Chimeric antigen receptor systems with adaptable receptor specificity

Abstract

The present invention relates to chimeric antigen receptors (CARs), particularly CARs with compatible receptor specificity (arCARs).The present invention also provides CAR polypeptides and other related molecules, polynucleotides, vectors, and cell compositions comprising them.Pharmaceutical compositions comprising the polypeptides, polynucleotides, vectors, or cells of the present disclosure, and their use in treating disease in subjects are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 127,587, filed December 18, 2020, which is incorporated by reference in its entirety. [Technical field]

[0002] This application relates to chimeric antigen receptors (CARs), in particular CARs with adaptable receptor specificity (arCARs), and their use in immunotherapy (e.g., adoptive cell therapy).

[0003] Reference to an Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing with the filename "SequenceListing_ST25.txt" and a creation date of December 15, 2021, and which is 103,886 bytes in size. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Adoptive cell therapy (ACT) typically involves isolating cells from a donor, culturing and / or manipulating the cells in vitro, and then transferring the cells into a patient for the treatment of disease. To enable the cells to target specific antigens, the cells are often engineered with chimeric antigen receptors (CARs). Traditional CARs have a fixed design; therefore, one type of CAR T cell can usually only target one antigen epitope. This inflexible design limits clinical applications and leads to very high manufacturing costs. Although there are various approaches for switch-CAR platforms, these antigen-specific CARs are typically made on a custom-made basis.

[0005] Thus, there remains a need for improved universal CAR platforms with built-in, convenient mechanisms for modulating the CAR to easily adapt the specificity of the CAR to improve therapies and methods for treating disease using adoptive cell therapy. Summary of the Invention [Means for solving the problem]

[0006] In one aspect, provided herein is a universal chimeric antigen receptor (arCAR) system having compatible receptor specificity components, the arCAR system comprising: (i) an immune effector cell having a chimeric antigen receptor comprising a first polypeptide comprising (a) an extracellular tag-binding domain, (b) a transmembrane domain, and (c) at least one intracellular signaling domain; and (ii) a second polypeptide comprising (a) an antigen-binding domain that binds to at least one antigen on a target cell and (b) a tag that is recognized by the tag-binding domain of the first polypeptide of the chimeric antigen receptor; Including, (i) the tag comprises an antibody or antigen-binding fragment thereof, or an alternative scaffold, and the tag-binding domain comprises an anti-idiotypic molecule that binds to the tag; or (ii) The tag comprises an anti-idiotypic molecule that binds to a tag binding domain, the tag binding domain comprising an antibody or antigen-binding fragment thereof, or an alternative scaffold.

[0007] In some embodiments, the anti-idiotypic molecule binds to at least one antigen-binding region of an antibody, antigen-binding fragment thereof, or surrogate scaffold, hi some embodiments, the anti-idiotypic molecule binds to at least one complementarity determining region (CDR) of an antibody or antigen-binding fragment thereof.

[0008] In some embodiments, the antigen-binding domain of the second polypeptide comprises an antibody or antigen-binding fragment thereof, or a surrogate scaffold. In some embodiments, the anti-idiotypic molecule is an anti-idiotypic antibody or antigen-binding fragment thereof, or an anti-idiotypic surrogate scaffold.

[0009] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody.

[0010] In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises a single domain antibody or a nanobody. In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises a VHH. In some embodiments, the extracellular tag binding domain and the tag each comprise a VHH. In some embodiments, the extracellular tag binding domain, the tag, and the antigen binding domain each comprise a VHH.

[0011] In some embodiments, one or more of the antigen-binding fragment, the extracellular tag-binding domain, the antigen-binding domain, and the tag comprise, at least in part, a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, and 110-133.

[0012] In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises an scFv.

[0013] In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0014] In some embodiments, the tag comprises an antibody or antigen-binding fragment thereof, or an alternative scaffold. In some embodiments, the antibody or antigen-binding fragment thereof, or an alternative scaffold binds to a polypeptide derived from a non-human source. In some embodiments, the polypeptide derived from a non-human source is respiratory syncytial virus (RSV) F protein, Listeria internalin, Cobra venom phospholipase A2, Ebola nucleoprotein, Herpes simplex virus (HSV) glycoprotein D, Lactococcus phage receptor binding protein (RBP), Geobacillus stearothermophilus, ricin, or hen egg white lysozyme.

[0015] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen. In some embodiments, the at least one antigen is associated with the same tumor or autoimmune disease. In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies. In some embodiments, the tumor antigen associated with glioblastoma is HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, or IL13Rα2. In some embodiments, the tumor antigen associated with ovarian cancer is FOLR1, FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFRα, or B7H4. In some embodiments, the tumor antigen associated with cervical cancer or head and neck cancer is GD2, MUC1, Mesothelin, HER2, or EGFR. In some embodiments, the tumor antigen associated with liver cancer is Claudin 18.2, GPC-3, EpCAM, cMET, or AFP.

[0016] In some embodiments, the transmembrane domain further comprises a hinge domain. In some embodiments, the transmembrane domain and / or the hinge domain are derived from CD8 or CD28.

[0017] In some embodiments, the at least one intracellular signaling domain comprises a CD3 zeta signaling domain.

[0018] In some embodiments, the at least one intracellular signaling domain comprises one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, OX40, CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, or 2B4 (CD244).

[0019] In various embodiments, the second polypeptide comprises an antigen-binding domain at the N-terminus and a tag at the C-terminus. In some embodiments, the second polypeptide comprises an antigen-binding domain at the C-terminus and a tag at the N-terminus.

[0020] In various embodiments, the second polypeptide is a soluble polypeptide.

[0021] In various embodiments of the arCAR described herein, the immune effector cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells or tumor infiltrating lymphocytes (TILs), dendritic cells or macrophages. In some embodiments, the immune effector cells are derived from induced pluripotent stem cells (iPSCs). In some embodiments, the immune effector cells are T cells or NK cells derived from induced pluripotent stem cells (iPSCs).

[0022] In various embodiments of the arCAR described herein, the arCAR further comprises one or more polypeptides, each of which comprises (a) an antigen-binding domain that binds to a unique antigen and (b) a tag that is recognized by the tag-binding domain of the first polypeptide.

[0023] In another aspect, provided herein is an arCAR system comprising two or more arCARs described herein, each arCAR comprising a unique pair of a tag and a tag-binding domain.

[0024] In another aspect, provided herein is a polynucleotide encoding a first polypeptide of the arCAR system described herein.

[0025] In another aspect, provided herein is a polynucleotide encoding a second polypeptide of the arCAR system described herein.

[0026] In another aspect, provided herein is a polynucleotide encoding the first and second polypeptides of the arCAR system described herein.

[0027] In some embodiments of the polynucleotides described herein, one or more of the antigen-binding fragment, the extracellular tag binding domain, the antigen-binding domain, and the tag are at least partially encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109.

[0028] In another aspect, provided herein is a recombinant vector comprising a polynucleotide described herein. In some embodiments, the vector is a plasmid.

[0029] In another aspect, provided herein is a host cell comprising a polynucleotide that expresses a first polypeptide of the arCAR system described herein.

[0030] In another aspect, provided herein is a host cell comprising a polynucleotide that expresses a second polypeptide of the arCAR system described herein.

[0031] In another aspect, provided herein is a pharmaceutical composition comprising an immune effector cell of the arCAR system described herein and a pharma- ceutically acceptable carrier and / or excipient.

[0032] In another aspect, provided herein is a pharmaceutical composition comprising a second polypeptide of the arCAR system described herein and a pharma- ceutically acceptable carrier and / or excipient.

[0033] In another aspect, provided herein is a kit comprising a pharmaceutical composition comprising an immune effector cell of the arCAR system described herein in combination with a pharmaceutical composition comprising a second polypeptide of the arCAR system.

[0034] In another aspect, provided herein is a method for preparing a host cell comprising a polynucleotide that expresses a first polypeptide of the arCAR system, comprising introducing into the cell a polynucleotide encoding the first polypeptide, or a recombinant vector comprising the polynucleotide.

[0035] In another aspect, provided herein is a method for preparing a host cell comprising a polynucleotide that expresses a second polypeptide of the arCAR system, comprising introducing into the cell a polynucleotide encoding the second polypeptide, or a recombinant vector comprising the polynucleotide.

[0036] In another aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising: (i) an immune effector cell comprising a chimeric antigen receptor comprising a first polypeptide of an arCAR as described herein; and (ii) a second polypeptide of the arCAR, or a polynucleotide encoding the second polypeptide, or a host cell comprising the second polypeptide. The method includes administering to a subject a therapeutically effective amount of

[0037] In some embodiments, the second polypeptide is administered before, after, or in conjunction with the immune effector cells.

[0038] In another aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising: (i) an immune effector cell comprising a first polypeptide of two or more arCARs, wherein each of the two or more arCARs comprises a unique pair of a tag and a tag-binding domain; and (ii) a second polypeptide of the two or more arCARs, or one or more polynucleotides encoding the second polypeptide, or one or more host cells comprising the second polypeptide. The method includes administering to a subject a therapeutically effective amount of

[0039] In some embodiments of the treatment method described herein, the immune effector cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell or a tumor infiltrating lymphocyte (TIL), a dendritic cell or a macrophage.In some embodiments, the immune effector cell is derived from an iPSC.In some embodiments, the immune effector cell constitutively expresses the first polypeptide.

[0040] In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the cancer is glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy.

[0041] In some embodiments, the immune effector cells and the second polypeptide are administered simultaneously, hi some embodiments, the immune effector cells and the second polypeptide are administered sequentially.

[0042] In some embodiments, the immune effector cells are autologous cells. In some embodiments, the immune effector cells are allogeneic cells.

[0043] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human. [Brief description of the drawings]

[0044] [Figure 1]1 shows a schematic diagram of an exemplary adaptive receptor specificity CAR (arCAR) of the present disclosure. The CAR binds to a "tag" on a soluble drug. The soluble drug can be an antibody or antibody fragment (e.g., VHH, scFv, Fab, mAb) or an alternative scaffold. Tumor specificity and activation of the CAR are driven by the soluble drug. [Figure 2A]

[0023] Figure 1 shows a schematic diagram of another exemplary arCAR of the present disclosure. VHHs are used in both the antigen-binding domain and tag in the soluble drug, as well as in the tag-binding domain (α-tag) of the arCAR. [Figure 2B] 1 shows the structural features of anti-idiotypic mAbs. [Figure 2C] 1 shows structural features of exemplary VHH constructs. [Figure 2D]

[0023] Figure 1 shows a schematic diagram of another exemplary arCAR of the present disclosure. Non-human primate (NHP) VHHs and anti-idiotypic (ID) NHP VHHs are used in the soluble drug and tag-binding domains of the arCAR. [Figure 3A] FIG. 1 shows a schematic diagram of another exemplary arCAR of the present disclosure. [Figure 3B] FIG. 1 shows a schematic diagram of another exemplary arCAR of the present disclosure. [Figure 4A]

[0023] Figure 1 shows a schematic diagram of another exemplary arCAR of the present disclosure. Herceptin scFv is fused to the CAR, and Herceptin anti-ID scFv (Her-anti-ID) is fused to an EGFR-targeting VHH (9G8). [Figure 4B] 1 shows that T cells expressing Herceptin scFv CAR can target EGFR+ cells in combination with 9G8-scFv69 fusion protein. [Diagram 5] 1 shows a vector map of the lentiviral vector used to deliver the CAR construct of the present disclosure. [Figure 6A] 1 shows vector maps of plasmids used to express the bridge constructs of the present disclosure. [Figure 6B] A gel electrophoresis image showing the purity of the bridged protein is shown. [Figure 7A] Schematic diagram of scFv69-CAR and two variants of the bridge protein: 9G8_Herceptin scFv and Herceptin scFv 9G8. [Figure 7B] Binding of scFv69-CAR to a bridge protein is shown. Samples in the graph appear in the following order: 9G8_Herceptin, Herceptin_9G8; [Figure 8A] EGFR expression in EGFR-transduced CHO cells. [Figure 8B] CAR expression in T cells is shown. [Figure 9A] 13 demonstrates that universal CAR-T cells exhibited CD25 activation when combined with the correct bridge protein. Samples in the graph appear in the order specified in the legend. [Figure 9B] 13 demonstrates that universal CAR-T cells did not exhibit CD25 activation when combined with a mismatch bridge protein. Samples in the graph appear in the order specified in the legend. [Figure 10A] Demonstrates that universal CAR-T cells exhibited cytotoxic activity when combined with the correct bridge protein. Samples in the graph appear in the order specified in the legend. [Figure 10B] 13 demonstrates that universal CAR-T cells did not exhibit cytotoxic activity when combined with a mismatch bridge protein. Samples in the graph appear in the order specified in the legend. [Figure 11A] 1 shows a vector map of the lentiviral vector used to deliver the VHH CAR of the present disclosure. [Figure 11B] 1 shows expression of VHH CARs in Jurkat cells. [Figure 11C] 1 shows the lack of tonic signaling at the surface of VHH CAR-transduced Jurkat cells. [Figure 11D] The FACs gating strategy used for CD69 detection in FIG. 11C is shown. [Figure 11E] Binding of anti-Chicken Hen Egg Lysozyme-VHH CAR to chicken or human derived lysozyme is shown. [Figure 11F] Alignment of the amino acid sequence of a VHH targeting Geobacillus stearothermophilus (SEQ ID NO: 96), a VHH targeting LYSO_CW_P01_B11 (SEQ ID NO: 102), and a VHH targeting LYSO_CW_P01_D04 (SEQ ID NO: 104) is shown. A consensus sequence (SEQ ID NO: 134) is shown above the aligned sequences. [Figure 12A] 1 shows the plasmid map p510 for proof-of-concept testing of arCAR. [Figure 12B] 1 shows the plasmid map p511 for proof-of-concept (POC) testing of arCAR. [Figure 12C] 1 shows the plasmid map p514 for proof-of-concept (POC) testing of arCAR. [Figure 12D] 1 shows the plasmid map p515 for proof-of-concept (POC) testing of arCAR. [Figure 12E] 1 shows the plasmid map p516 for proof-of-concept (POC) testing of arCAR. [Figure 12F] 1 shows the plasmid map p517 for proof-of-concept (POC) testing of arCAR. [Figure 12G] 1 shows the plasmid map p518 for proof-of-concept (POC) testing of arCAR. [Figure 12H] 1 shows the plasmid map p519 for proof-of-concept (POC) testing of arCAR. [Figure 12I] 1 shows the plasmid map p520 for proof-of-concept (POC) testing of arCAR. [Figure 12J] 1 shows the plasmid map p521 for proof-of-concept (POC) testing of arCAR. [Figure 12K]1 shows the plasmid map p522 for proof-of-concept (POC) testing of arCAR. [Figure 13] Figure 1 shows CAR and GFP expression in Nurkat cells (Nur77 reporter line of Jurkat cells) transduced with lentivirus (LV) containing humanized B11 (p1649) CAR and humanized D04 (p1648) CAR. Results for untransduced (UTD) Nurkat are also shown. CAR expression was measured by polyclonal anti-camelid VHH cocktail from Genscript (e.g. sufficient homology between humanized and camelid versions of VHH for robust detection). [Figure 14] Shown are B11 (p1649) and D04 (p1648) CAR Nurkat cells stained with biotinylated HEL detected with streptavidin-APC via flow cytometry. UTD are non-transduced Nurkat cells used as a negative control. [Figure 15] The outline of the phage display panning scheme is shown. The scheme for anti-idiotype discovery against D04 is shown as an example. Similarly, for anti-idiotype discovery against B11, the positions of the two VHHs are swapped in the diagram. [Figure 16A] The ELISA output (absorbance at 450 nm) of periplasmic extracts (PPE) of colonies obtained for each CNTY library is shown. Black bars represent the ELISA response against B11 protein and grey bars represent the response against D04 protein. [Figure 16B] The ELISA output (absorbance at 450 nm) of periplasmic extracts (PPE) of colonies obtained for each CNTY library is shown. Black bars represent the ELISA response against B11 protein and grey bars represent the response against D04 protein. [Figure 16C] The ELISA output (absorbance at 450 nm) of periplasmic extracts (PPE) of colonies obtained for each CNTY library is shown. Black bars represent the ELISA response against B11 protein and grey bars represent the response against D04 protein. [Figure 16D] The ELISA output (absorbance at 450 nm) of periplasmic extracts (PPE) of colonies obtained for each CNTY library is shown. Black bars represent the ELISA response against B11 protein and grey bars represent the response against D04 protein. [Figure 17A] FACS binding of anti-idiotypic VHH-Fc fusion constructs without an IgG-based linker to B11 CAR-expressing Nurkat cells is shown. Dashed boxes identify select proteins from Table 15 that were observed to bind to D04 in ELISA. Dotted boxes identify select proteins from Table 16 that were observed to bind to B11 in ELISA. Each protein was tested in both cell lines at a 3-fold dilution series from 50 nM to 0.02 pM (as indicated in the legend). Fc fusion proteins were detected via anti-Fc APC and the percentage of cells that were positive for this detection method is shown. Proteins that showed specific binding to the target cell line in the dilution series are marked with an asterisk under the protein ID. Controls include (i) no VHH-Fc added, and (ii) anti-VHH cocktail detection reagent (far right). Samples shown on the graph for each protein appear in the order specified in the legend. [Figure 17B]FACS binding of anti-idiotypic VHH-Fc fusion constructs without an IgG-based linker to D04 CAR-expressing Nurkat cells is shown. Dashed boxes identify select proteins from Table 15 that were observed to bind to D04 in ELISA. Dotted boxes identify select proteins from Table 16 that were observed to bind to B11 in ELISA. Each protein was tested in both cell lines at a 3-fold dilution series from 50 nM to 0.02 pM (as indicated in the legend). Fc fusion proteins were detected via anti-Fc APC and the percentage of cells that were positive for this detection method is shown. Proteins that showed specific binding to the target cell line in the dilution series are marked with an asterisk under the protein ID. Controls include (i) no VHH-Fc added, and (ii) anti-VHH cocktail detection reagent (far right). Samples shown on the graph for each protein appear in the order specified in the legend. [Figure 18] Binding of lead anti-idiotype biotinylated VHH-Fc to target and off-target CAR Nurkat cells detected via FACS with streptavidin-APC. Cell lines tested include B11, D04, P711, P712, P713, P716, and parental cells. B11 and D04 cells are CAR Nurkats expressing B11 and D04 VHH CARs, respectively. P711, P712, P713, P716 cells are off-target CAR Nurkats. Parental cells are untransduced non-CAR expressing Nurkats. Controls include biotinylated Protein A (non-specifically binds most VHHs), PROT786 (irrelevant biotinylated VHH-Fc protein), and no biotinylated protein added. Samples shown on the graph for each protein appear in the order specified in the legend. [Figure 19A]Shown are the results of an overnight stimulation assay using Nurkat cells incubated with 10 nM anti-idiotypic VHH Fc fusions via FACS. Shown are GFP expression of B11 and D04 CAR Nurkat cells and parental Nurkat cells after overnight co-culture with anti-idiotypic Fc fusion proteins. Controls include PMA / ionomycin (a TCR-crosslinker that results in robust Nurkat cell activation), biotinylated Protein A (btPROTA), and samples with no added protein. Samples shown on the graph for each protein appear in the order specified in the legend. [Figure 19B] Shown are the results of an overnight stimulation assay using Nurkat cells incubated with 10 nM anti-idiotypic VHH Fc fusions via FACS. Detection of biotinylated proteins via streptavidin-APC after overnight incubation is shown. Controls include PMA / ionomycin (a TCR-crosslinker that results in robust Nurkat cell activation), biotinylated Protein A (btPROTA), and a sample with no added protein. Samples shown on the graph for each protein appear in the order specified in the legend. [Figure 20A] The results of the same T cell killing assay are shown in which the bridge protein AMD109 was added at 5 nM. The percentage of targets killed is shown for either U87 or K562 target cells. [Figure 20B] The results of the same T cell killing assay are shown in which the bridge protein AMD112 was added at 5 nM. The percentage of targets killed is shown for either U87 or K562 target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Various publications, articles and patents are cited or described in the Background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0046] The present application provides, inter alia, a universal chimeric antigen receptor (arCAR) system with adaptable receptor specificity components and their use in immunotherapy (e.g., adoptive cell therapy). This arCAR platform provides a built-in, convenient mechanism for receptor modulation using tag polypeptide affinity, allowing multiple receptors to be presented in cell therapy. Such arCARs can enable fit-for-purpose cell therapy.

