T cells expressing HRS3-41BB, VH3Vk3-Cys-41BB, or VH5Vk3-Cys-41BB. (8B) Transduction efficiency of CD30CAR variants at day 11. (8C) Ratio of CD4 T cells to CD8 T cells in T cells transduced with CD30CAR variants at day 11. Each donor represents a unique donor consistent across 8A to 8C. (8D) CD30 expression detected by BerH8 and BY88 clones on total T cells over the culture period to demonstrate CD30 masking in cis by the CAR. (8E) Ratio of Tim3-, PD-1-, LAG3-expressing cells, and their combinations, among CD4 and CD8 T cells in the final product at day 11. [Figure 8C] Generation and characterization of T cells bearing a humanized CD30 CAR with a 4-1BB spacer. (8A) Fold expansion in cultures of untransduced T cells and T cells expressing HRS3-41BB, VH3Vk3-Cys-41BB, or VH5Vk3-Cys-41BB. (8B) Transduction efficiency of CD30CAR variants at day 11. (8C) Ratio of CD4 T cells to CD8 T cells in T cells transduced with CD30CAR variants at day 11. Each donor represents a unique donor consistent across 8A to 8C. (8D) CD30 expression detected by BerH8 and BY88 clones on total T cells over the culture period to demonstrate CD30 masking in cis by the CAR. (8E) Ratio of Tim3-, PD-1-, LAG3-expressing cells, and their combinations, among CD4 and CD8 T cells in the final product at day 11. [Figure 8D]Generation and characterization of T cells bearing a humanized CD30 CAR with a 4-1BB spacer. (8A) Fold expansion in cultures of untransduced T cells and T cells expressing HRS3-41BB, VH3Vk3-Cys-41BB, or VH5Vk3-Cys-41BB. (8B) Transduction efficiency of CD30CAR variants at day 11. (8C) Ratio of CD4 T cells to CD8 T cells in T cells transduced with CD30CAR variants at day 11. Each donor represents a unique donor consistent across 8A to 8C. (8D) CD30 expression detected by BerH8 and BY88 clones on total T cells over the culture period to demonstrate CD30 masking in cis by the CAR. (8E) Ratio of Tim3-, PD-1-, LAG3-expressing cells, and their combinations, among CD4 and CD8 T cells in the final product at day 11. [Figure 8E] Generation and characterization of T cells bearing a humanized CD30 CAR with a 4-1BB spacer. (8A) Fold expansion in cultures of untransduced T cells and T cells expressing HRS3-41BB, VH3Vk3-Cys-41BB, or VH5Vk3-Cys-41BB. (8B) Transduction efficiency of CD30CAR variants at day 11. (8C) Ratio of CD4 T cells to CD8 T cells in T cells transduced with CD30CAR variants at day 11. Each donor represents a unique donor consistent across 8A to 8C. (8D) CD30 expression detected by BerH8 and BY88 clones on total T cells over the culture period to demonstrate CD30 masking in cis by the CAR. (8E) Ratio of Tim3-, PD-1-, LAG3-expressing cells, and their combinations, among CD4 and CD8 T cells in the final product at day 11. [Figure 9A]In vitro antitumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (9A) Cytolysis of KM-H2 and HuT-78 cells was assessed using the xCELLigence Real-Time Cell Analysis system at an E:T ratio of 2:1 for four and five donors, respectively. (9B) Serial killing assay setup. See Example 1. (9C) Serial killing cell lysis for three donors. (9D) Total T cell counts at the end of each serial killing cell encounter. [Figure 9B] In vitro antitumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (9A) Cytolysis of KM-H2 and HuT-78 cells was assessed using the xCELLigence Real-Time Cell Analysis system at an E:T ratio of 2:1 for four and five donors, respectively. (9B) Serial killing assay setup. See Example 1. (9C) Serial killing cell lysis for three donors. (9D) Total T cell counts at the end of each serial killing cell encounter. [Figure 9C] In vitro antitumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (9A) Cytolysis of KM-H2 and HuT-78 cells was assessed using the xCELLigence Real-Time Cell Analysis system at an E:T ratio of 2:1 for four and five donors, respectively. (9B) Serial killing assay setup. See Example 1. (9C) Serial killing cell lysis for three donors. (9D) Total T cell counts at the end of each serial killing cell encounter. [Figure 9D]In vitro antitumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (9A) Cytolysis of KM-H2 and HuT-78 cells was assessed using the xCELLigence Real-Time Cell Analysis system at an E:T ratio of 2:1 for four and five donors, respectively. (9B) Serial killing assay setup. See Example 1. (9C) Serial killing cell lysis for three donors. (9D) Total T cell counts at the end of each serial killing cell encounter. [Figure 10A] In vitro on-target, off-tumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (10A) CD30 expression on HSPCs from two donors stimulated with 10 ng / mL FLT3L, SCF, and TPO for 12 days. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Effect of CD30 CAR T-cell exposure on HSPC subset viability. Two HSPC donors and two T-cell donors were combined to yield four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T-cell exposure on erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with a CD30-high KM-H2 target on cytolysis of CD30-low HSPCs. [Figure 10B]In vitro on-target, off-tumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (10A) CD30 expression on HSPCs from two donors stimulated with 10 ng / mL FLT3L, SCF, and TPO for 12 days. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Effect of CD30 CAR T-cell exposure on HSPC subset viability. Two HSPC donors and two T-cell donors were combined to yield four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T-cell exposure on erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with a CD30-high KM-H2 target on cytolysis of CD30-low HSPCs. [Figure 10C] In vitro on-target, off-tumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (10A) CD30 expression on HSPCs from two donors stimulated with 10 ng / mL FLT3L, SCF, and TPO for 12 days. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Effect of CD30 CAR T-cell exposure on HSPC subset viability. Two HSPC donors and two T-cell donors were combined to yield four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T-cell exposure on erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with a CD30-high KM-H2 target on cytolysis of CD30-low HSPCs. [Figure 10D]In vitro on-target, off-tumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (10A) CD30 expression on HSPCs from two donors stimulated with 10 ng / mL FLT3L, SCF, and TPO for 12 days. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Effect of CD30 CAR T-cell exposure on HSPC subset viability. Two HSPC donors and two T-cell donors were combined to yield four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T-cell exposure on erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with a CD30-high KM-H2 target on cytolysis of CD30-low HSPCs. [Figure 10E] In vitro on-target, off-tumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (10A) CD30 expression on HSPCs from two donors stimulated with 10 ng / mL FLT3L, SCF, and TPO for 12 days. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Effect of CD30 CAR T-cell exposure on HSPC subset viability. Two HSPC donors and two T-cell donors were combined to yield four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T-cell exposure on erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with a CD30-high KM-H2 target on cytolysis of CD30-low HSPCs. [Figure 10F]In vitro on-target, off-tumor activity of T cells carrying a humanized CD30 CAR with a 4-1BB spacer. (10A) CD30 expression on HSPCs from two donors stimulated with 10 ng / mL FLT3L, SCF, and TPO for 12 days. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Effect of CD30 CAR T-cell exposure on HSPC subset viability. Two HSPC donors and two T-cell donors were combined to yield four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T-cell exposure on erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with a CD30-high KM-H2 target on cytolysis of CD30-low HSPCs. [Figure 11A] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 11B] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 11C]In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 11D] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 11E] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 11F]In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 11G] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with peripheral T-cell lymphoma. (11A) Experimental setup for HuT-78 model. (11B) Tumor volume, tumor volume change, and endpoint tumor count. (11C) Twice-weekly IVIS monitoring to track T-cell biodistribution. (11D) Survival curve of mice after treatment. (11E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mouse plasma 8 days after treatment. [Figure 12A] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 12B]In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 12C] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 12D] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 12E-1] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 12E-2]In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 12F] In vivo persistence, efficacy, and toxicity of humanized CD30 CAR T cells in GM-CSF and IL-3-supplemented humanized mice xenografted with CD30-high NALM-6. (12A) Experimental setup for the NALM-6 model. (12B) Mouse weight changes. (12C) Twice-weekly IVIS monitoring to track NALM-6 disease. (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in mouse plasma 11 days after administration. [Figure 13A] Characterization of EBVST transduced to express humanized CD30 CAR. (13A) Expression and transduction efficiency of CD30 CAR in EBVST. (13B) Masking efficiency of CD30 CAR in EBVST. (13C) Cytotoxic potency and specificity of CD30 CAR EBVST using CD30+ KM-H2 cells and CD30- Daudi cells. [Figure 13B] Characterization of EBVST transduced to express humanized CD30 CAR. (13A) Expression and transduction efficiency of CD30 CAR in EBVST. (13B) Masking efficiency of CD30 CAR in EBVST. (13C) Cytotoxic potency and specificity of CD30 CAR EBVST using CD30+ KM-H2 cells and CD30- Daudi cells. [Figure 13C]Characterization of EBVST transduced to express humanized CD30 CAR. (13A) Expression and transduction efficiency of CD30 CAR in EBVST. (13B) Masking efficiency of CD30 CAR in EBVST. (13C) Cytotoxic potency and specificity of CD30 CAR EBVST using CD30+ KM-H2 cells and CD30- Daudi cells. [Example]