[0047] definition The term "chimeric antigen receptor" or "CAR" as used herein generally refers to a cell surface receptor that includes an extracellular target binding domain, a transmembrane domain, and a cytoplasmic domain that includes a signaling domain and optionally at least one costimulatory signaling domain, all in a combination not found together on a single protein in nature. This specifically includes receptors in which the extracellular and cytoplasmic domains are not found together on a single receptor protein in nature.

[0048] As used herein, the term "chimeric antigen receptor with compatible receptor specificity components" or "arCAR" refers to a two-component CAR system in which the extracellular target binding domain of the receptor can be coupled with a variety of different antigen binding moieties. Unless otherwise indicated, the term "chimeric antigen receptor" or "CAR" as used herein is meant to encompass the "chimeric antigen receptor with compatible receptor specificity components" or "arCAR" described herein. The arCAR system of the present disclosure can be used with immune effector cells such as lymphocytes, including T cells and natural killer (NK) cells, which can be derived from stem cells such as induced pluripotent stem cells (iPSCs).

[0049] The term "immune effector cell" as used herein refers to a cell differentiated into a form capable of regulating or effecting a specific immune response. Such cells can include mature lymphocytes suitable for therapy, including but not limited to cytotoxic T cells, helper T cells, natural killer cells, and tumor infiltrating lymphocytes (TIL), and may also include dendritic cells or macrophages.

[0050] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, such as fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, scFv fragments, recombinant IgG (rIgG) fragments, single-chain (or single-chain) antibody fragments (including single-chain variable fragments (scFv)), and single domain antibody (e.g., VHH, VNAR, sdAb, sdFv) fragments or nanobodies, Fd' fragments, Fd fragments, heavy chain variable regions, or isolated complementarity determining regions (CDRs). The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, tetrabodies, decabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, and includes antibodies of any class or subclass, including IgG and its subclasses (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgY, IgE, IgA, and IgD.

[0051] The term "anti-idiotypic molecule" refers to a molecule (e.g., peptide, protein, antibody or antibody fragment, surrogate scaffold) that specifically recognizes, specifically targets, and / or specifically binds to the idiotope of an antibody, such as an antigen-binding fragment. The idiotope of an antibody may include, but is not necessarily limited to, the complementarity determining regions (CDRs) of an antibody, the variable regions of an antibody, and / or residues in one or more of such variable regions and / or some portion or portions of such CDRs, and / or any combination of the foregoing. The CDRs may be one or more selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The variable regions of an antibody may be a heavy chain variable region, a light chain variable region, or a combination of a heavy chain variable region and a light chain variable region. A partial fragment or portion of the heavy and / or light chain variable region of an antibody may be a fragment comprising 2 or more, 5 or more, or 10 or more consecutive amino acids, e.g., about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 consecutive amino acids, within the heavy or light chain variable region of the antibody; an idiotope may comprise multiple non-contiguous stretches of amino acids. A partial fragment of the heavy and light chain variable region of an antibody may be a fragment comprising 2 or more, 5 or more, or 10 or more consecutive amino acids, e.g., about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 consecutive amino acids, within the variable region, and in some embodiments, comprises one or more CDRs or CDR fragments. A CDR fragment can be 2 or more, or 5 or more, consecutive or non-consecutive amino acids within a CDR. Thus, an antibody idiotope can be about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 consecutive amino acids including one or more CDRs or one or more CDR fragments within the heavy chain variable region or light chain variable region of an antibody.In another embodiment, the idiotope may be a single amino acid located in the variable region of an antibody, for example in a CDR site.

[0052] In some embodiments, an idiotope is any single antigenic determinant or epitope within the variable portion of an antibody. In some cases, it can overlap with the actual antigen-binding site of an antibody, and in some cases, it may include variable region sequences outside of the antibody's antigen-binding site. The set of individual idiotopes of an antibody is, in some embodiments, referred to as the "idiotype" of such an antibody.

[0053] As used herein, the term "antigen" refers to any molecule of agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) that can be bound by an antibody or antibody fragment, a T-cell receptor, or an alternative scaffold. Antigens can also elicit an immune response. Examples of immune responses can include, but are not limited to, antibody production, or activation of specific immunologically competent cells, or both. Those skilled in the art will understand that antigens need not be encoded by a "gene". It is readily apparent that antigens can be synthetically produced or derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or other biological components, organisms, subunits of proteins / antigens, dead or inactivated whole cells or fluids containing lysates.

[0054] The terms "vector", "expression vector", and "expression construct" are used interchangeably to refer to a configuration of matter that can be used to deliver a nucleic acid of interest inside a cell and mediate its expression in the cell. The most commonly used examples of vectors are autonomously replicating plasmids and viruses (e.g., adenovirus vectors, adeno-associated virus vectors (AAV), lentivirus vectors, Sindbis virus vectors, etc.). Expression constructs can be replicated in living cells or made synthetically. In one embodiment, the expression vector comprises a promoter operably linked to a polynucleotide (e.g., a polynucleotide encoding the first and / or second polypeptide of arCAR described herein), which controls the initiation of transcription by RNA polymerase and the expression of the polynucleotide. Typical promoters for expression in mammalian cells include, for example, the SV40 early promoter, the CMV immediate early promoter (see, for example, U.S. Patent Nos. 5,168,062 and 5,385,839; both of which are incorporated herein by reference in their entirety), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (AdMLP), the herpes simplex virus promoter, the mouse metallothionein gene promoter, and the U6 or H1 RNA pol III promoter. Non-limiting examples of promoters useful for expressing the first and / or second polypeptides of arCAR described herein in the methods of the present disclosure include, for example, the synapsin promoter (neuron-specific), the CamKIIa promoter (specific to excitatory neurons), the ubiquitin promoter, the CAG promoter, the CMV promoter, and the β-actin promoter. These and other promoters can be obtained from commercially available plasmids using techniques well known in the art. See, for example, Sambrook et al., supra. An enhancer element can be used in conjunction with the promoter to increase expression levels of the vector.Examples include the SV40 early gene enhancer as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer / promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, which is incorporated herein by reference in its entirety, and elements derived from human CMV as described in Boshart et al., Cell (1985) 41:521, which is incorporated herein by reference in its entirety, e.g., elements contained in the CMV intron A sequence.

[0055] Transcription terminator / polyadenylation signals may also be present in the expression vector. Examples of such sequences include, but are not limited to, sequences derived from SV40 as described in Sambrook et al. (infra), as well as bovine growth hormone terminator sequences (see, for example, U.S. Pat. No. 5,122,458, which is incorporated herein by reference in its entirety). In addition, 5'-UTR sequences may be placed adjacent to the coding sequence to enhance expression of the coding sequence. Such sequences include, for example, UTRs containing the Internal Ribosome Entry Site (IRES) present in the leader sequences of picornaviruses, such as the UTR of encephalomyocarditis virus (EMCV) (Jang et al. J. Virol. (1989) 63:1651-1660; which is incorporated herein by reference in its entirety). Other useful picornavirus UTR sequences include, for example, the polio leader sequence, the leader of Hepatitis A virus, and the IRES of Hepatitis C virus.

[0056] The term "host cell" refers to any cell that contains a heterologous nucleic acid. The heterologous nucleic acid may be a vector (e.g., an expression vector). For example, a host cell may be a cell from any organism that is selected, modified, transformed, grown, used or engineered in any way for the production of a substance by the cell, for example, for the expression by the cell of a gene, DNA or RNA sequence, protein or enzyme. An appropriate host may be determined. For example, a host cell may be selected based on the vector backbone and the desired outcome. As an example, a plasmid or cosmid may be introduced into a prokaryotic host cell for replication of some types of vectors. Bacterial cells, such as but not limited to DH5α, JM109, and KCB, SURE® Competent Cells, and SOLOPACK Gold Cells, may be used as host cells for replication and / or expression of vectors. Additionally, bacterial cells, such as E. coli LE392, may be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500 and YPH501), insect and mammalian cells. Examples of mammalian eukaryotic host cells for replicating and / or expressing vectors include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos and PC12.

[0057] The host cells of the present disclosure include T cells and natural killer cells that contain a DNA or RNA sequence encoding a CAR and / or express a CAR on the cell surface. The host cells can be used to enhance the activity of T cells, the activity of natural killer cells, the treatment of cancer, and the treatment of autoimmune diseases.

[0058] The terms "T cells" and "T lymphocytes" are interchangeable and are used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells. Also included are "NKT cells," which refers to a specialized population of T cells that express a semi-invariant (or semi-invariant) αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+ and CD8- cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CDId. NKT cells can have either protective or harmful effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma-delta T cells (γδ T cells)," which refers to a specialized population that is a small subset of T cells that have a distinct TCR on their surface; unlike most T cells, whose TCR is composed of two glycoprotein chains called α- and β-TCR chains, the TCR in γδ T cells is composed of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immune regulation, are an important source of IL-17, and have been found to induce robust CD8+ cytotoxic T cell responses. In addition, "regulatory T cells" or "Treg" refers to T cells that suppress aberrant or excessive immune responses and play a role in immune tolerance. Treg cells are typically transcription factor Foxp3-positive CD4+ T cells, and can also include transcription factor Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0059] The terms "natural killer cells" and "NK cells" are used interchangeably and synonymously herein. As used herein, NK cells refer to differentiated lymphocytes with a CD16+CD56+ and / or CD57+TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that fail to express "self" MHC / HLA antigens by activation of specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and to release protein molecules called cytokines that stimulate or inhibit immune responses.

[0060] In certain embodiments of the present disclosure, cells containing the nucleic acid constructs of the present disclosure are identified in vitro or in vivo by including a marker in the expression vector. Such a marker provides an identifiable change to the cells, allowing easy identification of cells containing the expression vector. Typically, the inclusion of a drug selection marker aids in the cloning and selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be used. Fluorescent markers (e.g., green fluorescent protein (GFP), EGFP, or Dronpa) or immunological markers may also be used. Further examples of selection markers are well known to those skilled in the art.

[0061] As used herein, the term "variant" in the context of a protein or polypeptide (e.g., an arCAR polypeptide or domain thereof) refers to: (a) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide; (b) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the nucleotide sequence encoding the polypeptide; (c) a polypeptide encoded by a nucleotide sequence having 0%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to said polypeptide; (d) a polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and substitutions) relative to said polypeptide. (d) a polypeptide encoded by a nucleic acid capable of hybridizing under high stringency, medium stringency, or typical stringency hybridization conditions to a nucleic acid encoding the polypeptide; (e) a polypeptide encoded by a nucleotide sequence capable of hybridizing under high stringency, medium stringency, or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide.

[0062] Percent sequence identity can be determined using any method known to those skilled in the art. In certain embodiments, percent identity is determined using the "Best Fit" or "Gap" program of the sequence analysis software package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, medium, and typical stringent conditions) is described, see, for example, U.S. Patent Application Publication No. 2005 / 0048549 (e.g., paragraphs 72-73).

[0063] In the context of this disclosure, the terms "treat", "treatment" and the like, in relation to any of the disease conditions listed herein, mean preventing, alleviating or relieving at least one symptom associated with such condition, or slowing or reversing the progression of such condition, or halting, delaying the onset (i.e., the period before clinical symptoms of the disease) and / or reducing the risk of development or worsening of the disease.

[0064] As used herein, the term "therapeutically effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce a desired activity (e.g., alleviation of symptoms associated with cancer or autoimmune disease) when administered to a subject in need thereof. It should be noted that when a combination of active ingredients (or active ingredients) is administered, the effective amount of the combination may or may not include the amount of each ingredient that would have been effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the condition being treated, the particular drug(s) used, the mode of administration, and the like. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0065] The phrase "pharmacologically acceptable" as used in connection with the compositions of the present disclosure refers to molecular entities and other components of the composition that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human).Preferably, as used herein, the term "pharmacologically acceptable" means approved by a regulatory agency of the U.S. Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more specifically, humans.

[0066] As used herein, the term "combination" of the composition of the present disclosure with at least an additional therapeutic agent means that at least two, but any desired combination of drugs can be delivered simultaneously or sequentially.When used to treat various diseases, it is contemplated that the compositions and methods of the present disclosure can be utilized with other therapeutic methods / drugs suitable for the same or similar disease. Such other therapeutic methods / drugs can be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. The appropriate therapeutically effective dosage of each drug can be reduced due to additive or synergistic effects.

[0067] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water or aqueous solutions, such as saline solution and aqueous dextrose and glycerol solutions, are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of binders (for compressed pills), glidants, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0068] As used herein, an "individual" or "subject" or "animal" refers to a human, a veterinary animal (e.g., a cat, a dog, a cow, a horse, a sheep, a pig, etc.), or an experimental animal model of a disease or disorder (e.g., cancer or an autoimmune disease). In a preferred embodiment, the subject is a human.

[0069] The term "associated with" is used to encompass any correlation, coincidence, and any causal relationship.

[0070] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, according to the practice of the art. Alternatively, "about" can mean within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, even more preferably within 5%, and most preferably within 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. When a particular value is described in the present application and claims, unless otherwise specified, the term "about" is implicit and, in this context, means within an acceptable error range for the particular value.

[0071] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0072] In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization [BD Hames & SJ Higgins eds. (1985)]; Transcription and Translation [BD Hames & SJ Higgins, eds. (1984)]; Animal Cell Culture [RI Freshney, ed. (1986)]; Immobilized Cells and Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, FM et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include those described in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985); U.S. Patent No. 5,071,743; Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419; Hogrefe, Strategies 14. 3: 74-75 (2001); U.S. Patent Nos. 5,702,931, 5,780,270, and 6,242,222; Angag and Schutz, Biotech. 30: 486-488 (2001); Wang and Wilkinson, Biotech. 29: 976-978 (2000); Kang et al., Biotech. 20: 44-46 (1996); Ogel and McPherson, Protein Engineer. 5: 467-468 (1992); Kirsch and Joly, Nucl. Acids. Res. 26: 1848-1850 (1998); Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996); Boles and Miogsa, Curr. Genet. 28: 197-198 (1995); Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993); Tessier and Thomas, Meths. Molec. Biol. 57: 229-237; and Pons et al., Meth. Molec. Biol. 67: 209-218.

[0073] Chimeric antigen receptors with compatible receptor specificity components (arCARs) In one aspect, provided herein is a universal chimeric antigen receptor (arCAR) system having compatible receptor specificity components, the arCAR system comprising: (i) an immune effector cell having a chimeric antigen receptor comprising a first polypeptide comprising (a) an extracellular tag-binding domain, (b) a transmembrane domain, and (c) at least one intracellular signaling domain; and (ii) a second polypeptide comprising (d) an antigen-binding domain that binds to at least one antigen on a target cell and (b) a tag that is recognized by the tag-binding domain of the first polypeptide of the chimeric antigen receptor; Including, (i) the tag comprises an antibody or antigen-binding fragment thereof, or an alternative scaffold, and the tag-binding domain comprises an anti-idiotypic molecule that binds to the tag; or (ii) The tag comprises an anti-idiotypic molecule that binds to a tag binding domain, the tag binding domain comprising an antibody or antigen-binding fragment thereof, or an alternative scaffold.

[0074] In some embodiments, the anti-idiotype molecule binds to at least one antigen-binding or non-framework region of an antibody or antigen-binding fragment thereof.

[0075] In some embodiments, the tag comprises an antibody or antigen-binding fragment thereof or an surrogate scaffold that binds to a polypeptide derived from a non-human source. Alternatively, the tag binding domain comprises an antibody or antigen-binding fragment thereof or an surrogate scaffold that binds to a polypeptide derived from a non-human source.

[0076] In some embodiments, the anti-idiotypic molecule binds to at least one complementarity determining region (CDR) of an antibody or antigen-binding fragment thereof.

[0077] In some embodiments, the anti-idiotypic molecule is an anti-idiotypic antibody or an antigen-binding fragment thereof, or a surrogate scaffold.

[0078] In some embodiments, the antigen-binding domain of the second polypeptide comprises an antibody or antigen-binding fragment thereof, or an surrogate scaffold.

[0079] In various embodiments, antibodies or antibody fragments suitable for use in the arCAR system of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single chain Fvs (scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPs™"), intrabodies, minibodies, single domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAbs®), and epitope-binding fragments of any of the above. Antibodies and / or antibody fragments may be derived from mouse antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0080] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab ’ In some embodiments, the antigen-binding fragment is a F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is a scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0081] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or a nanobody.

[0082] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0083] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0084] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH.

[0085] In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises an scFv.

[0086] In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.

[0087] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise an scFv.

[0088] In some embodiments, the antibodies or antigen-binding fragments (e.g., VHH, scFv) used herein are humanized. Humanized proteins have the potential to reduce the risk of immunogenicity.

[0089] Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high affinity and specific binding of target biological substances, can also be used in the arCARs of the present disclosure. Such scaffolds have been shown to produce molecules with improved characteristics, such as higher stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that may be used in the arCAR system of the present disclosure include engineered tenascin type III domains derived from tenascin (e.g., Centyrin™); engineered gamma B-crystallin-derived scaffolds or engineered ubiquitin-derived scaffolds (e.g., Affilins); engineered fibronectin type III (10Fn3) domains derived from fibronectin (e.g., monobodies, AdNectins™, or AdNexins™); engineered ankyrin repeat motif-containing polypeptides (e.g., DARPins™); engineered A domains derived from low density lipoprotein receptor (LDLR-A) (e.g., Avimers™); lipocalins (e.g., anticalins); engineered proteases Kunitz domains from inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); engineered Z domains from Protein A (Affibodies™); Sac7d-derived polypeptides (e.g., Nanoffitins® or affitins); engineered SH2 domains from Fyn (e.g., Fynomers®); CTLD3 (e.g., tetranectin); thioredoxin (e.g., peptide aptamers); KALBITOR®; beta sandwich (e.g., iMab); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimetics; as well as engineered counterparts of any of the above that retain their binding functionality (Woern A, Pluckthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004); Binz H et al., Nat Biolechnol 23: 1257-68 (2005); Hey T et al., Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Skerra, Current Opin. in Biotech., 2007 18: 295-304; Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85 (2011); each of which is incorporated by reference in its entirety).

[0090] In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0091] Molecules derived from non-human sources can be derived from any non-human organism, including, but not limited to, viruses, bacteria, yeast, fungi, plants, and non-human animals.

[0092] In some embodiments, the molecule derived from a non-human source is a polypeptide. In some embodiments, the source of the protein is derived from a non-human polypeptide that has no human homologue.

[0093] In some embodiments, the polypeptide derived from a non-human source is respiratory syncytial virus (RSV) F protein, Listeria internalin, Cobra venom phospholipase A2, Ebola nucleoprotein, Herpes simplex virus (HSV) glycoprotein D, lactococcal phage receptor binding protein (RBP), Geobacillus stearothermophilus, ricin, or hen egg white lysozyme.

[0094] In some embodiments, the first polypeptide of the arCAR of the present disclosure comprises a leader sequence. The leader sequence may be located at the N-terminus of the extracellular tag-binding domain. The leader sequence may be optionally cleaved from the extracellular tag-binding domain during cell processing and localization of the CAR to the cell membrane. Various leader sequences known to those skilled in the art may be used as leader sequences. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte macrophage colony-stimulating factor (GMCSF), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, mouse Ig-κ signal peptide, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from human immunoglobulin heavy chain (HC).

[0095] In some embodiments, the leader sequence is derived from GMCSF. In one embodiment, the GMCSF leader sequence comprises the amino acid sequence set forth in SEQ ID NO:1, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1.

[0096] In some embodiments, the leader sequence is derived from mouse Ig-κ signal peptide. In one embodiment, the mouse Ig-κ signal peptide comprises the amino acid sequence set forth in SEQ ID NO:61, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:61.

[0097] In some embodiments, the first polypeptide of an arCAR of the disclosure comprises a transmembrane domain fused in frame between the extracellular tag-binding domain and the cytoplasmic domain.

[0098] The transmembrane domain may be derived from a protein that contributes to the extracellular tag-binding domain, a protein that contributes to the signaling domain or the co-signaling domain, or from a completely different protein. In some cases, the transmembrane domain may be selected or modified by amino acid substitution, deletion, or insertion to minimize interaction with other members of the arCAR complex. In some cases, the transmembrane domain may be selected or modified by amino acid substitution, deletion, or insertion to avoid binding of proteins that naturally associate with the transmembrane domain. In some embodiments, the transmembrane domain contains additional amino acids that allow flexibility and / or optimal distance between the domains connected to the transmembrane domain.

[0099] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains that are particularly useful in the present disclosure may be derived from (i.e., may include at least the transmembrane regions of) the α, β or ζ chains of the T cell receptor (TCR), NKG2D, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan and / or valine may be found at each end of a synthetic transmembrane domain.

[0100] In some embodiments, it is desirable to utilize transmembrane domains of the zeta, eta or FcεR1 gamma chains that contain cysteine ​​residues capable of disulfide bonding, so that the resulting chimeric protein can form disulfide-linked dimers with itself or with unmodified versions of the zeta, eta or FcεR1 gamma chains or related proteins. In some cases, the transmembrane domains are selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it is desirable to use transmembrane domains of zeta, eta or FcεR1 gamma and -β, MB1 (Igα), B29 or CD3-γ, zeta or eta to retain physical association with other members of the receptor complex.

[0101] In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:24, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:24.

[0102] In some embodiments, the first polypeptide of the arCAR system of the present disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, and the tag-binding domain, linker, and transmembrane domain are in-frame with each other.

[0103] The term "spacer region" as used herein generally refers to any oligopeptide or polypeptide that functions to link a tag-binding domain to a transmembrane domain. Spacer regions can be used to provide more flexibility and accessibility to the tag-binding domain. Spacer regions can contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. Spacer regions can be derived from all or a portion of naturally occurring molecules, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or from all or a portion of an antibody constant region. Alternatively, the spacer region can be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or can be a completely synthetic spacer region sequence. Non-limiting examples of spacer regions that can be used in accordance with the present disclosure include a portion of the human CD8 alpha chain, a partial extracellular domain of CD28, an FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, Ig hinge, or functional fragments thereof. In some embodiments, additional linking amino acids are added to the spacer region to ensure that the antigen binding domain is at an optimal distance from the transmembrane domain. In some embodiments, if the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.

[0104] In some embodiments, the spacer region comprises a hinge domain. The hinge domain can be derived from CD8α, CD28 or immunoglobulin (IgG). For example, the IgG hinge can be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof.

[0105] In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or a functional fragment thereof. In certain embodiments, the IgG hinge is derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof. In certain embodiments, the hinge domain comprises an immunoglobulin CH1, CH2, CH3 and / or hinge region. In certain embodiments, the hinge domain comprises an immunoglobulin core hinge region. The term "core hinge" may be used interchangeably with the term "short hinge" (also known as "SH"). Non-limiting examples of suitable hinge domains are core immunoglobulin hinge regions, including EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgG1, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 59) from IgG3, and ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see Wypych et al., JBC 2008 283(23): 16194-16205; incorporated herein by reference in its entirety for all purposes). In certain embodiments, the hinge domain is a fragment of an immunoglobulin hinge.

[0106] In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21. In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22.

[0107] In some embodiments, the transmembrane domain and / or hinge domain are derived from CD8 or CD28. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD28.

[0108] In some aspects, the first polypeptide of the arCAR of the present disclosure comprises a cytoplasmic domain that comprises at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more costimulatory signaling domains.

[0109] The cytoplasmic domain is responsible for activating at least one of the normal effector functions of the host cell (e.g., T cell) in which the arCAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Usually, the entire signaling domain is present, but in many cases it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the signaling domain sufficient to transmit the effector function signal.

[0110] Non-limiting examples of signaling domains that can be used in the arCARs of the present disclosure include, for example, signaling domains derived from DAP10, DAP12, Fcε receptor Iγ chain (FCER1G), FcR β, NKG2D, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, or CD79B.

[0111] In some embodiments, the cytoplasmic domain comprises a CD3 zeta signaling domain. In one embodiment, the CD3 zeta signaling domain comprises an amino acid sequence set forth in SEQ ID NO:6, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6.

[0112] In some embodiments, the cytoplasmic domain further comprises one or more costimulatory signaling domains, in some embodiments, the one or more costimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, or HVEM.

[0113] In one embodiment, the costimulatory signaling domain is derived from 41BB. In one embodiment, the 41BB costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:8, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8.

[0114] In one embodiment, the costimulatory signaling domain is derived from IL2Rb. In one embodiment, the IL2Rb costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO:9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9.

[0115] In some embodiments, the costimulatory signaling domain is derived from CD40. In some embodiments, the CD40 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10.

[0116] In one embodiment, the costimulatory signaling domain is derived from OX40. In one embodiment, the OX40 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO:11, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:11.

[0117] In some embodiments, the costimulatory signaling domain is derived from CD80. In some embodiments, the CD80 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12.

[0118] In some embodiments, the costimulatory signaling domain is derived from CD86. In some embodiments, the CD86 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13.

[0119] In some embodiments, the costimulatory signaling domain is derived from CD27. In some embodiments, the CD27 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0120] In one embodiment, the costimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.

[0121] In one embodiment, the costimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16.

[0122] In one embodiment, the costimulatory signaling domain is derived from DAP10. In one embodiment, the DAP10 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17.

[0123] In one embodiment, the costimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP12 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18.

[0124] In one embodiment, the costimulatory signaling domain is derived from 2B4 (CD244). In certain embodiments, the 2B4 (CD244) costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

[0125] In some embodiments, the costimulatory signaling domain is derived from CD28. In some embodiments, the CD28 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO:20, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20.

[0126] In some embodiments, the arCAR of the present disclosure comprises one costimulatory signaling domain. In some embodiments, the arCAR of the present disclosure comprises two or more costimulatory signaling domains. In some embodiments, the arCAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.

[0127] In some embodiments, the signal transduction domain and the costimulatory signal transduction domain can be arranged in any order. In some embodiments, the signal transduction domain is upstream of the costimulatory signal transduction domain. In some embodiments, the signal transduction domain is downstream of the costimulatory signal transduction domain. When more than one costimulatory domain is included, the order of the costimulatory signal transduction domains can be swapped.

[0128] Non-limiting exemplary CAR regions and sequences are provided in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0129] In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen. The antigen-binding domain of the second polypeptide may bind to multiple antigens or multiple epitopes in the antigen. For example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more antigens. As another example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more epitopes in the same antigen.

[0130] The selection of antigen binding domains may depend on the type and number of antigens that define the surface of target cells. For example, antigen binding domains may be selected to recognize antigens that function as cell surface markers on target cells associated with a particular disease state. In an embodiment, the arCAR of the present disclosure can be genetically modified to target tumor antigens of interest by engineering a desired antigen binding domain that specifically binds to the antigen (e.g., on tumor cells). Non-limiting examples of cell surface markers that may serve as targets for antigen binding domains in the arCAR of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0131] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen.