[0353] In the examples below, we describe the generation and characterization of novel CAR constructs. Example 1: Materials and Methods 1.1 Plasmid constructs and retrovirus production The murine HRS3 scFv and its humanized versions were cloned into the pSFG retroviral vector upstream of wild-type IgG1 Fc, either an OX40- or 4-1BB-derived spacer, followed by the CD28 transmembrane domain, CD28, and CD3ζ signaling domains. A truncated form of the CD30 molecule consisting of only the CD30 extracellular domain was cloned into the pSFG retroviral vector.

[0354] [Table 5-1]

[0355] [Table 5-2]

[0356] [Table 5-3]

[0357] Retroviruses carrying CD30 CAR or truncated CD30 were produced in HEK293VG or RD114 packaging cell lines (BioVec Pharma, Quebec, Canada) by transient transfection with pSFG vectors using PEIpro transfection reagent (Polyplus, Illkirch, France). At 48 and 72 hours posttransfection, the retrovirus-containing medium was collected and concentrated 10-fold using a RetroX concentrator (Takara Bio Inc., Kusatsu, Shiga Prefecture, Japan). Retroviruses were either used immediately or flash-frozen and stored at -80°C.

[0358] The stable RD114 retroviral packaging cell line, which produces high titers of GFP-firefly luciferase (GFP-FFLuc) viral particles, was kindly provided by Dr. Masataka Suzuki (Baylor Center for Gene Therapy, Baylor College of Medicine).

[0359] 1.2 Donors Concentrated leukoreduced blood transfusion products collected from consented healthy donors using the Spectra Optia® apheresis system according to the CMNC collection protocol and frozen in ACD-A anticoagulant were purchased from HemaCare (Northridge, California, USA). Frozen leukopaks were thawed, and PBMCs were extracted by gradient centrifugation using Ficoll-Paque PLUS (Cytiva, MA, USA). PBMCs were either used immediately for experiments or frozen at 30–50 × 10 cells in CryoStor® CS10 cell freezing medium (STEMCELL Technologies, Cambridge, Massachusetts, USA). 6 The samples were either frozen in smaller aliquots of 1000 ml / cryovial.

[0360] Cord blood CD34+ cells isolated from cord blood mononuclear cells via positive immunomagnetic separation from consenting donors were purchased from Lonza (Walkersville, MD, USA). 6 Frozen vials of cells were thawed and CD34+ cells were either used immediately for experiments or thawed at 5 × 10 per cryovial in CryoStor® CS10 Cell Freezing Medium (STEMCELL Technologies, Cambridge, MA, USA). 4 Aliquots of cells were frozen.

[0361] 1.3 CAR T cell production For activated T cell (ATC) transduction, PBMCs were thawed and plated on cell culture plates precoated with anti-CD3 / CD28. Cultures were grown in 10% FBS, 45% Advanced RPMI, and 45% Click medium at 37°C with 5% CO2 to generate ATCs. After two days of culture, IL-7 and IL-15 were added to the cell culture. On day three of culture, ATCs were transduced by spinfection with a retrovirus containing the indicated scFv and CD30.CAR with a spacer. After 24 hours, the retrovirus was washed out, and the ATCs were cultured with occasional medium changes to replenish IL-7 and IL-15. For use in in vivo solid tumor models, ATCs were further transduced with a retroviral vector encoding GFP-firefly luciferase (GFP-FFLuc) on day four of culture, allowing for IVIS tracking of ATCs in surviving mice. Eleven days after transduction, ATCs were frozen using CryoStor according to the manufacturer's manual or injected into mice. For humanized mouse studies, ATCs were generated from CD34- cord blood cells instead of PBMCs and later injected into humanized mice reconstituted with CD34+ cord blood cells from the same donor.

[0362] For EBV-specific T cell transduction, CD45RA depletion of PBMCs (RAD-PBMCs, optional) was performed by negative selection using CD45RA MACS Beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Total PBMCs or RAD-PBMCs were incubated at 1 × 10 with viral peptides from an overlapping peptide library (15-mers with 11 amino acid overlaps) from JPT Technologies (Berlin, Germany). 6 T cells were cultured at 1000 cells / well. Five days later, cells were transduced with a humanized CD30 CAR construct using RetroNectin (Takara Bio, Kusatsu, Shiga, Japan) as described above. Four days after transduction, T cells were subsequently stimulated with irradiated costimulatory cells expressing markers such as CD80, CD86, and 4-1BB. After 7–8 days, VSTs were harvested and either frozen or used for cell assays.