[0132] In some embodiments, the antigen binding domain binds to at least one tumor antigen. In some embodiments, the antigen binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors.

[0133] In some embodiments, the antigen binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.

[0134] In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies. Non-limiting examples of tumor antigens associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2. Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFRα, Nectin-4, and B7H4. Non-limiting examples of tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, Mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigens associated with liver cancer include claudin 18.2, GPC-3, EpCAM, cMET, and AFP.Non-limiting examples of tumor antigens associated with hematological malignancies include CD19, CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70.Non-limiting examples of tumor antigens associated with bladder cancer include nectin-4 and SLITRK6.Non-limiting examples of tumor antigens associated with kidney cancer include CD70 and FOLR1.

[0135] Further examples of antigens that the antigen binding domain may target include alpha-fetoprotein, A3, antigen specific for the A33 antibody, Ba733, BrE3 antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138. , colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic These include, but are not limited to, gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placenta growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, angiogenesis markers, oncogene markers, or oncogene products.

[0136] In one embodiment, the antigen targeted by the antigen binding domain is CD19. In one embodiment, the antigen binding domain comprises an anti-CD19 scFv. In one embodiment, the anti-CD19 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2. In certain embodiments, the anti-CD19 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 7, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7.

[0137] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells that produce "self"-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder, such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis (granulomatosis with polyangiitis), myasthenia gravis, Hashimoto's thyroiditis, hyperthyroidism (Graves' disease or Graves' disease), chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease, or ulcerative colitis.

[0138] In some embodiments, autoimmune antigens that may be targeted by the arCARs disclosed herein include platelet antigens, myelin protein antigens, Sm antigens in snRNPs, pancreatic islet cell antigens, rheumatoid factors, and anti-citrullinated proteins, citrullinated proteins and peptides (e.g., CCP-1, CCP-2 (cyclic citrullinated peptides)), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factors, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage (e.g., collagen II, IX and XI), circulating serum proteins (e.g., RF (IgG, IgM), fibrinogen, plasminogen, ferritin), nuclear components (e.g., RA33 / hnRNP, A2, Sm, eukaryotic translation elongation factor 1α1), stress proteins (e.g., HSP-65, -70, -90, BiP), inflammatory / immune factors (e.g., B7-H1, IL-1α, and IL-8), enzymes (e.g., calpastatin, α-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase), receptors (e.g., lipocortin 1), neutrophil nuclear proteins (e.g., lactoferrin and 25-35 kD nuclear protein), granule proteins (e.g., bactericidal permeability increasing protein, These include, but are not limited to, ribosomal protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, pancreatic islet cell antigens, rheumatoid factors, histones, ribosomal P protein, cardiolipin, vimentin), nucleic acids (e.g., dsDNA, ssDNA, and RNA), ribonucleopartides and proteins (e.g., Sm antigens (including but not limited to SmD and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro(SSA), and La(SSB) antigens).

[0139] Non-limiting exemplary antigen targets are provided in Tables 2-4.

[0140] [Table 2-1]

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

[0141]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

[0142]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

[0143] In various embodiments, the scFv fragments used in the arCAR system of the present disclosure may include a linker between the VH and VL domains. The linker may be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should have an appropriate length to link the VH and VL such that the VH and VL form the correct conformation relative to each other and retain the desired activity, such as binding to an antigen. The linker may be about 5-50 amino acids in length. In some embodiments, the linker is about 10-40 amino acids in length. In some embodiments, the linker is about 10-35 amino acids in length. In some embodiments, the linker is about 10-30 amino acids in length. In some embodiments, the linker is about 10-25 amino acids in length. In some embodiments, the linker is about 10-20 amino acids in length. In some embodiments, the linker is about 15-20 amino acids in length. Exemplary linkers that can be used are Gly-rich linkers, Gly- and Ser-containing linkers, Gly- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0144] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises an amino acid sequence as set forth in SEQ ID NO:3, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:3. In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker comprises an amino acid sequence as set forth in SEQ ID NO:25, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:25.

[0145] Other linker sequences may include portions of the hinge region, CL or CH1 of an immunoglobulin from any immunoglobulin heavy or light chain isotype. Exemplary linkers that may be used include any of SEQ ID NOs: 26-56 in Table 1. Additional linkers are described, for example, in International Publication No. WO2019 / 060695, which is incorporated herein by reference in its entirety.

[0146] In some embodiments, the second polypeptide comprises an antigen-binding domain at the N-terminus and a tag at the C-terminus.In some embodiments, the second polypeptide comprises an antigen-binding domain at the C-terminus and a tag at the N-terminus.

[0147] In some embodiments, the second polypeptide is a soluble polypeptide.

[0148] In some embodiments, the arCAR system further comprises one or more additional polypeptides, each comprising (a) an antigen-binding domain that binds to a unique antigen, and (b) a tag that is recognized by the tag-binding domain of the first polypeptide.

[0149] In another aspect, provided herein is an arCAR system comprising two or more arCARs described herein, each arCAR comprising a unique pair of a tag and a tag-binding domain.

[0150] In a further aspect, provided herein is a polypeptide of arCAR as described herein.In one aspect, provided herein is a first polypeptide of arCAR as described herein.In one aspect, provided herein is a second polypeptide of arCAR as described herein.

[0151] Without wishing to be bound by theory, the arCAR system of the present disclosure may have a variety of advantages, including: High degree of specificity where anti-ID molecules / antibody fragments minimize off-target interactions, especially in combination with non-human protein targets for antibody specificity. Specificity for the binding loop on the tag avoids undesired interactions with similar framework / scaffold proteins. True-ID can also be used to neutralize binding to the target. · The inherent ability to develop a range of affinities to optimize and modulate the interaction of the "tag" polypeptide with the CAR. The system is unlimited in possible pairings allowing the development of numerous optimized CAR / "tag" polypeptide pairs, each with unique specificities enabling fit-for-purpose therapies including modulation of binding affinity, logic gates, multiple specificities per cell and multiple soluble drugs per treatment. This approach is "platform agnostic" and allows for the incorporation of a different scaffold (e.g. replacing scFv with VHH) if immunogenicity or other limitations to one scaffold arise.

[0152] Polynucleotides and Vectors In another aspect, provided herein is a polynucleotide encoding one or more polypeptides in the arCAR system of the present disclosure.

[0153] In some embodiments, the present disclosure provides a polynucleotide that encodes the first polypeptide of the arCAR system.In some embodiments, the present disclosure provides a polynucleotide that encodes the first polypeptide of two or more arCAR systems.In some embodiments, each of the two or more arCAR systems comprises a unique pair of tag and tag binding domain.

[0154] In some embodiments, the present disclosure provides a polynucleotide that encodes the second polypeptide of the arCAR system of the present disclosure.In some embodiments, the present disclosure provides a polynucleotide that encodes the second polypeptide of two or more arCAR systems of the present disclosure.In some embodiments, each of the two or more arCAR systems comprises a unique pair of tag and tag binding domain.

[0155] In some embodiments, the present disclosure provides a polynucleotide that encodes both polypeptides of the arCAR system.In some embodiments, the polynucleotide encodes both polypeptides of two or more arCAR systems of the present disclosure.In some embodiments, each of the two or more arCAR systems comprises a unique pair of tag and tag binding domain.

[0156] Polynucleotides can contain any type of nucleotide, including but not limited to DNA and RNA, they can be single-stranded or double-stranded, they can be synthetic or partially derived from natural sources, and they can contain natural, non-natural or modified nucleotides. Polynucleotides can contain naturally occurring or non-naturally occurring internucleotide bonds, or both types of bonds.

[0157] In some embodiments, the polynucleotides described herein are DNA molecules. In various embodiments, the polynucleotides described herein are RNA molecules.

[0158] In one aspect, the disclosure provides a recombinant vector comprising a polynucleotide described herein.

[0159] In some embodiments, the recombinant vector comprises a polynucleotide encoding the first polypeptide of the arCAR system of the present disclosure.In some embodiments, the recombinant vector comprises a polynucleotide encoding the first polypeptide of two or more arCARs of the present disclosure.In some embodiments, each of the two or more arCARs comprises a unique pair of tag and tag-binding domain.

[0160] In some embodiments, the recombinant vector comprises a polynucleotide that encodes the second polypeptide of the arCAR system of the present disclosure.In some embodiments, the recombinant vector comprises a polynucleotide that encodes the second polypeptide of two or more arCARs of the present disclosure.In some embodiments, each of the two or more arCARs comprises a unique pair of tag and tag binding domain.

[0161] In some embodiments, the recombinant vector comprises a polynucleotide that encodes both polypeptides of the arCAR system of the present disclosure. In some embodiments, the recombinant vector comprises a polynucleotide that encodes both polypeptides of two or more arCARs of the present disclosure. In some embodiments, each of the two or more arCARs comprises a unique pair of tag and tag-binding domain.

[0162] The recombinant vector may be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and expansion, or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, bicistronic IRES vectors (e.g., from Clontech) can be used to contain both the polynucleotides encoding the first and second polypeptides of the arCAR system described herein.

[0163] In some embodiments, the recombinant vector is a non-viral vector. The viral vector can be a plasmid or a transposon (such as a PiggyBac- or SleepingBeauty transposon). In one embodiment, the vector is a plasmid.

[0164] In some embodiments, the recombinant vector is a viral vector.Suitable viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, alphavirus vectors, adenoviral vectors, adeno-associated viral vectors (AAV), herpes virus vectors, vaccinia vectors, and fowlpox virus vectors.In some embodiments, the viral vector has the natural or engineered ability to transform host cells (e.g., T cells).

[0165] Recombinant vectors can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0166] Recombinant vectors may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for recombinant expression vectors include, for example, neomycin / G418 resistance gene, puromycin resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resistance gene.

[0167] Vectors useful in the context of this disclosure can be "naked" nucleic acid vectors (i.e., vectors with little or no proteins, sugars and / or lipids encapsulating them) or vectors complexed with other molecules. Other molecules that can be suitably combined with vectors include, but are not limited to, viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.

[0168] Vector DNA can be introduced into host cells (e.g., immune effector cells) through conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into cells, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, gene gun, or electroporation.

[0169] In various embodiments, the polynucleotide encoding the arCAR polypeptide (e.g., the first or second polypeptide) is operably linked to at least a regulatory element. The regulatory element can mediate the expression of the arCAR polypeptide (e.g., the first or second polypeptide) in the host cell. Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyadenylation (polyA) tails, IRES elements, response elements, and termination signals. In an embodiment, the regulatory element regulates the expression of the arCAR polypeptide. In an embodiment, the regulatory element increases the expression of the arCAR polypeptide (e.g., the first or second polypeptide). In an embodiment, the regulatory element increases the expression of the arCAR polypeptide (e.g., the first or second polypeptide) upon activation of the host cell. In an embodiment, the regulatory element decreases the expression of the arCAR polypeptide (e.g., the first or second polypeptide). In an embodiment, the regulatory element decreases the expression of the arCAR polypeptide (e.g., the first or second polypeptide) upon activation of the host cell.

[0170] Modified host cells In one aspect, provided herein is a host cell engineered to express one or more of the polypeptides of the arCAR of the disclosure.

[0171] In some embodiments, the present disclosure provides a host cell that is an immune effector cell comprising the first polypeptide of the arCAR system of the present disclosure.In some embodiments, the present disclosure provides a host cell that comprises two or more first polypeptides of the arCAR of the present disclosure.In some embodiments, each of the two or more arCARs comprises a unique pair of tag and tag-binding domain.

[0172] In some embodiments, the present disclosure provides a host cell comprising the second polypeptide of the arCAR of the present disclosure.In some embodiments, the present disclosure provides a host cell comprising two or more second polypeptides of the arCAR of the present disclosure.In some embodiments, each of the two or more arCARs comprises a unique pair of tag and tag binding domain.

[0173] In some embodiments, provided herein is a host cell comprising both the first and second polypeptides of an arCAR of the disclosure.

[0174] In some embodiments, provided herein is a host cell comprising a polynucleotide or recombinant vector described herein.

[0175] In various embodiments, the cell having a chimeric antigen receptor comprising the first polypeptide is an immune effector cell. In some embodiments, the cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a tumor infiltrating lymphocyte (TIL), a dendritic cell, or a macrophage.

[0176] In some embodiments, the immune effector cell is derived from a stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), or an embryonic stem cell (ESC). In some embodiments, the cell is an iPSC. In an aspect, the cell is a T cell derived from an iPSC. In one embodiment, the cell is a NK cell derived from an iPSC.

[0177] In one aspect, provided herein is a method for preparing the engineered host cells described herein.

[0178] In one embodiment, a method for preparing an engineered host cell includes introducing a polynucleotide encoding a first polypeptide of an arCAR of the disclosure, or a recombinant vector comprising the polynucleotide, into the cell.

[0179] In one embodiment, a method for preparing an engineered host cell includes introducing a polynucleotide encoding a second polypeptide of an arCAR of the disclosure, or a recombinant vector comprising the polynucleotide, into the cell.

[0180] In one embodiment, a method of preparing an engineered host cell comprises introducing into the cell a polynucleotide encoding both the first and second polypeptides of an arCAR of the disclosure, or a recombinant vector comprising the polynucleotide.

[0181] In various embodiments, the engineered cells constitutively express the first polypeptide of the arCAR described herein. In various embodiments, the engineered cells inducibly express the first polypeptide of the arCAR described herein.

[0182] In various embodiments, the engineered cells constitutively express the second polypeptide of the arCAR described herein. In various embodiments, the engineered cells inducibly express the second polypeptide of the arCAR described herein.

[0183] In various embodiments, the host cells may be autologous / autogenic ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). In some embodiments, the host cells are obtained from a mammalian subject. In some embodiments, the host cells are obtained from a primate subject. In some embodiments, the host cells are obtained from a human subject.

[0184] In certain embodiments, immune cells such as lymphocytes are used. Lymphocytes can be obtained from sources such as, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumor. Lymphocytes can also be generated by differentiation of stem cells. In certain embodiments, lymphocytes can be obtained from blood collected from a subject using techniques commonly known to those skilled in the art, such as sedimentation, e.g., FICOLL™ separation.

[0185] In certain embodiments, immune cells from the circulating blood of a subject are obtained by apheresis. Apheresis machines typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis are washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing. The cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. The washing step can be performed by methods known to those skilled in the art, including, but not limited to, using a semi-automated flow-through centrifuge (e.g., Cobe 2991 cell processor, or Baxter CytoMate). After washing, the cells can be resuspended in various biocompatible buffers, cell culture media, or other saline solutions with or without buffers.

[0186] In an embodiment, T lymphocytes can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and further depleting monocytes. As an example, cells can be sorted by centrifugation through a PERCOLL™ gradient. In an embodiment, after isolation of PBMCs, both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naive, memory, and effector T cells, either before or after activation, expansion, and / or genetic modification.

[0187] In certain embodiments, T lymphocytes can be enriched.For example, certain subpopulations of T lymphocytes expressing one or more markers, such as, but not limited to, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD27, CD28, CD34, CD36, CD45RA, CD45RO, CD56, CD62, CD62L, CD122, CD123, CD127, CD235a, CCR7, HLA-DR or combinations thereof can be enriched using either positive or negative selection techniques.In certain embodiments, the T lymphocytes for use in the compositions of the present disclosure do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0188] In certain embodiments, NK cells can be enriched. For example, specific subpopulations of T lymphocytes expressing one or more markers, such as, but not limited to, CD2, CD16, CD56, CD57, CD94, CD122, or combinations thereof, can be enriched using either positive or negative selection techniques.

[0189] In some embodiments, pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), are used to generate host cells, such as NK cells or T lymphocytes. Human iPSCs and human ESCs can be generated by various methods known in the art. For example, PSCs (e.g., iPSCs or ESCs) can be modified with the arCAR of the present disclosure by contacting cells with a polynucleotide or recombinant vector that encodes the polypeptide of arCAR, and the engineered PSCs can be used to generate or generate immune cells (e.g., T cells) that contain the arCAR of the present disclosure.

[0190] iPSCs can be generated directly from adult cells (somatic cells). iPSCs can be generated or created by introducing a specific set of pluripotency-associated genes or "reprogramming factors" into a given cell type. Reprogramming factors include, but are not limited to, OCT4 (also known as "POU5FL"), SOX2, cMYC, and KLF4, also known as Yamanaka factors; see Takahashi, K; Yamanaka, S (2006). Cell 126 (4): 663-76. Each of the reprogramming factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even unrelated genes such as lineage specifiers. Upon introduction of the reprogramming factors, the cells begin to form colonies resembling PSCs, which can be isolated based on their morphology, conditions that select for their growth, or via expression of surface markers or reporter genes. In certain embodiments, the PSCs used in the methods of the present invention are subject-specific.

[0191] Techniques are known for generating PSCs from various somatic cells by reprogramming using Yamanaka factors (OCT4, SOX2, KLF4, cMYC). For example, reprogramming of mature lymphocytes into iPSCs has been achieved for mouse B cells (Hanna et al., (2008) Cell, 133, pp. 250-264; Wada et al., (2011) Int. Immunol., 23, pp. 65-74), for mouse T cells and mature NK T cells (Watarai et al., (2010) J. Clin. Invest., 120, pp. 2610-2618), and for human T cells (Loh et al., (2010) Cell Stem Cell, 7, pp. 15-19; Seki et al., (2010) Cell Stem Cell, 7, pp. 11-14). iPSCs can be generated from human T cells using total peripheral blood mononuclear cells (PBMCs) or CD3+ cells as the cell source population (Loh et al., (2010) Cell Stem Cell, 7, pp. 15-19; Seki et al., (2010) Cell Stem Cell, 7, pp. 11-14; Staerk et al. (2010) Cell Stem Cell, 7, pp. 20-24; Brown et al, (2010) PloS One 5, e11373).

[0192] To reach a sufficient therapeutic dose of the host cell composition, the host cell is often subjected to one or more rounds of stimulation / activation. In certain embodiments, the method of preparing the host cell for administration to a subject comprises stimulating and activating the host cell in the presence of one or more stimulating signals or agents (e.g., compounds, small molecules, e.g., small organic molecules, nucleic acids, polypeptides, or fragments, isoforms, variants, analogs, or derivatives thereof). In certain embodiments, the method of preparing the host cell for administration to a subject comprises stimulating and activating the host cell in the presence of one or more stimulating signals or agents, and then proliferating.

[0193] Immune cells (e.g., T lymphocytes and NK cells) can be activated by inducing changes in their biological state, in which the cells express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be caused by primary stimulatory signals. Costimulatory signals amplify the magnitude of the primary signal and suppress cell death after the initial stimulation, resulting in a more sustained activation state and therefore a higher cytotoxic potential.

[0194] T cells can generally be activated using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated by reference herein in its entirety. In certain embodiments, T cells are activated by binding to an agent that activates CD3ζ.

[0195] In some embodiments, CD2 binding agents can be used to provide a primary stimulatory signal to T cells. For example, but not limited to, CD2 agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, such as T1 1.3 antibody combined with T1 1.1 or T1 1.2 antibody (Meuer, SC et al. (1984) Cell 36:897-906), and 9.6 antibody (which recognizes the same epitope as T11.1) combined with 9-1 antibody (Yang, SY et al. (1986) J. Immunol. 137:1097-1100). Other antibodies that bind to the same epitope as any of the above antibodies can also be used.

[0196] In some embodiments, immune cells are activated by administering phorbol myristate acetate (PMA) and ionomycin. In some embodiments, host cells are activated by administering an appropriate antigen to induce activation and then expansion. In some embodiments, PMA, ionomycin, and / or an appropriate antigen are administered together with CD3 to induce activation and / or expansion.

[0197] Generally, the activator used in the present disclosure includes, but is not limited to, antibodies, fragments thereof, and proteinaceous binding molecules with antibody-like functions. Examples of (recombinant) antibody fragments are bivalent antibody fragments such as Fab fragments, Fv fragments, single chain Fv fragments (scFv), (Fab)2'-fragments, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490). A bivalent antibody fragment may be a (Fab)2'-fragment, or a bivalent single-chain Fv fragment, while a monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

[0198] In certain embodiments, one or more binding sites of the CD3ζ agent may be bivalent proteinaceous artificial binding molecules, such as dimeric lipocalin muteins (i.e., duocalins). In certain embodiments, the receptor binding reagent may have a single second binding site (i.e., monovalent). Examples of monovalent agents include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single chain Fv fragments (scFv), including bivalent single chain Fv fragments.

[0199] Agents that specifically bind to CD3 include, but are not limited to, anti-CD3 antibodies, bivalent antibody fragments of anti-CD3 antibodies, monovalent antibody fragments of anti-CD3 antibodies, and proteinaceous CD3 binding molecules with antibody-like binding properties. Proteinaceous CD3 binding molecules with antibody-like binding properties can be aptamers, muteins based on lipocalin family polypeptides, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers. They can also be attached to beads.

[0200] In certain embodiments, the activating agent (e.g., CD3 binding agent) can be present at a concentration of about 0.1 to about 10 μg / ml. In certain embodiments, the activating agent (e.g., CD3 binding agent) can be present at a concentration of about 0.2 μg / ml to about 9 μg / ml, about 0.3 μg / ml to about 8 μg / ml, about 0.4 μg / ml to about 7 μg / ml, about 0.5 μg / ml to about 6 μg / ml, about 0.6 μg / ml to about 5 μg / ml, about 0.7 μg / ml to about 4 μg / ml, about 0.8 μg / ml to about 3 μg / ml, or about 0.9 μg / ml to about 2 μg / ml. In certain embodiments, the activating agent (e.g., CD3 binding agent) is administered at a concentration of about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μM, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μM, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or 10 μg / ml. In certain embodiments, the CD3 binding agent can be present at a concentration of 1 μg / ml.

[0201] NK cells can generally be activated using methods such as those described in, for example, U.S. Patent Nos. 7,803,376, 6,949,520, 6,693,086, 8,834,900, 9,404,083, 9,464,274, 7,435,596, 8,026,097, 8,877,182; U.S. Patent Application Publication Nos. 2004 / 0058445, 2007 / 0160578, 2013 / 0011376, 2015 / 0118207, 2015 / 0037887; and PCT Patent Application Publication No. WO2016 / 122147. each of which is incorporated herein by reference in its entirety.

[0202] In some embodiments, NK cells are activated by, for example, but not limited to, inhibition of an inhibitory receptor on NK cells (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E or LILRB5 receptor).

[0203] In certain embodiments, the NK cells are activated by, for example, but not limited to, feeder cells (e.g., native K562 cells, or K562 cells genetically modified to express 4-1BBL and a cytokine (e.g., IL15 or IL21)).

[0204] In other embodiments, interferons, or macrophage-derived cytokines, can be used to activate NK cells, for example, interferons including, but not limited to, interferon alpha and interferon gamma, and cytokines including, but not limited to, IL-15, IL-2, IL-21.

[0205] In certain embodiments, the NK activator can be present at a concentration of about 0.1 to about 10 μg / ml. In certain embodiments, the NK activator can be present at a concentration of about 0.2 μg / ml to about 9 μg / ml, about 0.3 μg / ml to about 8 μg / ml, about 0.4 μg / ml to about 7 μg / ml, about 0.5 μg / ml to about 6 μg / ml, about 0.6 μg / ml to about 5 μg / ml, about 0.7 μg / ml to about 4 μg / ml, about 0.8 μg / ml to about 3 μg / ml, or about 0.9 μg / ml to about 2 μg / ml. In certain embodiments, the NK activator is administered at a concentration of about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μM, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μM, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or about 10 μg / ml. In certain embodiments, the NK activator can be present at a concentration of 1 μg / ml.

[0206] In certain embodiments, the activator is attached to a solid support, such as, but not limited to, beads, absorbent polymers present in a culture plate or well, or other matrices, such as, but not limited to, sepharose or glass; it may be expressed (such as in a native or recombinant form) on the cell surface of a native or recombinant cell line by means known to those of skill in the art.

[0207] In certain embodiments, the host cells can be genetically modified after stimulation / activation. In certain embodiments, the host cells are modified within 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours of stimulation / activation. In certain embodiments, the cells are modified within 16-24 hours after stimulation / activation. In certain embodiments, the host cells are modified within 24 hours. In certain embodiments, the host cells can be genetically modified prior to stimulation / activation.

[0208] In order to genetically modify host cells to express the arCAR of the present disclosure, arCAR polynucleotide or recombinant vector must be introduced into host cells.Polynucleotide can be introduced via viral or non-viral delivery method.The method suitable for polynucleotide delivery for use with the present method includes any method known to those skilled in the art that can introduce polynucleotide into organelle, cell, tissue or organism.

[0209] In various embodiments, genetic modification is performed ex vivo. Various methods are available for transfecting cells and tissues taken from a subject via ex vivo modification. For example, retroviral gene transfer in vitro can be used to genetically modify cells taken from a subject, and the cells are transferred back to the subject. For example, see Wilson et al., Science, 244:1344-1346, 1989 and Nabel et al., Science, 244(4910):1342-1344, 1989; both of which are incorporated herein by reference in their entirety. In certain embodiments, host cells can be taken from a subject and transfected ex vivo with a polynucleotide or recombinant vector of the present disclosure. In certain embodiments, host cells obtained from a subject can be transfected or transduced with a polynucleotide or recombinant vector of the present disclosure, and then administered back to the subject.

[0210] In some embodiments, host cells can be transduced via retroviral transduction.References describing retroviral transduction of genes include U.S. Patent No. 5,399,346 to Anderson et al.; U.S. Patent No. 4,650,764 to Temin et al.; U.S. Patent No. 4,980,289 to Temin et al.; Markowitz et al., J. Virol. 62:1120 (1988); U.S. Patent No. 5,124,263 to Temin et al.; International Patent Application Publication No. WO95 / 07358; and Kuo et al., Blood 82:845 (1993), each of which is incorporated herein by reference in its entirety.

[0211] Another suitable method of gene transfer includes injection. In some embodiments, the polynucleotide or recombinant vector can be delivered to cells, tissues, or organisms via one or more injections (e.g., needle injection). Non-limiting methods of injection include injection of a composition (e.g., a saline-based composition). The polynucleotide or recombinant vector can also be introduced by direct microinjection. Non-limiting sites of injection include subcutaneous, intradermal, intramuscular, intranodal (allows direct delivery of antigen to lymphatic tissue), intravenous, intraprostatic, intratumoral, intralymphatic (allows direct administration of DCs), and intraperitoneal. It is understood that proper preparation of the injection site (e.g., shaving the injection site to observe proper needle placement) is necessary.

[0212] Electroporation is another suitable method for gene transfer.See, for example, Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol., 6, 716-718; both of which are incorporated herein by reference in their entirety for all purposes.Electroporation involves exposing a suspension of cells and DNA to a high voltage electric discharge.In some embodiments, cell wall degrading enzymes, such as pectin degrading enzymes, can be used to make host cells more susceptible to genetic modification by electroporation than untreated cells.See, for example, U.S. Patent No. 5,384,253, which is incorporated herein by reference in its entirety for all purposes.

[0213] Methods of electroporation for use with the present disclosure include, for example, those described in Sardesai, NY, and Weiner, DB, Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7:120-127 (2011), both of which are incorporated by reference in their entirety for all purposes.

[0214] Nucleic acid vaccines can be used to introduce arCAR polynucleotide or vector into host cells.Such vaccines include, but are not limited to, non-viral vectors, "naked" DNA and RNA, and viral vectors.The methods of genetically modifying cells with these vaccines and optimizing the expression of genes contained in these vaccines are known to those skilled in the art.

[0215] Additional methods of gene transfer include liposome-mediated transfection (e.g., polynucleotides entrapped in lipid complexes suspended in an excess of aqueous solution; see, e.g., Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. pp. 87-104). Also, polynucleotides complexed with Lipofectamine or Superfect; DEAE-dextran (e.g., DEAE-dextran followed by polyethylene glycol is used to deliver polynucleotides to cells. See, e.g., Gopal, TV, Mol Cell Biol. 1985 May; 5(5):1188-90); calcium phosphate (e.g., calcium phosphate precipitation is used to introduce polynucleotides into cells. See, e.g., Graham and van der Eb, (1973) Virology, 52, 456-467; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and Rippe et al., Mol. Cell Biol., 10:689-695, 1990); sonication loading (introduction of polynucleotides by direct sonic loading. See, e.g., Fechheimer et al., (1987) Proc. See Nat'l Acad. Sci. USA, 84, 8463-8467); microprojectile bombardment (e.g., one or more particles can be coated with at least one polynucleotide and delivered to a cell by a propelling force.See, e.g., U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and International Patent Application No. WO 94 / 09699; Klein et al., (1987) Nature, 327, 70-73, Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568-9572); and receptor-mediated transfection (e.g., selective uptake of macromolecules by receptor-mediated endocytosis occurs in target cells using cell type-specific distribution of various receptors. See, e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., (1997) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO 0273085; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993; Nicolau et al., (1987) Methods Enzymol., 149, 157-176); it is intended that each reference cited herein be incorporated by reference in its entirety for all purposes.

[0216] In further embodiments, the host cell is genetically modified using gene editing with homology directed repair (HDR). Homologous recombination repair (HDR) is a mechanism used by cells to repair double-stranded DNA breaks. In HDR, a donor polynucleotide with homology to the double-stranded DNA break site is used as a template to repair the broken DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to DNA. Thus, new nucleic acid material can be inserted or copied into the target DNA break site. Double-stranded DNA breaks in host cells can be induced by site-specific nucleases. The term "site-specific nuclease" as used herein refers to a nuclease that can specifically recognize and cleave a nucleic acid (DNA or RNA) sequence. Site-specific nucleases suitable for use in the present disclosure include, but are not limited to, RNA-guided endonucleases (e.g., CRISPR-associated (Cas) proteins), zinc finger nucleases, TALEN nucleases, or mega-TALEN nucleases. For example, a site-specific nuclease (e.g., Cas9+ guide RNA) capable of inducing a double-stranded break in a target DNA sequence can be introduced into a host cell along with a donor polynucleotide encoding a polypeptide of an arCAR of the disclosure.

[0217] After the host cells have been activated and transduced, the cells are grown in culture.

[0218] The T cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months, or more, for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.

[0219] Agents that can be used to expand T cells can include interleukins such as IL-2, IL-7, IL-15, or IL-21 (see, e.g., Cornish et al. 2006, Blood. 108(2):600-8; Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22):12670-12674; Battalia et al, 2013, Immunology, 139(1):109-120). Other illustrative examples of agents that can be used to expand T cells are agents that bind to CD8, CD45, or CD90, e.g., αCD8, αCD45, or αCD90 antibodies. Illustrative examples of T cell populations include antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells (an illustrative example of a memory T cell is a CD62L / CD8 / specific central memory T cell), or regulatory T cells (an illustrative example of a Treg is a CD4+CD25+CD45RA+ Treg cell).

[0220] Further that may be used to expand T cells include methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041; each of which is incorporated herein by reference in its entirety.