[0363] 1.4 Transduction efficiency and phenotyping by flow cytometry and antibody staining Flow cytometry in this study was performed using an Aurora cytometer (Cytek Biosciences) or a FACSymphony A3 cell analyzer (BD Biosciences). Up to 200k T cells were stained with Live / Dead™ NIR viability dye (Thermo Fisher) and assessed for surface epitope presentation using fluorescently labeled monoclonal antibodies against CD3 (clone SK7, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BioLegend), CD56 (clone B159, BD Biosciences), CD19 (clone SJ25C1, BioLegend), CD30 (clone BerH8, BD Biosciences; clone BY88, BioLegend), PD-1 (clone EH12.1, BD Biosciences), Tim3 (clone 7D3, BD Biosciences), and LAG3 (clone 11C3C65, BioLegend). CD30 CAR expression was measured using recombinant human CD30 protein fused to a His tag (10777-H08H, Sino Biological) followed by PE-conjugated anti-His (clone J095G46, BioLegend), or biotinylated recombinant human CD30 protein (ACROBiosystems, CD0-H82E6) followed by PE-conjugated streptavidin (BD Biosciences, 554061), or FITC-labeled recombinant human CD30 protein (ACROBiosystems, CD0-HF2H3). Flow data were analyzed and gated using FlowJo v10.8.1 for Windows.

[0364] 1.5 Cytotoxicity assay Before evaluating the efficacy of CAR through cytotoxicity assay, the cell culture medium is replaced with assay medium containing 2% RPMI and 2% FBS. Cytotoxicity assay was performed using xCelligence® Real-Time Cell Analysis System (Agilent) at 5% CO2 and 37°C. Target cells are added onto the PET plate with anti-CD40 antibody anchored according to the manufacturer's manual (Agilent). 24 hours after target cell adhesion, CAR T cells are seeded onto the PET plate at a CAR T:target cell ratio of 0.2:1, 1:1, or 5:1. The PET plate is returned to the xCelligence system, and cytotoxicity is monitored for 48 hours.

[0365] 1.6 Sequential Killing Efficacy Assay KM-H2 cells were used as the target cell line with an initial E:T ratio of 1:2. For encounter 1, 50,000 effector and 100,000 target cells were placed per well of a 96-well plate. Three sets of this setup were prepared. After 48 hours, cells from set 2 were harvested and added to two new sets of 100,000 KM-H2 target cells per well to set up encounter 2. After another 48 hours, cells from set 1 were harvested and added to a new set of 100,000 KM-H2 target cells per well for encounter 3. To distinguish target cells from different encounters, KM-H2 cells for encounters 2 and 3 were labeled with lipophilic membrane dyes PKH67 and PKH26 (Sigma-Aldrich), respectively. Forty-eight hours after each encounter, one set of cells was stained with Live / Dead™ NIR viability dye (Thermo Fisher) and fluorescently labeled monoclonal antibodies against CD3 (clone UCHT1, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), CD40 (clone 5C3, BioLegend), and CD30 (clone BerH8, BD Biosciences) to enumerate the target cell population remaining at each round of 48-hour culture. See Figure 9B for a schematic of the assay setup. Cell lysis was calculated as (KM-H2 counts in target-only control wells - KM-H2 counts in assay wells) / KM-H2 counts in target-only control wells.

[0366] 1.7 Cytokine release assay Prior to assessing cytokine release, the cell culture medium is changed to 2% assay medium containing RPMI and 2% FBS. Target cells are added to a 96-well flat-bottom plate. CAR T cells are plated on top of the target cells at a CAR T:target cell ratio of 1:1. The plate is incubated at 37°C with 5% CO2 for 24 hours. After 24 hours, cell-free medium is collected by centrifugation at 500g for 5 minutes. Cytokines released into the medium, including TNFα, GMCSF, and IFNγ, are assayed using BioLegends' ELISA kits.

[0367] 1.8 Luminex Multiplex Assays Cytokine levels in co-cultured cell samples or plasma were determined by FLEXMAP 3D® (Luminex) using Milliplex™ Human High Sensitivity T Cell Panel Premix 13-plex (Millipore, HSTCMAG28SPMX13). Analysis was performed using Bio-plex Manager software (Bio-Rad).

[0368] 1.9 In vitro safety assays using hematopoietic stem and progenitor cells (HSPCs) CD34+ HSPCs were stimulated with 10 ng / mL each of Flt3-ligand (FLT3L), stem cell factor (SCF), and thrombopoietin (TPO) (all from Miltenyi Biotec) at 5,000–20,000 cells per 200 μL per well of a 96-well U-bottom plate for the indicated time periods.

[0369] In cocultures of CD34+ HSPCs and T cells, CD34+ cells were isolated by T cell depletion using magnetic beads conjugated to anti-CD3 antibodies (Miltenyi Biotec). Following the gating strategy described by Hombach et al., HSPC subsets were analyzed by flow cytometry after staining with Live / Dead™ NIR viability dye (Thermo Fisher Scientific) and fluorescently labeled monoclonal antibodies against CD34 (clone 561, BD Biosciences), CD133 (clone 7, BioLegend), CD45RA (clone HI100, BD Biosciences), CD38 (clone HIT2, BD Biosciences), and CD10 (clone HI10a, BD Biosciences).

[0370] Erythroid and myeloid developmental potential was assessed using the StemMACS™ HSC-CFU Assay Kit (Miltenyi Biotec, 130-125-042). Cells were labeled with fluorescently conjugated antibodies against CD14, CD15, and CD235a as part of the StemMACS kit antibody cocktail, and colony types were identified according to the manufacturer's protocol. Colony types include colony-forming units (CFU-G) for granulocytes and macrophages (CFU-M) and burst-forming units (BFU-E) for erythroids. In the case of more primitive progenitors, CFU-GM give rise to both granulocytes and macrophages, while CFU-GEMM differentiate into all three cell populations.