[0221] In certain embodiments, the agent used for expansion (e.g., IL-2) is administered at about 20 units / ml to about 200 units / ml. In certain embodiments, the agent used for expansion (e.g., IL-2) is administered at about 25 units / ml to about 190 units / ml, about 30 units / ml to about 180 units / ml, about 35 units / ml to about 170 units / ml, about 40 units / ml to about 160 units / ml, about 45 units / ml to about 150 units / ml, about 50 units / ml to about 140 units / ml, about 55 units / ml to about 130 units / ml, about 60 units / ml to about 120 units / ml, about 65 units / ml to about 110 units / ml, about 70 units / ml to about 100 units / ml, about 75 units / ml to about 95 units / ml, or about 80 units / ml to about 90 units / ml. In certain embodiments, the agent used for expansion (e.g., IL-2) is at about 20 units / ml, about 25 units / ml, about 30 units / ml, 35 units / ml, 40 units / ml, 45 units / ml, about 50 units / ml, about 55 units / ml, about 60 units / ml, about 65 units / ml, about 70 units / ml, about 75 units / ml, about 80 units / ml, about 85 units / ml, about 90 units / ml, about 95 units / ml, about 100 units / ml, about 105 units / ml, about 11 0 units / ml, about 115 units / ml, about 120 units / ml, about 125 units / ml, about 130 units / ml, about 135 units / ml, about 140 units / ml, about 145 units / ml, about 150 units / ml, about 155 units / ml, about 160 units / ml, about 165 units / ml, about 170 units / ml, about 175 units / ml, about 180 units / ml, about 185 units / ml, about 190 units / ml, about 195 units / ml, or about 200 units / ml. In certain embodiments, the agent used for expansion (e.g., IL-2) is administered at about 5 mg / ml to about 10 ng / ml. In some embodiments, the agent used for expansion (e.g., IL-2) is administered at about 5.5 ng / ml to about 9.5 ng / ml, about 6 ng / ml to about 9 ng / ml, about 6.5 ng / ml to about 8.5 ng / ml, or about 7 ng / ml to about 8 ng / ml. In some embodiments, the agent used for expansion (e.g., IL-2) is administered at about 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, or 10 ng / ml.

[0222] The NK cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months, or more, for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.

[0223] Agents that can be used for NK cell expansion can include agents that bind to CD16 or CD56, such as αCD16 or αCD56 antibodies. In certain embodiments, the binding agent comprises an antibody (see, e.g., Hoshino et al, Blood. 1991 Dec. 15; 78(12):3232-40). Another agent that can be used for NK cell expansion is IL-15 (see, e.g., Vitale et al. 2002. The Anatomical Record. 266:87-92; incorporated herein by reference in its entirety for all purposes).

[0224] Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium (MEM), RPMI Medium 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, and / or cytokines in sufficient amounts for growth and expansion).

[0225] Examples of other additives for host cell expansion include, but are not limited to, detergents, pH buffers such as Plasmanate, HEPES, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol, antibiotics (e.g., penicillin and streptomycin), which are included only in the experimental cultures and not in the cultures of cells injected into the subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0226] In certain embodiments in which PSCs (e.g., iPSCs or ESCs) are used as the starting cell population, the method may further comprise differentiating the PSCs into immune cells such as T cells. Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lineages include those described in the art, for example, those described in International Patent Application Publication Nos. WO2017 / 078807 and WO2019 / 112899, the disclosures of which are incorporated herein by reference. iPSC-derived hematopoietic lineage cells may include, but are not limited to, definitive hemogenic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem and progenitor cells, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0227] In some embodiments, the present disclosure also provides a method for purifying the arCAR polypeptide of the present disclosure. In some embodiments, the method comprises purifying the second polypeptide of arCAR from a host cell modified to express the second polypeptide of arCAR. In some embodiments, the second polypeptide of arCAR is a soluble protein.

[0228] Pharmaceutical Compositions In one aspect, the composition comprises one or more of the arCAR polypeptides, polynucleotides, vectors comprising same, and cell compositions as disclosed herein. In some embodiments, the compositions of the present disclosure include, but are not limited to, pharmaceutical compositions.

[0229] In one aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide of arCAR described herein and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises a second polypeptide of arCAR described herein and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises two or more second polypeptides of arCAR described herein and a pharmaceutically acceptable carrier and / or excipient.

[0230] In another aspect, the disclosure provides a pharmaceutical composition comprising a polynucleotide or recombinant vector described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises a polynucleotide encoding a second polypeptide of an arCAR described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises a polynucleotide encoding two or more second polypeptides of an arCAR described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises two or more polynucleotides each encoding a second polypeptide of an arCAR described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises a recombinant vector comprising a polynucleotide encoding a second polypeptide of an arCAR described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises a recombinant vector comprising a polynucleotide encoding a second polypeptide of an arCAR described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises a recombinant vector comprising a polynucleotide encoding two or more second polypeptides of an arCAR described herein and a pharma- ceutically acceptable carrier and / or excipient. In various embodiments, each of the two or more arCARs comprises a unique pair of a tag and a tag-binding domain.

[0231] In another aspect, the disclosure provides a pharmaceutical composition comprising the modified host cell described herein and a pharma- ceutically acceptable carrier and / or excipient.

[0232] Examples of pharmaceutical carriers include, but are not limited to, sterile liquids, such as water and oils (including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.) Water or saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions.

[0233] Compositions comprising the modified host cells disclosed herein may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0234] Compositions comprising the modified host cells disclosed herein may include one or more of the following: sterile diluents such as water for injection, saline (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents that may serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose.

[0235] In some embodiments, the composition is formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural or intramuscular administration. Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials. Injectable pharmaceutical compositions are preferably sterile. In some embodiments, the composition is reconstituted from a lyophilized formulation before administration.

[0236] In some embodiments, the modified host cells may be mixed with a substance to which they adhere or permeate, such as, but not limited to, nanoparticles, prior to their administration.

[0237] Treatment In some aspects, provided herein are methods of using the arCAR of the present disclosure for the treatment of disease.

[0238] In one aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising: (i) an immune effector cell comprising a chimeric antigen receptor comprising a first polypeptide of an arCAR described herein; and (ii) a second polypeptide of the arCAR, or a polynucleotide encoding the second polypeptide, or a host cell comprising the second polypeptide. The method includes administering to a subject a therapeutically effective amount of

[0239] In some embodiments, the method further comprises administering one or more additional polypeptides, each of which comprises (a) an antigen-binding domain that binds to a unique antigen and (b) a tag that is recognized by the tag-binding domain of the first polypeptide.

[0240] In another aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising: (i) an immune effector cell comprising a chimeric antigen receptor comprising a first polypeptide of two or more arCARs, wherein the two or more arCARs are selected from the arCAR systems described herein; and (ii) a second polypeptide of the two or more arCARs, or one or more polynucleotides encoding the second polypeptide, or one or more host cells comprising the second polypeptide. The method includes administering to a subject a therapeutically effective amount of

[0241] In various embodiments of the treatment methods described herein, the cell is an immune effector cell. In some embodiments, the cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, or a tumor infiltrating lymphocyte (TIL). In some embodiments, the cell is a T cell. In some embodiments, the cell is derived from an iPSC. In various embodiments, the cell constitutively expresses the first polypeptide.

[0242] In various embodiments of the methods of treatment described herein, the host cells and / or polypeptides are administered as a pharmaceutical composition that also includes a pharma- ceutically acceptable carrier and / or excipient as described herein.

[0243] In various embodiments of the treatment methods described herein, the disease is cancer. The terms "cancer", "malignant tumor", "neoplasm", "tumor" and "carcinoma" are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled and / or autonomous growth, and thus exhibit aberrant growth phenotypes characterized by a marked loss of control of cell growth. In general, the target cells for treatment in this application include pre-cancerous (e.g., benign) cells, malignant cells, pre-metastatic cells, metastatic cells and non-metastatic cells. The teachings of the present disclosure may be relevant to any cancer. To give some non-limiting examples, in some embodiments, the teachings of the present disclosure are applied to one or more cancers, such as hematopoietic cancers, including, for example, leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissues, squamous cell carcinoma of the mouth, pharynx, larynx and lung, liver cancer, genitourinary cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, endometrial cancer, and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancer, breast cancer, gastrointestinal cancer and nervous system cancer, benign lesions such as papilloma. In some embodiments, the cancer treated by the method of the present disclosure is a solid tumor. In some embodiments, the cancer treated by the method of the present disclosure is a hematological malignancy. etc.

[0244] In some embodiments, the cancer treated by the method of the present disclosure is glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies. In some embodiments, the hematological malignancies are leukemia (e.g., acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML)), myeloma, or lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma (NHL)).

[0245] In various embodiments of the methods of treatment described herein, the disease is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, myasthenia gravis, Hashimoto's disease, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease, or ulcerative colitis.

[0246] When multiple polypeptides with unique antigen-binding specificities are administered, the method can be used to target multiple antigens (or multiple epitopes in the same antigen) in the same disease (e.g., tumor or autoimmune disease), or multiple antigens in different diseases (e.g., tumor or autoimmune disease).

[0247] In some embodiments, the engineered immune effector cells and the second polypeptide, or the polynucleotide encoding said second polypeptide, or the host cell comprising said second polypeptide, are administered simultaneously.

[0248] In some embodiments, the engineered immune effector cells and the second polypeptide, or the polynucleotide encoding said second polypeptide, or the host cell comprising said second polypeptide, are administered sequentially. For example, the second polypeptide, or the polynucleotide encoding said second polypeptide, or the host cell comprising said second polypeptide may be administered before or after administration of the engineered host cell.

[0249] In some embodiments, the immune effector cells are autologous to the subject receiving the treatment. In some embodiments, the immune effector cells are allogeneic to the subject receiving the treatment. If the immune effector cells are isolated from a donor, the method may further include a method of preventing graft-versus-host disease (GVHD) and host cell rejection.

[0250] In some embodiments, additional steps can be performed prior to administration to a subject. For example, immune effector cells can be expanded in vitro after contacting (e.g., transducing or transfecting) with a polynucleotide or recombinant vector described herein (e.g., a polynucleotide or recombinant vector encoding a first polypeptide of an arCAR), but prior to administration to a subject. In vitro expansion can be performed for 1 or more days, e.g., 2 or more days, 3 or more days, 4 or more days, 6 or more days, or 8 or more days, prior to administration to a subject. Alternatively, or in addition, in vitro expansion can continue for 21 days or less, e.g., 18 days or less, 16 days or less, 14 days or less, 10 days or less, 7 days or less, or 5 days or less, prior to administration to a subject. For example, in vitro expansion can be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days prior to administration to a subject.

[0251] In some embodiments, during in vitro expansion, immune effector host cells can be stimulated with antigen (e.g., TCR antigen). Antigen-specific expansion can optionally be supplemented with expansion under conditions that non-specifically stimulate lymphocyte proliferation, such as, for example, anti-CD3 antibody, anti-Tac antibody, anti-CD28 antibody, or phytohemagglutinin (PHA). Expanded host cells can be administered directly to a subject or frozen for future use, i.e., for later administration to a subject.

[0252] In some embodiments, the immune effector host cells are treated ex vivo with interleukin-2 (IL-2) prior to infusion into the subject, and / or the subject is treated with IL-2 after infusion. Additionally, in some embodiments, the patient can undergo preparative lymphodepletion (temporary removal of the immune system) prior to administration of the modified host cells. The combination of IL-2 treatment and preparative lymphodepletion can enhance the persistence of the modified host cells.

[0253] In some embodiments, immune effector host cells are transduced or transfected with a nucleic acid encoding a cytokine, which may be engineered to provide constitutive, regulatable, or temporally controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that act to enhance survival of T lymphocytes during contraction and can promote the formation and survival of memory T cells.

[0254] In some embodiments, the compositions (e.g., host cells or polypeptides) of the present disclosure are administered in a therapeutically effective amount. The dosage of the compositions administered in the methods of the present disclosure varies widely depending on the subject's physical parameters, frequency of administration, mode of administration, clearance rate, etc. The initial dose may be higher, followed by a lower maintenance dose. To maintain an effective dosage level, the dose may be administered less frequently, such as weekly or biweekly, or may be divided into smaller doses and administered daily, twice weekly, etc. A variety of doses will be effective to achieve in vivo persistence of modified host cells. A variety of doses will be effective to improve in vivo effector functions of modified host cells.

[0255] In some embodiments, a composition comprising modified host cells prepared by the methods described herein comprises at least 10 2 ~10 10 cells / kg body weight, 10 5 ~10 9 cells / kg body weight, 10 5 ~10 8 cells / kg body weight, 10 5 ~10 7 cells / kg body weight, 10 7 ~10 9 cells / kg body weight, or 10 7 ~10 8 The composition may be administered at a dose of cells / kg body weight, including all integer values ​​within these ranges. The number of modified host cells will depend on the intended therapeutic use of the composition.

[0256] The modified host cells may be administered multiple times at the dosages listed above. The modified host cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing treatment.

[0257] It is also contemplated that when used to treat various diseases / disorders, the compositions and methods of the present disclosure may be utilized with other therapeutic methods / medicines suitable for the same or similar disease / disorder. Such other therapeutic methods / medicines may be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. The appropriate therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0258] In some embodiments, the composition of the present disclosure (e.g., host cell or polypeptide) is administered before, substantially simultaneously with, or after the administration of an additional therapeutic agent, such as a cancer therapeutic agent. The additional cancer therapeutic agent can be, for example, a chemotherapeutic agent, a biological agent, surgery, gene therapy, or radiation therapy. In some embodiments, the subject to which the composition is administered does not undergo a treatment sufficient to cause immune cell depletion, such as lymphodepleting chemotherapy or radiation therapy.

[0259] In some embodiments of any of the above treatment methods, the method further includes administering to the subject one or more additional compounds selected from the group consisting of immunosuppressants, biologics, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).

[0260] In some embodiments, the methods and compositions of the disclosure can be combined with other therapies that block inflammation (eg, via blockade of IL1, INFα / β, IL6, TNF, IL23, etc.).

[0261] The methods and compositions of the present disclosure can be combined with other immune modulating treatments, such as, for example, therapeutic vaccines (including but not limited to, GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to, agents that block CTLA4, PD1, LAG3, TIM3, etc.), or checkpoint activators (including but not limited to, agents that enhance 4-1BB, OX40, etc.). The methods of the present disclosure can also be combined with other treatments that have the ability to modulate NKT function or stability, including but not limited to, CD1d, CD1d fusion proteins, CD1d dimers or larger polymers of CD1d unloaded or loaded with antigen, CD1d-chimeric antigen receptor (CD1d-CAR), or any other of the five known CD1 isomers present in humans (CD1a, CD1b, CD1c, CDle). The methods of the present disclosure can also be combined with other treatments, such as midostaurin, enasidenib, or combinations thereof.

[0262] The therapeutic method of the present disclosure can also be combined with additional immunotherapy and therapy.For example, when used to treat tumor, the composition of the present disclosure can be used in combination with conventional therapy, such as surgery, radiation therapy, chemotherapy, or combinations thereof, depending on the type of tumor, the condition of the patient, other health problems, and various factors.In certain aspects, other therapeutic agents useful for combined tumor therapy with the inhibitor of the present disclosure include antiangiogenic agents. Many antiangiogenic agents have been identified and are known in the art, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TEMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38 Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, and those listed in Carmeliet and Jain (2000). In one embodiment, the engineered host cells of the present disclosure can be used in combination with a VEGF antagonist or a VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof (e.g., the anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0263] Non-limiting examples of chemotherapeutic compounds that can be used in the combination therapy of the present disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacytidine, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide ... Sulfamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, Hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide These include, but are not limited to, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0264] These chemotherapeutic compounds can be classified according to their mechanism of action, for example, into the following groups: antimetabolites / anticancer agents, e.g., pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogues, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, e.g., natural products such as the vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents such as the taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilone, and navelbine. disruptor), epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, prilosporin, camycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16);antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase, which systematically metabolizes L-asparagine and removes cells that do not have the ability to synthesize asparagine themselves);antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, thrombolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents (anti-migratory agents, such as cyclosporine, ... agent); antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin ); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disrupting agents. ;

[0265] In various embodiments of the methods described herein, the subject is a mammal. In some embodiments, the subject is a human. The subject may be a juvenile or adult of any age or gender. EXAMPLES

[0266] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate the disclosed embodiments. However, it should be understood that the present application is not limited to the exemplary embodiments shown below.