[0371] 1.10 Extranodal NK-T cell lymphoma model mouse In the extranodal NK-T cell lymphoma model, 2 × 10 6 SNK-6 cells were cultured in NOD-scid IL2Rγma null (NSG) mice were injected subcutaneously into the right flank. On day 15, mice were randomized into treatment groups stratified by tumor volume. On day 16, 4 × 10 6Mice were intravenously injected with GFP-luciferase-tagged ATC. IL-2 (1000 IU per mouse) was delivered via intraperitoneal injection to all mice. A mouse Fc receptor-blocking antibody (Bio X Cell, Cat#:BE0307) was administered to mice bearing the IgG1 CH2CH3 spacer CD30 CAR at a dose of 8 μg of drug per gram of body weight every other day. The mouse Fc receptor-blocking antibody was injected to prevent nonspecific interactions between the IgG1 CH2CH3 domain and mouse Fc receptor-bearing immune cells. The biodistribution of T cells was assessed using an IVIS imaging system (Perkin Elmer). Regions of interest (ROIs) were drawn on the mice, and mean radiance (p / sec / cm) was measured using Living Image® 4.7.4 software. 2 / sr) was quantified. Mice were sacrificed on day 36 and blood, spleen, liver, and tumors were collected for end-point flow cytometry analysis.

[0372] 1.11 IL-3 / GM-CSF supplemented humanized mice To generate humanized mice, irradiated NSG pups were cultured with 1 x 10 humanized NSG pups derived from HLA-typed cord blood donors. 5 CD34 + At week 16, mice with >20% hCD45 reconstitution were delivered with human IL-3 / GM-CSF-encoding plasmids via hydrodynamic tail vein injection to support bone marrow cell reconstitution. Humanized mice were pre-bled via cheek for assessment of baseline human cytokine levels in serum.

[0373] 1.12 Peripheral T-cell lymphoma model mouse 1×10 6 HuT-78 was subcutaneously injected into the right flank of humanized mice. On day 4 or 6, 1 × 10 6 pcs or 4 x 10 6CAR and luciferase dual-transduced ATC T cells were intravenously injected into mice as indicated in the figure. For CD30CAR spacer studies, IL-2 and mouse FcR blockade injections were administered in a similar regimen as in the NK / T lymphoma model. Cheek bleeds were performed 6 days after treatment to assess cytokine levels in plasma 6 days after ATC treatment. For CD30CAR scFv studies, an additional cheek bleed was performed 6 days after treatment. T cell biodistribution was assessed using an IVIS imaging system (Perkin Elmer). Regions of interest (ROIs) were drawn on the mice, and mean radiance (p / sec / cm2 / sr) was quantified using Living Image® 4.7.4 software. Mice were sacrificed, and blood, spleen, liver, and tumor were collected for endpoint flow cytometry analysis. Bone marrow and lungs were also collected for CD30CAR scFv studies.

[0374] 1.13 CD30-positive NALM-6 systemic tumor model Nalm6-CD30 expressing GFP-FFLuc was generated by transducing Nalm6 with a retroviral vector encoding truncated CD30 and GFP-FFLuc. Nalm6 expressing both CD30 and GFP were selected twice, generating >99% double-positive clones. 2 × 10 6 Nalm6-CD30+ cells stably expressing GFP-firefly luciferase (GFP-FFLuc) were intravenously injected into humanized mice. Tumor engraftment was monitored weekly using an IVIS imaging system (Perkin Elmer). Mice were bled via the cheek 9 days before T cell treatment to assess plasma cytokine levels after tumor engraftment. For CD30CAR spacer studies, mice were intraperitoneally injected with 35 mg of human IVIG (Sigma-Aldrich, I4506) and, in some treatment groups, with 8 μg / g of mouse FcR blocker (Bio X Cell, Cat#:BE0307) 2 days before ATC treatment. 20 or 21 days after NALM-6 injection, 5 × 10 6Mice were administered T cells via retro-orbital injection. Body weight and temperature were monitored daily, and tumor burden was tracked twice weekly via IVIS. Cheek blood was collected 2 and 7 days after treatment for the CD30CAR spacer study and 3 and 6 days after treatment for the CD30CAR scFv study. At the end of the experiment, mice were sacrificed, and blood, spleen, liver, and bone marrow were collected for flow cytometry analysis.

[0375] Example 2: Results 2.1 Novel OX40- and 41-BB-derived spacers retain in vitro killing efficacy with lower cytokine production The spacer domain can significantly affect the safety and efficacy of CAR T cells. Spacers derived from the IgG Fc domain have commonly been used in the initial design of CARs. However, the use of wild-type Fc domains can lead to undesirable interactions with Fc receptor-expressing cells. A highly flexible linker can facilitate recognition of sterically challenging epitopes, while linker length can optimize the synaptic distance between CAR T cells and target cells. Optimizing this synaptic distance can enhance CAR signaling by eliminating phosphatases such as CD45, and cytotoxicity by promoting efficient delivery of cell-killing mediators such as perforin and granzymes. Therefore, we are optimizing CD30 CARs by investigating novel spacers. We designed four spacers of different lengths and rigidities derived from various immune receptors: OX40, 4-1BB, CD44, and CD96. The OX40 and 4-1BB spacers are relatively short, less glycosylated, and therefore more flexible, whereas the CD44 and CD96 spacers are highly glycosylated and therefore more rigid. None of these sequences are known to interact with their cognate ligands or any other known receptors, and are therefore theoretically inactive. Finally, they do not contain unpaired cysteine residues and are not known to dimerize. For comparison, we included the IgG2 CH2CH3 spacer, which is comparable in length and rigidity to the IgG1 spacer but has lower affinity for Fc receptors.

[0376] The alternative spacers were cloned into the HRS-3 CD30 CAR pSFG retroviral vector. Expression of transduced CAR T cells with various spacers was assessed by flow cytometry (Figure 1A). All CARs were equally efficient in activated T cells, regardless of the spacer domain. Next, the efficiency of various CARs to recognize and kill target cells was also compared using an assay with CD30+ KM-H2 cells at an effector-to-target ratio of 1:1. In addition to the CD96 spacer, the alternative spacers showed killing efficiencies comparable to those of the original IgG1 spacer (Figure 1B). Furthermore, we first investigated the basal levels of cytokine secretion of CAR T cells with various spacers. In the absence of stimulation, elevated cytokine secretion suggests CAR instability and, therefore, tonic activation of CAR T cells. None of the CARs with alternative spacers showed a significant increase in basal secretion of GM-CSF, interferon-γ, or TNF-α, suggesting that CAR stability was not compromised with the novel spacers (Figure 1C). However, upon activation of CAR T cells with CD30+KM-H2, cytokine secretion by CAR T cells expressing the OX40, 41-BB, and CD96 spacers was significantly reduced in all three donors tested (Figure 1C).