[0267] Example 1. Development of a novel arCAR with a VHH tag Identify novel VHHs with specificity for non-human proteins using phage display technology or by immunization. Select candidate VHHs for production and quality control analysis. Further test the candidate VHHs for specificity and cross-reactivity. Use the lead VHHs to generate anti-VHHs.

[0268] Anti-VHHs with specificity for the lead tag VHH are identified using phage display technology or generated by immunization in llamas or other camelids. Several commercially available phage campaigns can be used, including tungsten, VHH, and SuperHuman, as well as scFv. Candidate anti-VHHs that bind to the CDRs or other non-framework regions of the tag VHH are selected.

[0269] The soluble tagged VHH is expressed and produced in CHO cells. The soluble tagged VHH is purified by protein A chromatography. The anti-VHH is incorporated into the CAR construct as an extracellular tag-binding domain. The lentiviral construct is transduced into T cells to express the CAR that contains the anti-VHH domain. The anti-VHH is also tested for specificity and cross-reactivity.

[0270] In vitro assays will be established to evaluate T cell activation via soluble tagged VHHs and anti-tag CARs by assessing cytokine production and cytotoxicity, respectively. Lead arCAR constructs will be selected for further analysis.

[0271] Example 2. Development of arCAR with existing VHH tags Select an existing VHH that binds to a non-human protein. Non-limiting examples of VHHs that can be used in constructing the arCAR of the present disclosure include VHHs targeting RSV F protein, Listeria monocytogenes internalin, cobra venom phospholipase A2, Ebola virus nucleoprotein, HSV glycoprotein D, Racotococcus phage RBP, Geobacillus stearothermophilus, ricin (e.g., V5E4), and hen egg white lysozyme. VHHs can also be engineered to eliminate binding by scrambling the CDRs. Select candidate VHHs for production and quality control analysis. Use the selected VHHs to generate anti-VHHs.

[0272] Anti-VHHs with specificity for the lead tag VHH are identified using phage display technology or by immunization in llamas. Several commercially available phage campaigns can be used, including tungsten, VHH and SuperHuman, as well as scFv. Candidate anti-VHHs that bind to the CDRs or other non-framework regions of the tag VHH are selected.

[0273] The soluble tagged VHH is expressed and produced in CHO cells. The soluble tagged VHH is purified by protein A chromatography. The anti-VHH is incorporated into the lentiviral CAR construct as an extracellular tag-binding domain. The lentiviral construct is transduced into T cells to express the CAR containing the anti-VHH domain. The anti-VHH is also tested for specificity and cross-reactivity.

[0274] In vitro assays will be established to evaluate T cell activation via soluble tagged VHHs and anti-tag CARs by assessing cytokine production and cytotoxicity, respectively. Lead arCAR constructs will be selected for further analysis.

[0275] Example 3. arCAR with BCMA / anti-BCMA VHHs To test the arCAR concept, several approaches will be used, which are listed below.

[0276] To test protein-first concepts:protein interactions in the arCAR platform, an anti-BCMA antibody is fused to a tumor-targeting domain antibody. BCMA is fused to the transmembrane domain of the CAR. An exemplary arCAR incorporating BCMA / anti-BCMA antibodies is shown in FIG. 3A.

[0277] In another set of experiments, scFvs derived from Herceptin mAb sequences are developed. The sequences identified for anti-Herceptin idiotypic mAbs are converted to scFv format. These known sequences are incorporated into the arCAR platform to demonstrate the concept. The Herceptin scFv is fused to the CAR and the Herceptin anti-ID scFv is fused to a soluble tumor targeting domain antibody (e.g., anti-CD19 scFv or VHH). An exemplary arCAR incorporating Herceptin / Herceptin scFv is shown in Figure 3B. This arCAR is tested in vitro for potential cytotoxicity.

[0278] Several antibodies with specificity for non-human proteins have been identified in the literature. Such examples include H6dyx, hP3-3, hAnti-Z, hHSV GD, hLP RBP, hVHH4, hSbsB, and hV5E4. These are constructed in scFv or VHH format for characterization. Candidates are selected based on biophysical properties. Anti-ID campaigns are then performed against these binders to develop a diverse affinity panel that allows proof-of-concept for the benefits of modulating arCAR affinity.

[0279] Example 4. arCAR with Herceptin / anti-Herceptin scFv In a proof-of-concept study, an scFv derived from Herceptin (Trastuzumab) mAb was developed. Another scFv (scFv69) was developed from an anti-Herceptin idiotypic mAb. These sequences were assembled into different CAR expression constructs (Table 5), and then each was packaged into a lentiviral vector and expressed via viral transduction in either Jurkat cells or primary T cells. CAR expression was detected by flow cytometry and binding to EGFR soluble protein. CAR-expressing cells were used in combination with CHO cells overexpressing EGFR or cell lines with native cell surface expression of EGFR to demonstrate EGFR-specific cytotoxicity. Figure 4A shows an exemplary arCAR in which Herceptin scFv was fused to the CAR and Herceptin anti-ID scFv was fused to an EGFR-targeting VHH (9G8). FIG. 4B shows that T cells expressing Herceptin scFv CAR bound to 9G8-scFv69 fusion protein can target EGFR+ cells.

[0280] Table 5 shows the CAR expression constructs that were made, containing either Herceptin scFv or anti-Herceptin idiotype scFv69 as the extracellular tag binding domain. Figure 5 shows the vector map of the lentiviral vector used to deliver the CAR construct. Table 6 shows Herceptin scFv and scFv69 (Herceptin anti-ID).

[0281] Jurkat cells were transduced with lentiviral vectors carrying the CAR constructs listed in Table 5 to assess CAR expression and CD69 activation. To measure CD69 modulation as a readout for activation, CAR-expressing Jurkat cells were co-cultured with EGFR-positive cells for 24 and 48 hours. After co-culture, cells were stained for CD69 levels using anti-human CD69-BV421 antibody and measured with an Intellicyte flow cytometer. CAR constructs were also expressed in T cells to assess cytotoxicity. To perform this assay, T cells were isolated using the Pan T cell Isolation Kit (Miltenyi Cat. No. 130-096-535) according to the manufacturer's instructions. Cells were resuspended in RPMI (10% FBS) containing 30 units / ml IL2 and activated overnight with Dynabeads™ Human T-Activator CD3 / CD28 (ThermoFisher, Cat. No. 11132D). The next day, T cells were transduced with a lentiviral plasmid containing a CAR expression cassette using TransDux™ MAX Lentiviral Transduction Reagent (SBI, Cat. No. LV860A-1) (Figures 12A-12K). T cells were expanded for 7 days, and cytotoxicity assays were set up on day 7. Target cells labeled with Recon Cell Trace Violet were co-cultured with CAR-positive T cells at various ratios for 24 or 48 h. After 48 h, cell viability was checked with an Intellicyte flow cytometer.

[0282] [Table 5]

[0283] [Table 6-1] [Table 6-2] [Table 6-3]

[0284] Four fusion protein (or bridge protein) constructs (Table 7) were assembled containing 9G8 VHH (Table 6) fused to either Herceptin scFv or anti-Herceptin idiotype scFv69 via a (G4S)3 linker (SEQ ID NO:25). Figure 6A shows the vector map of the plasmid used to clone the bridge constructs described in Table 7.

[0285] Plasmids carrying the bridge constructs listed in Table 6 were transfected into EXPi-293 cells for protein expression using Expifectamine according to the manufacturer's instructions. Soluble bridge proteins were purified. The clarified supernatant was loaded onto a HiTrap MabSelect SuRe column at a flow rate of 1 ml / min. The column was washed with 20 column volumes of PBS to remove unbound proteins. The column was eluted with 10 cV of 0.1 M Na acetate (pH 3.5) and the sample was neutralized with 2.5 M Tris (pH 6.5). The protein samples were polished by SEC to remove high molecular weight species. Figure 6B shows a gel electrophoresis image showing the purity of the bridge proteins purified from each construct.

[0286] [Table 7]

[0287] A binding assay for the binding of bridge protein to CAR-transduced Jurkat cells was performed to detect the binding between Jurkat cells transduced with scFv69-CAR and two variants of bridge protein (9G8_Herceptin scFv and Herceptin scFv 9G8, as shown in Figure 7A). After incubating the cells and performing a staining assay as follows, the binding of bridge protein was detected with Intellicyte. CAR-transduced Jurkat cells were incubated with bridge protein for 30 minutes. After incubation, the cells were washed and then incubated with biotinylated EGFR for 30 minutes. After a washing step, PE-labeled streptavidin was incubated with the cells. Bridge protein-bound CAR-transduced Jurkat cells were determined by flow cytometry to be 65% positive for 9G8_Herceptin scFv and 45% positive for Herceptin scFv 9G8 (Figure 7B). Binding of the bridge proteins was also detected in the opposite configurations: scFv69_9G8 bridge and Herceptin CAR, and 9G8_scFv69 bridge and Herceptin CAR (data not shown).

[0288] Cytotoxic activity was evaluated between target cells and CAR-expressing T cells. Expression of EGFR was detected in EGFR-transduced CHO cells as assessed by flow cytometry (Figure 8A). CAR expression was also evaluated in T cells. The highest expression was observed in CARs with a long Ig-hinge format for both HER-CAR (p514) and scFv69-CAR (p515) (Figure 8B).

[0289] To set up the cytotoxicity assay, target cells (EGFR-transduced CHO and parental CHO) were plated at 10,000 cells / well and labeled with CellTrace™ Violet (CTV). CAR T cells were plated at 100,000 cells / well for an approximate CAR+ effector to target cell ratio (E:T) of about 5:1. CAR-T cells were co-cultured with CHO-EGFR+ cells for 48 hours. The controls used in the assay are shown in Table 8. Bridge proteins 9G8-Herceptin (p522) and 9G8-scFv69 (p519) at 10, 1, 0.1, and 0 nM were used.

[0290] [Table 8]

[0291] Universal CAR-T cells showed CD25 activation when combined (or paired) with the correct bridge protein, but not with the mismatch bridge protein (Figures 9A, 9B). Furthermore, universal CAR-T cells showed cytotoxic activity when combined with the correct bridge protein, but not with the mismatch bridge protein (Figures 10A, 10B).

[0292] Example 5. Identification of VHHs for the universal CAR platform VHH binders were selected as potential targets for anti-idiotype discovery to form specific and unique binding pairs that could function interchangeably as "tag" and "anti-tag". A list of VHHs known to bind non-human proteins was identified. Additional VHHs that bind Hen Egg Lysozyme (HEL) were also obtained. These VHHs and the generated plasmids are provided in Table 9, and the sequences are provided in Table 10 (underlined sequences indicate the CDR positions).

[0293] [Table 9]

[0294] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5]

[0295] Lentiviral CAR expression constructs were assembled containing the VHHs listed in Table 9. Figure 11A shows the vector map of the lentiviral vector used to deliver the VHH CAR. The lentiviral vector was then used to transduce Jurkat cells. Robust CAR expression in Jurkat cells was observed for 10 / 14 VHH CARs (Figure 11B). All candidate VHHs were expressed at very high levels in Jurkat cells as CARs. To determine whether there is tonic signaling or non-specific activation of these VHHs when present in the CAR, the transduced Jurkat cells were incubated with human embryonic kidney cells (HEK) overnight and evaluated the next day for the T cell activation marker CD69. Low or no non-specific activation in Jurkat cells was observed for all VHH CAR constructs (Figures 11C, 11D), indicating little or no signaling through the CAR without target-driven activation. The three anti-lysozyme VHH CARs bound chicken lysozyme and did not cross-react with human (Figure 11E), demonstrating specificity for the chicken protein.

[0296] Based on these data, LYSO_CW_P01_B11(B11) VHH and LYSO_CW_P01_D04(D04) VHH were selected for further consideration due to their high expression as CARs, low to no tonic signaling and non-specific activity, and ideal biophysical characteristics (demonstrated by their robust expression as soluble proteins). These VHHs had good expression as either CARs or bridge proteins.

[0297] [Table 11]

[0298] Figure 11F shows the alignment of amino acid sequences of VHHs targeting Geobacillus stearothermophilus, LYSO_CW_P01_B11 and LYSO_CW_P01_D04. A consensus sequence was generated based on the aligned sequences.

[0299] As a final characterization of the specificity of these VHHs, CAR Jurkat cells transduced with B11 and D04 VHHs, as well as other anti-HEL VHHs from the candidate panel, were tested for binding to biotinylated chicken and human lysozyme to confirm the absence of binding to human lysozyme protein. Both B11(p242) and D04(p244) VHHs show binding to chicken lysozyme without cross-reactivity to human lysozyme protein (Figure 11E).

[0300] Example 6. Humanization of B11 and D04 VHHs The B11 and D04 proteins are derived from a camelid VHH phage display library at Distributed Bio (San Francisco, CA). To limit the immunogenic potential of these VHHs, they were humanized in the framework regions of the VHHs. From this example onwards, reference to B11 and D04 is a reference to the humanized VHHs.

[0301] [Table 12]

[0302] Humanized B11 and D04 underwent similar characterization steps as the camelid versions to ensure that humanization of the framework regions did not impair their biophysical characteristics, their ability to be expressed as CARs, or their properties when present on a CAR.

[0303] [Table 13]

[0304] Each VHH CAR was transduced into Jurkat cells as described above. Additionally, each VHH CAR was transduced into Nurkat cells, the Nur77 reporter line of Jurkat cells (Nurkat). This cell line contains a GFP reporter that responds to Nur77, a transcription factor that is activated by CD3z stimulation and serves as a reporter of CAR activation. Thus, activation via CAR in Nurkat cells causes the cells to express GFP. Figure 13 shows CAR and GFP expression in Nurkat cells transduced with lentiviruses containing humanized B11 (p1649) CAR and humanized D04 (p1648) CAR. UTD cells are non-transduced Nurkat cells. CAR expression was measured using a polyclonal anti-camelid VHH cocktail (Genscript). These results indicated that the expression of B11 VHH and D04 VHH as CARs was not affected by the humanization of these sequences. In addition, the lack of GFP expression in these CAR Nurkats indicates that there is little tonic signaling mediated by the humanized version of CAR.

[0305] To confirm that the binding properties of each VHH are unchanged as a result of humanization, we tested whether these humanized VHH CARs can still bind to HEL. Figure 14 shows the results of an assay in which B11 (p1649) and D04 (p1648) CAR Nurkat were stained with biotinylated HEL and detected with streptavidin-APC via flow cytometry. UTD is non-transduced Nurkat as a negative control. Both B11 and D04 retained their ability to bind to HEL after humanization.

[0306] Example 7. Phage display panning of anti-idiotypic VHHs against B11 and D04 The humanized B11 and D04 VHHs were used in phage library panning to identify specific anti-ID VHHs for each. The synthetic VHH phage library consists of four sub-libraries distinguished primarily by the length of their CDR3 sequences, with library 1 (CNTY1) having the shortest CDR length and libraries 3 and 4 (CNTY3 and CNTY4) having the longest CDR lengths.

[0307] One panning scheme used is shown in Figure 15. In this method, anti-idiotypic VHHs against D04 were discovered by first depleting the library against B11-Fc protein. This was done to remove phages that bind to the framework regions of the VHHs and the Fc portion of the protein. This depleted phage output was then panned with biotinylated D04-Fc protein. Biotinylated D04 with phage binders could be isolated via streptavidin. The output phage were amplified in bacteria and used in further panning rounds with increasingly stringent panning conditions. Alternatively, VHHs displayed by the phage could be tested by ELISA for binding to D04 or B11 proteins, sequenced, and cloned into expression vectors for more stringent testing. Using this phage library panning approach, VHH binders against both D04 and B11 were identified.