[0377] Next, we investigated the effect of CD30 antigen density on the efficacy of CD30 CARs expressing novel spacers. CD30-negative Raji cells were retrovirally transduced to express truncated CD30 proteins. FAC sorting and single-cell cloning were performed, identifying three distinct clones expressing various levels of CD30 (Figure 2A). CAR T cells expressing either the IgG1, OX40, or 4-1BB spacers were co-incubated with various CD30-expressing Raji clones, untransduced Raji, or the positive control cell line KM-H2 at an effector-to-target ratio of 1:1 (Figure 2B). All CARs expressing alternative spacers exhibited similar cytotoxicity efficiency as Raji clones expressing various levels of CD30 (Figure 2C), suggesting that differences in dimerization ability do not impair the sensitivity of CARs with alternative spacers to CD30 density. We compared the secretion of GM-CSF, interferon-γ, or TNF-α cytokines by CARs with various spacers stimulated with various CD30+ target cells. Similar to our previous observations using KM-H2 cells, cytokine secretion by CAR T cells expressing the OX40, 41-BB, and CD96 spacers was all significantly lower when using Raji cells expressing various levels of CD30 (Figure 2C).

[0378] We next cloned the 4-1BB spacer into humanized VH3VK3 and VH5VK3 CARs with and without the Cys23 mutation. We show that humanized CD30 CARs with the 4-1BB spacer were well expressed in transduced primary T cells (Figure 3A) and similarly induced potent cytotoxicity when used with CD30-expressing KM-H2 cells at an effector-to-target ratio of 1:1 (Figure 3B). Resting transduced cells expressing humanized CARs with the 4-1BB spacer also preserved their hypotonic signal in the absence of antigen stimulation (Figure 3C).

[0379] 2.2 CARs with novel OX40- and 41-BB-derived spacers do not interact nonspecifically with monocytes or NK cells CARs carrying wild-type IgG1-derived spacers readily interact with Fc receptor-expressing cells, such as monocytes and NK cells. The novel spacers are not known to interact with any receptors or ligands. Next, we investigated the possibility that the novel spacers might interact with other immune cells. CD14+ monocytes were isolated from donor PBMCs and incubated with CAR T cells carrying various spacers at a T cell:monocyte ratio of 1:5. The cytotoxicity of CARs carrying the novel OX40- and 41-BB-derived spacers against monocytes was assessed by flow cytometry 48 or 96 hours after incubation. At 48 hours, one set of CAR T cells was restimulated with fresh monocytes at a T cell:monocyte ratio of 1:5. Cytotoxicity against monocytes was assessed by flow cytometry 48 hours after restimulation (Figure 4A). We observed that CD30 CAR T cells bearing the IgG1 CH2CH3 domain induced high monocyte cytolysis at both 48 and 96 hours of incubation, and 48 hours after restimulation. In contrast, monocyte cytolysis by CD30 CAR T cells bearing IgG2, OX40, 4-1BB, CD96, and CD44 spacer domains was low or absent at all of the above time points (Figure 4B). Furthermore, monocyte cytolysis by CD30 CAR T cells bearing the IgG1 CH2CH3 domain was partially attenuated in the presence of a human Fc receptor-blocking antibody (Figure 4B), suggesting that the interaction between the IgG1 CH2CH3 domain and Fc receptors is important for monocyte cytolysis.

[0380] Interaction between CD30 CAR T cells bearing the IgG1 CH2CH3 domain and monocytes resulted in increased exhaustion compared with their counterparts bearing the OX40 and 4-1BB spacer domains, as evidenced by a higher proportion of CD30 CAR T cells bearing the IgG1 CH2CH3 spacer that coexpressed three immune checkpoint molecules (PD-1, Lag-3, and Tim-3) (Figure 4C). Interaction between CD30 CAR T cells bearing the IgG1 CH2CH3 domain and monocytes also resulted in the production of vast amounts of proinflammatory cytokines (G-CSF, GM-CSF, IL-6, IL-1α, and TNF-α) in cocultures, in contrast to the small amounts detected in cocultures of monocytes with CD30 CAR T cells bearing the OX40 and 4-1BB spacer (Figure 4D).

[0381] Further experiments to evaluate the spacer-mediated interaction between CD30 CAR T cells and natural killer (NK) cells (Figure 4E) revealed that CD30 CAR T cells bearing the IgG1 CH2CH3 domain induced cytolysis of NK cells (Figure 4F), particularly the CD16 high fraction (Figure 4G). In contrast, CD30 CAR T cells bearing the OX40 and 4-1BB spacer domains induced little NK cell cytolysis, sparing most CD16 high NK cells (Figures 4F and 4G).

[0382] 2.3 CD30 CAR T cells with a novel spacer improved persistence and efficacy against human NK-T lymphoma We next performed studies in immunodeficient NSG mice to evaluate the effect of different spacers on the in vivo persistence and efficacy of CD30 CAR T cells against extranodal NK-T cell lymphoma (Figure 5A, Example 1).

[0383] The presence of the IgG1 CH2CH3 linker in CAR-T cells has been described to result in CAR-T cell interaction with mouse Fc receptor-expressing immune cells in immunocompromised mice, resulting in pulmonary sequestration and impaired antitumor efficacy (19). This phenomenon is less applicable to hosts with an intact ability to produce physiological amounts of immunoglobulins, as IgG may occupy Fc receptors on immune cells, reducing their availability for interaction with the IgG1 CH2CH3 domain. To circumvent this phenomenon, which may be specific to severely immunocompromised hosts, a mouse Fc receptor-blocking antibody (clone 2.4.G2) was administered to mice receiving CD30 CAR T cells bearing the IgG1 CH2CH3 domain.

[0384] After tumor engraftment, mice were randomized to receive luciferase-expressing CD30 CAR T cells with various spacers. IVIS monitoring 1 day after treatment revealed that all CD30 CAR T cells initially trafficked to the lungs. However, CD30 CAR T cells with an IgG1 CH2CH3 spacer rapidly disappeared in the animals within 4 days after treatment, consistent with published data (19) that IgG1 CH2CH3 domain interactions with Fc receptor-expressing mouse cells can significantly impair the in vivo longevity of CAR T cells (Figure 5B). Administration of a mouse FcR-blocking antibody to mice receiving CD30 CAR T cells with an IgG1 CH2CH3 spacer extended CAR T cell persistence but did not completely prevent lung trapping and cell elimination (Figure 5B). CD30 CAR T cells with an IgG1 CH2CH3 spacer also failed to migrate to tumors, even in the presence of a mouse FcR-blocking antibody (Figure 5B and Figure 5C). On the contrary, CD30 CAR T cells with IgG2, OX40, 41BB, and CD96 spacers persisted and expanded well in animals, clearly outnumbering IgG1 CH2CH3 spacer CD30 CAR T cells for up to 20 days after treatment (Figure 5B). Compared with CD30 CAR T cells with IgG1 CH2CH3 spacers, IgG2, OX40, 41BB, and CD96 spacer CD30 CAR T cells showed improved migration and expansion within tumors in situ, and these effects were observed for up to 20 days after treatment (Figures 5B and 5C).

[0385] Mice were sacrificed on day 20 for endpoint flow analysis. Higher numbers of CD30 CAR T cells bearing IgG2, OX40, 41BB, and CD96 domains were observed in tumor samples at endpoint, consistent with the IVIS results (Figure 5D). Also, significantly higher populations of CD30 CAR T cells bearing the IgG2, OX40, 41BB, and CD96 spacer were detected in the blood, spleen, liver, and systemic tissue of mice (Figure 5E). Finally, SNK-6 cells, defined as the viable CD3-, CD45+, and CD30+ cell population in the tumor, were observed to be less abundant in mice receiving CD30 CAR T cells with the OX40, 41BB, CD96, and CD44 spacer (Figure 5F).

[0386] 2.4 In vivo persistence and efficacy of CD30 CAR T cells with OX-40 and 4-1BB spacers in humanized mice xenografted with human peripheral T-cell lymphoma To further investigate the effect of spacers on the in vivo persistence, expansion, antitumor activity, and safety of CD30 CAR T cells, we conducted studies in a system that can better recapitulate human immune responses. We utilized humanized mice reconstituted with human CD34+ cells for a model of peripheral T-cell lymphoma. After tumor engraftment, the mice were randomized to receive luciferase-expressing CD30 CAR T cells generated from the CD34-negative fraction of the same donor used for mouse humanization. The CD30 CAR T cells were also transduced with luciferase to enable in vivo cell tracking (Figure 6A; see Example 1 for details). For the same reasons as above, we also administered a murine Fc receptor-blocking antibody to the group of mice receiving IgG1 CH2CH3 CD30 CAR T cells. Due to promising readouts from previous in vitro experiments, we chose to focus on CD30 CAR T cells with IgG1 CH2CH3, OX40, and 41BB spacers.

[0387] Similar to the observations in the SNK6 model, all CD30 CAR T cells initially trafficked to the lungs on day 1. By 10 days after treatment, CD30 CAR T cells bearing the OX40 and 41BB spacers were observed to have undergone systemic expansion with good tumor localization (Figures 6B and 6C). In contrast, CD30 CAR T cells bearing the IgG1 CH2CH3 domain exhibited limited systemic survival and tumor localization. Coadministration of an Fc receptor-blocking antibody did not rescue this poor persistence of IgG1 CH2CH3 spacer CD30 CAR T cells (Figures 6B and 6C). Not surprisingly, significantly better tumor control was observed in mice receiving OX40 and 41BB spacer CD30 CAR T cells, as evidenced by the lower number of viable HuT-78 cells detected in the primary tumor at endpoint (Figure 6D).

[0388] Plasma collected 6 days after treatment was assessed for cytokine levels using Luminex®. Plasma levels of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), granulocyte-macrophage-colony stimulating factor (GM-CSF), and interleukin 2 (IL-2) remained low in all treatment arms (Figure 6E), demonstrating that the in vivo activity and expansion of OX40 and 41BB spacer CD30 CAR T cells does not induce systemic production of proinflammatory cytokines (Figure 6E).

[0389] 2.5 CD30 CAR with a novel 41-BB spacer improves safety profile in high tumor burden disease compared with IgG1 CH2CH3 spacer High tumor burden is a known risk factor for the development of cytokine release syndrome (CRS) associated with CAR T-cell therapy in patients with hematological malignancies ( 20 , 21 ). Myeloid cells, including monocytes and macrophages, have been depicted as key players in the development of CRS and neurotoxicity after CAR T-cell therapy ( 22 , 23 ).

[0390] With this in mind, we sought to investigate the safety of a CD30 CAR with an IgG1 CH2CH3 and 41-BB spacer in humanized NSG mice xenografted with CD30-transduced NALM-6 cells. Note that the mice used in this model were supplemented with human GM-CSF and interleukin-3 (IL-3) after humanization to support stable engraftment of myeloid lineages (see Methods and Materials). Disease progression was monitored by bioluminescence imaging of CD30+NALM-6 cells transduced with firefly luciferase. Treatment was administered when the systemic tumor burden in the mice was high (Figure 7A).

[0391] We hypothesized that interaction between the IgG1 CH2CH3 spacer and Fc receptor-expressing immune cells in human subjects would be minimized because physiological levels of circulating immunoglobulins may occupy Fc receptors and reduce their availability for interacting with the IgG1 CH2CH3 linker on CAR T cells. To establish a model that recapitulates patient conditions, we treated all humanized mice with human intravenous immunoglobulin (IVIG) prior to CAR T cell treatment. Humanized mice randomized to receive CD30 CAR T cells with the IgG1 CH2CH3 spacer were also treated with a mouse Fc receptor-blocking antibody to prevent interaction with mouse Fc receptor-expressing immune cells. Mice were randomized to receive no treatment, untransduced T cells + human IVIG, CD30 CAR T cells with the IgG1 CH2CH3 spacer + human IVIG + mouse Fc receptor-blocking antibody, or CD30 CAR T cells with the 4-1BB spacer + human IVIG (Figure 7A). Plasma IgG levels were assessed 3 days after IVIG administration, a time point after IVIG distribution to the extravascular compartment and IgG levels had reached steady state. Administration of human IVIG significantly increased plasma IgG levels from baseline (Figure 7B).

[0392] Body weight changes were minimal in untreated mice or mice treated with untransduced T cells, CD30 CAR T cells with a 4-1BB spacer. On the contrary, mice given IgG1 CH2CH3 spacer CD30 CAR T cells underwent progressive weight loss of up to 30% of basal body weight (Figure 7C). Due to debilitating weight loss and rapid deterioration in mice given CD30 CAR T cells with an IgG1 CH2CH3 spacer (Figure 7C), we terminated the experiment on day 9. While tumor control after 9 days of treatment appeared improved in mice given CD30 CAR T cells with either spacer, this difference compared to untreated or untransduced T cell treatment was not significant (Figure 7D). In our experience, tumor control using the NALM-6 leukemia model can take up to two weeks. Therefore, early termination of the experiment prevented us from understanding whether differences in efficacy might exist between CD30 CAR T cells with IgG1 CH2CH3 or 4-1BB spacers. We did not observe any differences in CD30 CAR T cell populations with IgG1 CH2CH3 or 4-1BB spacers at the endpoint, suggesting that the combination of human IVIG and mouse Fc receptor-blocking antibodies reduces interactions between human and mouse Fc receptor-expressing immune cells (Figure 7E). We noted that endogenous human T cells, B cells, NK cells, and myeloid cells in the mouse femur, liver, and spleen were similar across all treatment groups (Figure 7F), indicating that the unfavorable safety profile observed with CD30 CAR T cells with IgG1 CH2CH3 spacers in mice is unrelated to changes in immune cell populations. Along with dramatic weight loss, we noted highly elevated levels of human IL-6, IL-8, IL-10, IFN-γ, TNF-α, and GM-CSF in mice receiving CD30 CAR T cells with an IgG1 CH2CH3 spacer. Among these cytokines, IL-6, IL-8, TNF-α, and GM-CSF have frequently been implicated as soluble mediators of CRS after CAR T-cell therapy (22, 23).In comparison, the levels of these cytokines remained low in mice from other treatment groups. We note that these important observations of elevated systemic levels of multiple inflammatory cytokines and rapid worsening in IgG1 CH2CH3 spacer CD30 CAR T-treated mice fulfill the unified criteria for CRS (24).