[0308] This panning scheme was repeated four times with decreasing target antigen concentrations to select for high affinity binders (200 nM for rounds 1 and 2, 50 nM for round 3, and 10 nM for round 4). Output phages from the final round of panning were used to transform bacteria into single colonies that were screened as periplasmic extracts (PPE) in ELISA against both target and off-target VHHs (Figure 16).

[0309] PPE samples that showed specific responses to target proteins and little responses to off-target proteins were selected for further characterization. Six unique B11 binders and 18 unique D04 binders were identified. These unique sequences were cloned into Fc fusion expression vectors for further testing. The sequences and identifiers of these proteins are shown in Table 14.

[0310] [Table 14-1] [Table 14-2]

[0311] Example 8. Specificity and affinity testing of anti-idiotypic VHHs To determine anti-idiotypic specificity and binding, the Fc fusion proteins were tested by FACS against Nurkat cells expressing either the B11 VHH CAR or the D04 VHH CAR.

[0312] As shown in Figures 17A-17B, several anti-idiotypes showed robust and specific binding to VHHs on CARs at a range of concentrations with negligible binding to off-target CARs (e.g., AMD71 (for B11) and AMD69, AMD77, AMD82, AMD84, AMD87 and AMD88 (for D04)). Several binders were also noted (AMD72 and AMD74) that showed non-specific binding, as indicated by binding to both cell lines. Of these, AMD71, AMD77, AMD82, AMD84, AMD87 and AMD88 were selected for further specificity testing. Biotinylated anti-idiotype VHH-Fc fusion proteins were arrayed against various other VHH CAR-Nurkat and then any binding to those cell lines was detected via FACS.

[0313] Figure 18 shows the binding of lead anti-idiotype biotinylated VHH-Fc to target and off-target CAR Nurkat cells detected via FACS using streptavidin-APC. Cell lines tested include B11, D04, P711, P712, P713, P716, and parental cells. B11 and D04 cells are CAR Nurkat cells expressing B11 VHH CAR and D04 VHH CAR, respectively. P711, 712, 713, 716 cells are off-target CAR Nurkat cells. Parental cells are non-transduced non-CAR expressing Nurkat cells. Controls include biotinylated protein A, which binds non-specifically to most VHHs, PROT786, an irrelevant biotinylated VHH-Fc protein, and no biotinylated protein added. The lead VHH had robust binding to the target VHH while showing negligible binding to the off-target VHH CAR and the parental Nurkat.

[0314] In some cases, the specific binding events that form the basis of the universal CAR "tag" and "anti-tag" interactions are an important aspect of the platform's safety profile. Furthermore, the binding events of lysozyme VHH with anti-idiotype alone should not be sufficient to trigger CAR-induced activation. To determine whether the binding of B11 or D04 to their soluble anti-idiotypes would trigger CAR-induced activation without fusion of tumor antigen-targeting VHH to the soluble protein, overnight stimulation assays were performed in which lead soluble anti-idiotype Fc fusion proteins were incubated with their target CAR Nurkat and off-target CAR Nurkat as well as parental Nurkat. Figures 19A-19B show the results of overnight stimulation assays using Nurkat cells incubated with 10 nM anti-idiotype VHH Fc fusions via FACS. Figure 19A shows GFP expression of B11 and D04 CAR Nurkat cells and parental Nurkat cells after overnight co-culture with anti-idiotype Fc fusion protein. Figure 19B shows detection of biotinylated protein via streptavidin-APC after overnight incubation. Controls include PMA / ionomycin (a TCR-crosslinker that results in robust Nurkat cell activation), biotinylated protein A, and a sample with no added protein. None of the anti-idiotype leads caused observable activation in Nurkats as measured by GFP expression compared to PMA / ionomycin induction. The anti-idiotype was also still detectable after overnight incubation, indicating that if the anti-idiotype caused CAR activation, the concentration of antigen used in the assay was sufficient to cause activation over the time course of the experiment that was observable.

[0315] Affinity measurements were obtained by Octet for anti-ID binders to both B11-his tagged VHH and D04-his tagged VHH.

[0316] [Table 15]

[0317] [Table 16]

[0318] The anti-idiotypic proteins exhibited affinities ranging from 2 nM (AMD77 for D04) to 3.7 μM (AMD87 for D04). To validate the specificity of the two highest affinity binders AMD77 (2 nM) and AMD88 (72 nM) over Octet, we tested each protein for binding to itself, its target VHH, off-target VHH, serum-derived total human IgG, and Fc-y fragments.

[0319] [Table 17]

[0320] Each sample (either AMD77 or AMD88) was tested against the analytes D04-his, AMD77, AMD88, serum derived IgG, fc-y fragment, PROT939 (off-target VHH-fc soluble), and D04-fc. Response values ​​are shown in Table 17. Both AMD77 and AMD88 showed minimal responses to human IgG, Fc, and off-target VHH-Fc PROT939 while maintaining strong responses to D04-his and D04-Fc. AMD88 showed responses to itself and to AMD77. AMD77 did not associate with itself.

[0321] Example 9. Design of bridge proteins The bridge proteins include VHH arCAR binders and tumor-targeting VHHs, specifically anti-CD70 and anti-EGFR in this example. These were constructed with and without Fc domains that function as half-life extenders in vivo. To achieve universal CAR specificity, bridge proteins were designed that incorporate a selected anti-idiotype against D04 paired with the D04 CAR, or D04 on a soluble bridge protein paired with an anti-ID against D04 as the CAR.

[0322] [Table 18-1] [Table 18-2]

[0323] [Table 19]

[0324] [Table 20]

[0325] Example 10. T cell cytotoxicity with D04 CAR and huD04 CNTY1 D10 bridge To determine whether the universal platform can mediate cytotoxicity by T cells, we created bridge proteins consisting of a VHH (huD04_CNTY1_D10) derived from the AMD88 protein linked in tandem by a Whitlow linker to the EGFR-targeting VHH 9G8 fused to an IgG1 Fc fragment (Table 19). Each bridge protein was tested in a T cell cytotoxicity assay using primary T cells expressing the D04 CAR.

[0326] [Table 21]

[0327] Killing assays were performed by co-culturing CAR T cells with CellTrace Violet-labeled EGFR-positive cells for 48 hours at an effector-to-target ratio of 1:2, adjusted for % CAR positivity of T cells, with either 5 nM bridge AMD109 (Figure 20A) or AMD112 (Figure 20B).

[0328] As shown in Figures 20A-20B, when the bridge and CAR are "matched" (e.g., an anti-idiotypic VHH is present on the bridge protein and its target VHH is present on the CAR), robust cytotoxicity of EGFR-positive cells is observed, as in the case of P1650 CAR-expressing T cells with D04 on the CAR (huD04_CNTY1_D10, anti-idiotype to D04 on the soluble bridge protein). This cytotoxic activity is comparable to the 9G8 CAR. When the CAR and bridge are mismatched, as in the case with CAR AMD94 or NONS F12 CAR, no cytotoxicity is observed. * * *

[0329] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0330] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

Claims

1. A universal chimeric antigen receptor (arCAR) system having compatible receptor specificity components, the arCAR system comprising: (i) an immune effector cell having a chimeric antigen receptor, the chimeric antigen receptor comprising: (a) an extracellular tag-binding domain; (b) a transmembrane domain; and (c) at least one intracellular signaling domain; an immune effector cell comprising a first polypeptide comprising (ii) a second polypeptide, (a) an antigen-binding domain that binds to at least one antigen on a target cell; (b) a tag recognized by the tag binding domain; a second polypeptide comprising: Including, (i) the tag comprises an antibody, an antigen-binding fragment thereof, or a surrogate scaffold, and the tag binding domain comprises an anti-idiotypic molecule that binds to the tag; or (ii) the tag comprises an anti-idiotypic molecule that binds to a tag binding domain, the tag binding domain comprising an antibody, an antigen-binding fragment thereof, or an alternative scaffold; The arCAR system, wherein the anti-idiotypic molecule binds to at least one antigen-binding region of the antibody, antigen-binding fragment thereof, or surrogate scaffold.

2. A universal chimeric antigen receptor (arCAR) system comprising two or more compatible receptor specificity components, the arCAR system comprising: (i) an immune effector cell comprising two or more chimeric antigen receptors, each of the two or more chimeric antigen receptors comprising: (a) an extracellular tag-binding domain; (b) a transmembrane domain; and (c) at least one intracellular signaling domain; an immune effector cell comprising a first polypeptide comprising (ii) two or more second polypeptides, each of which comprises: (a) an antigen-binding domain that binds to at least one antigen on a target cell; (b) a tag recognized by the tag binding domain; two or more second polypeptides comprising: Including, each of the two or more chimeric antigen receptors comprises a unique extracellular tag binding domain, and each of the two or more second polypeptides comprises a unique tag recognized by the unique extracellular tag binding domain; (i) the tag comprises an antibody, an antigen-binding fragment thereof, or a surrogate scaffold, and the tag binding domain comprises an anti-idiotypic molecule that binds to the tag; or (ii) the tag comprises an anti-idiotypic molecule that binds to a tag binding domain, the tag binding domain comprising an antibody, an antigen-binding fragment thereof, or an alternative scaffold; The arCAR system, wherein the anti-idiotypic molecule binds to at least one antigen-binding region of an antibody, an antigen-binding fragment thereof, or an alternative scaffold.

3. 3. The arCAR system of claim 1 or 2, wherein the antibody, antigen-binding fragment thereof, or surrogate scaffold binds to a polypeptide derived from a non-human source.

4. 4. The arCAR system of claim 3, wherein the polypeptide derived from a non-human source is respiratory syncytial virus (RSV) F protein, Listeria monocytogenes internalin, cobra venom phospholipase A2, Ebola virus nucleoprotein, herpes simplex virus (HSV) glycoprotein D, Lactococcus phage receptor binding protein (RBP), Geobacillus stearothermophilus, ricin, or hen egg white lysozyme.

5. The arCAR system of any one of claims 1 to 4, wherein the antigen-binding domain of the second polypeptide comprises an antibody, an antigen-binding fragment thereof, or an surrogate scaffold.

6. The arCAR system of any one of claims 1 to 5, wherein the anti-idiotype molecule is an anti-idiotype antibody, an antigen-binding fragment thereof, or an anti-idiotype surrogate scaffold.

7. The arCAR system of any one of claims 1 to 6, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody. (i) at least one of the extracellular tag-binding domain, the antigen-binding domain, and the tag comprises a VHH; or (ii) at least one of the extracellular tag binding domain, the antigen binding domain, and the tag comprises an scFv; The arCAR system described in any one of claims 1 to 7.

9. The arCAR system of any one of claims 1 to 8, wherein the alternative scaffold is Affilin or Centyrin. (i) the tag comprises a polypeptide sequence of SEQ ID NO: 104 or 112, and the extracellular tag-binding domain comprises a polypeptide sequence selected from SEQ ID NOs: 113-129; or (i') the tag comprises a polypeptide sequence selected from SEQ ID NOs: 113-129 and the extracellular tag binding domain comprises the polypeptide sequence of SEQ ID NO: 104 or 112; or (ii) the tag comprises a polypeptide sequence of SEQ ID NO: 102 or 111, and the extracellular tag binding domain comprises a polypeptide sequence selected from SEQ ID NOs: 130-133; or (ii') the extracellular tag binding domain comprises a polypeptide sequence of SEQ ID NO: 102 or 111, and the tag comprises a polypeptide sequence selected from SEQ ID NOs: 130-133. (i) the tag comprises a polypeptide sequence of SEQ ID NO:112 and the extracellular tag-binding domain comprises a polypeptide sequence of SEQ ID NO:129; or (ii) the tag comprises a polypeptide sequence of SEQ ID NO: 129 and the extracellular tag binding domain comprises a polypeptide sequence of SEQ ID NO:

112.

12. The arCAR system of any one of claims 1 to 11, wherein the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen.

13. 13. The arCAR system of claim 12, wherein the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy.

14. The tumor antigen associated with glioblastoma is HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, or IL13Rα2; The tumor antigen associated with ovarian cancer is FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, or B7H4; the tumor antigen associated with cervical cancer or head and neck cancer is GD2, MUC1, mesothelin, HER2, or EGFR; The arCAR system of claim 13, wherein the tumor antigen associated with liver cancer is claudin 18.2, GPC-3, EpCAM, cMET, or AFP.

15. The arCAR system of any one of claims 1 to 14, wherein the anti-idiotype molecule, antibody, and / or antibody fragment is humanized.

16. The arCAR system described in any one of claims 1 to 15, wherein the first polypeptide further comprises a spacer region between the extracellular tag binding domain and the transmembrane domain, the spacer region comprising a hinge domain.

17. The arCAR system of claim 16, wherein the transmembrane domain and / or hinge domain is derived from CD8 or CD28.

18. The arCAR system of any one of claims 1 to 17, wherein the at least one intracellular signaling domain comprises a CD3ζ signaling domain.

19. The arCAR system of any one of claims 1 to 18, wherein the at least one intracellular signaling domain comprises one or more costimulatory signaling domains.

20. 20. The arCAR system of claim 19, wherein the one or more costimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, OX40, CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, or 2B4 (CD244).

21. (i) the second polypeptide comprises an antigen-binding domain at its N-terminus and a tag at its C-terminus; or (ii) the second polypeptide comprises an antigen-binding domain at its C-terminus and a tag at its N-terminus.

22. The arCAR system of any one of claims 1 to 21, wherein the second polypeptide is a soluble polypeptide.

23. The arCAR system of any one of claims 1 to 22, wherein the immune effector cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell or a tumor infiltrating lymphocyte (TIL), a dendritic cell, or a macrophage.

24. 24. The arCAR system of claim 23, wherein the immune effector cells are derived from induced pluripotent stem cells (iPSCs).

25. 25. The arCAR system of claim 24, wherein the immune effector cells are T cells or NK cells derived from induced pluripotent stem cells (iPSCs).

26. A pharmaceutical composition comprising the immune effector cell of any one of claims 1 to 25 and a pharma- ceutically acceptable carrier and / or excipient; and A pharmaceutical composition comprising a second polypeptide according to any one of claims 1 to 25 and a pharma- ceutically acceptable carrier and / or excipient. Including kit.

27. 1. A kit for use in a method of treating a disease in a subject in need thereof, comprising: (i) an immune effector cell comprising a chimeric antigen receptor comprising the first polypeptide of the arCAR of any one of claims 1 to 25; and (ii) a second polypeptide of the arCAR, a polynucleotide encoding the second polypeptide, or a host cell comprising the second polypeptide. Including the kit.

28. 28. The kit of claim 27, wherein the second polypeptide is administered before, after, or in conjunction with immune effector cells.

29. 29. The kit of claim 27 or 28, wherein the immune effector cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell or a tumor infiltrating lymphocyte (TIL), a dendritic cell, or a macrophage.

30. The kit of any one of claims 27 to 29, wherein the immune effector cells are derived from iPSCs.

31. The kit of any one of claims 27 to 30, wherein the immune effector cells constitutively express the first polypeptide.

32. The kit according to any one of claims 27 to 31, wherein the disease is cancer or an autoimmune disease.

33. 33. The kit of claim 32, wherein the cancer is glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or a hematological malignancy.

34. the immune effector cells and the second polypeptide are administered simultaneously; or The kit of any one of claims 27 to 33, wherein the immune effector cells and the second polypeptide are administered sequentially.

35. The kit of any one of claims 27 to 34, wherein the immune effector cells are autologous or allogeneic cells.

36. The kit of any one of claims 27 to 35, wherein the subject is a human.

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