[0393] 2.6 Generation and characterization of T cells bearing humanized CD30 CAR with a 4-1BB spacer Next, we evaluated the phenotype and function of T cells transduced with CAR constructs containing the 4-1BB spacer domain and the VH3Vk3-Cys or VH5Vk3-Cys antigen-binding domain. Untransduced or CD30 CAR ATCs were generated from PBMCs (four donors) and from cord blood-derived CD34- cells (three donors) for use in experiments involving humanized mice using the same cord blood donor. VH3Vk3-Cys-41BB and VH5Vk3-Cys-41BB CD30 CAR ATCs expanded similarly to, or even better than, HRS3-41BB CD30 CAR ATCs (Figure 8A). The CD30 CAR variants had high transduction efficiencies, up to 98% (Figure 8B). Where variation was observed between constructs, HRS3-41BB CD30 CAR T cells had a slightly lower frequency of CAR+ cells. Most donors generated relatively more CD4 T cells than CD8 T cells, and CD4 / CD8 ratios were roughly comparable between CD30 CAR variants (Figure 8C).

[0394] Upon activation, T cells upregulate CD30, which can bind and activate CD30 CAR-expressing cells, potentially resulting in fratricide. CD30 CAR binding to CD30 in cis may help protect cells from unintentionally activating their CD30 CAR-bearing siblings and potentially from fratricide. To assess the extent of CD30 masking by CAR molecules, we used two distinct commercially available anti-CD30 clones, BerH8 and BY88. When T cells were transduced with HRS3-IgG1, CD30 expression was virtually undetectable by BerH8, suggesting that BerH8 binds to a similar site on CD30 as the parent HRS3 scFv. In contrast, staining for CD30 with BY88 yielded detectable populations in both untransduced cells and CD30 CAR T cells. Before transduction (day 0), CD30 expression detected by both BerH8 and BY88 was comparable in ATCs and remained similar in untransduced T cells during expansion (Figure 8D). In CD30CAR-transduced cells, CD30 detection by BerH8 was undetectable as early as 3 days after transduction. In contrast, staining with BY-88 revealed that cell surface CD30 expression often increased sharply early and gradually declined to less than 10% in most conditions by the time of harvest (day 11), demonstrating that all scFv constructs effectively masked CD30 in cis.

[0395] To assess the degree of activation and / or exhaustion of CD30CAR-expressing cells, we stained cells with antibodies against PD-1, Tim3, and LAG3 and analyzed the proportion of cells expressing one or a combination of these proteins in the three donors (Figure 8E). CD4 T cells primarily contained Tim3 mono-positive cells, with low proportions (approximately 5-20%) of cells expressing both Tim3 and PD-1 or neither marker. CD8 T cells were similarly predominantly Tim3 mono-positive, particularly for donor 5056. The remaining cells consisted of cells bearing neither marker and Tim3-LAG3 dual expressers. Within each donor, the frequencies of these populations were comparable across the various CD30CAR variants. The proportion of cells expressing all three proteins was very low, suggesting that cells were not ultimately exhausted at the end of the expansion process.

[0396] 2.7 In vitro antitumor activity of T cells bearing humanized CD30 CAR We evaluated the cytolytic function of T cells transduced with humanized CD30CAR variants by co-culturing T cells with two target tumor cell lines, KM-H2 and HuT-78, both of which express high levels of CD30, at an E:T ratio of 2:1. All CD30CAR variants achieved nearly 100% lysis of KM-H2 targets by 60 hours in three of four donors tested (Figure 9A). When HuT-78 was used as the target cell, there was more heterogeneity in the killing potency of the CD30CAR variants, both in terms of maximum cytolysis and kinetics of cytolysis. Most constructs achieved at least 60% cytolysis of HuT-78 cells by 60 hours.

[0397] Effective tumor clearance in vivo is predicated on the ability of CAR T cells to maintain killing activity throughout successive rounds of target encounters. To model such a scenario in vitro, a flow cytometry-based serial killing assay was established in which CAR T cells underwent three consecutive target encounters, each lasting 48 hours (Figure 9B; see Example 1.6). To distinguish target cells from various encounters, KM-H2 cells were labeled with lipophilic membrane dyes PKH67 and PKH26 dyes for encounters 2 and 3, respectively. The HRS3 and VH3Vk3-Cys CD30CAR variants demonstrated good serial killing efficacy in the three donors tested, whereas VH5Vk3-Cys maintained cytolytic function through three consecutive rounds of target encounters only in one donor, 8584 (Figure 9C).

[0398] T cell expansion was accompanied by target cell lysis; HRS3 and VH3Vk3-Cys cells expanded well in all three donors (Figure 9D), whereas VH5Vk3-Cys cells expanded only in donor 8584. HRS3-bearing cells steadily increased in number with each successive target encounter. VH3Vk3-Cys cells showed a peak in cell count at the end of encounter 2 but declined after encounter 3 without any apparent effect on killing efficacy.

[0399] 2.8 In vitro on-target, extratumoral activity of T cells bearing a humanized CD30 CAR with a 4-1BB spacer In addition to being expressed on malignant and activated lymphocytes, CD30 can also be upregulated on hematopoietic stem and progenitor cells (HSPCs) upon cytokine stimulation, albeit at lower levels than on lymphoma cells (21). This raises a potential safety concern if CD30CAR T cells unintentionally attack CD30-expressing HSPCs in the recipient. While one group observed minimal killing of HSPCs by HRS3scFv CAR T cells (21), we wanted to ensure that the same was true for our humanized CD30CAR variant.

[0400] We first evaluated CD30 expression on umbilical cord blood CD34+ cells from two donors using the same cytokine stimulation protocol used by Hombach et al. CD30 expression peaked on days 2–3 of stimulation for both donors after culture in 10 ng / mL FLT3L, SCF, and TPO (Figure 10A), but did not reach high levels on KM-H2 cells (Figure 10B). CD30 surface expression has been reported to be heterogeneous on HSPC subsets (23), and this was found to be true for the two donors assayed (Figure 10C). After 2 days of stimulation, CD30 was upregulated on multipotent progenitor cells (MPPs) and lymphoid-primed pluripotent cells (LMPPs), and to a lesser extent on multilymphoid-primed progenitors (MLPs), but not on erythroid-myeloid-primed progenitors (EMPs). These data are consistent with the trends reported by Hombach et al. (23) Therefore, we used HSPCs 2 days after cytokine stimulation to capture the population at its peak of CD30 expression.

[0401] To determine whether CD30CAR variants have any effect on the viability of progenitor cell populations, CD34+ HSPCs were exposed to effector cells for 24 hours, followed by isolation of HSPCs for further culture in the same cytokine cocktail of 10 ng / mL FLT3L, SCF, and TPO for 9 days, after which the cells were analyzed by flow cytometry (Figure 10D, see Example 1). Encountering any of the CD30CAR variants altered the composition of the progenitor cell population compared to untransduced T cells, suggesting some cytolysis of progen...

Claims

1. A chimeric antigen receptor (CAR) comprising: (i) an antigen-binding domain that specifically binds to a target antigen; (ii) a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 100, 99, 101, and 102; (iii) a transmembrane domain; and (iv) a signaling domain comprising an amino acid sequence containing an immunoreceptor tyrosine-based activation motif (ITAM).

2. The CAR of claim 1, comprising a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:

100.

3. The CAR of claim 1 or 2, wherein the antigen-binding domain specifically binds to a target antigen selected from CD30, CD19, CD20, CD22, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

4. The CAR of any one of claims 1 to 3, wherein the antigen-binding domain specifically binds to CD30.

5. the antigen-binding domain is (i) the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 2 HC-CDR2 having the amino acid sequence of SEQ ID NO: 3 HC-CDR3 having the amino acid sequence of SEQ ID NO:4 a heavy chain variable (VH) region incorporating (ii) the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 10 LC-CDR2 having the amino acid sequence of SEQ ID NO: 11 LC-CDR3 having the amino acid sequence of SEQ ID NO: 12 A light chain variable (VL) region incorporating The CAR according to any one of claims 1 to 4, comprising:

6. the antigen-binding domain is a VH region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 1, 45, 52, 17, 21, 24, 26, and 28; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 9, 49, 54, 30, 35, 38, 41, 43, 147, 148, 149, 150, 151, or 152; The CAR according to any one of claims 1 to 5, comprising:

7. the antigen-binding domain is (i) a VH region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 1, and a VL region having an amino acid sequence that has at least 70% amino acid sequence identity to SEQ ID NO: 9; or (ii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 45, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 42; or (iii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 54; or (iv) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 30; or (v) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 21, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 35; or (vi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 38; or (vii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 41; or (viii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 43; or (ix) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 147; or (x) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 148; or (xi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 21, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 149; or (xii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 150; or (xiii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 151; or (xiv) a VH region having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO:

152. The CAR according to any one of claims 1 to 7, comprising:

8. The CAR of any one of claims 5 to 7, wherein the antigen-binding domain is or comprises a single-chain variable fragment (scFv) comprising a VH region and a VL region.

9. 9. The CAR of any one of claims 1 to 8, wherein the transmembrane domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 103, 104 or 105.

10. 10. The CAR of any one of claims 1 to 9, wherein the signaling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 106, 107, or 108.

11. 11. The CAR of any one of claims 1 to 10, wherein the signaling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:

111.

12. SEQ ID NOs: 124, 144, 190, 210, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 145, 146, 183, 184, 185, 186, 187, 188, 189 12. The CAR of claim 1, comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity with any one of the following: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 211 or 212.

13. 13. A nucleic acid or nucleic acids, optionally isolated, encoding the CAR of any one of claims 1 to 12.

14. 14. An expression vector or vectors comprising the nucleic acid or nucleic acids of claim 13.

15. A cell comprising a CAR according to any one of claims 1 to 12, a nucleic acid or nucleic acids according to claim 13, or an expression vector or expression vectors according to claim 14.

16. 16. A method comprising culturing the cell of claim 15 under conditions suitable for expression of a CAR by the cell.

17. A composition comprising a CAR described in any one of claims 1 to 12, a nucleic acid or multiple nucleic acids described in claim 13, an expression vector or multiple expression vectors described in claim 14, or a cell described in claim 15, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

18. 18. A CAR according to any one of claims 1 to 12, a nucleic acid or nucleic acids according to claim 13, an expression vector or expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17, for use in a method of drug treatment or prevention.

19. 18. A CAR according to any one of claims 1 to 12, a nucleic acid or nucleic acids according to claim 13, an expression vector or expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17, for use in the treatment or prevention of cancer.

20. The cancer is a cancer expressing the target antigen for CAR, CD30-positive cancer, EBV-associated cancer, blood cancer, myeloid hematologic malignancy, hematopoietic malignancy, lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T-cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, E BV-associated lymphoma, EBV-positive B-cell lymphoma, EBV-positive diffuse large B-cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection / AIDS, oral hairy leukoplakia, Burkitt lymphoma, post-transplant lymphoproliferative disorder, central nervous system lymphoma, anaplastic large cell lymphoma, T-cell lymphoma, ALK-positive anaplastic T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, NK-T-cell lymphoma, extranodal NK-T-cell lymphoma, thymoma, multiple myeloma, solid tumors, epithelial cell carcinoma, gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, oropharyngeal cancer, oropharyngeal cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, colorectal cancer, colon cancer, colon cancer, cervical cancer, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer 20. The CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell, or composition for use according to claim 19, wherein the CAR is selected from the group consisting of alveolar lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer, ovarian cancer, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor, or testicular embryonal carcinoma.

21. 18. A CAR according to any one of claims 1 to 12, a nucleic acid or nucleic acids according to claim 13, an expression vector or expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17, for use in the treatment or prevention of a disease or condition characterized by an alloreactive immune response.

22. 22. The CAR, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition for use according to claim 21, wherein the disease or condition characterized by an alloreactive immune response is selected from the group consisting of a disease or condition associated with allogeneic transplantation, graft-versus-host disease (GVHD) or graft rejection.

23. 18. Use of the CAR of any one of claims 1 to 12, the nucleic acid or nucleic acids of claim 13, the expression vector or vectors of claim 14, the cell of claim 15, or the composition of claim 17 to deplete or increase killing of cells expressing the target antigen for the CAR.

24. 16. An in vitro complex, optionally isolated, comprising the CAR of any one of claims 1 to 12, or the cell of claim 15, bound to the target antigen for the CAR.

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