Engineered cytokine receptors for tunable adoptive cell therapy

Abstract

Receptor cytokine switches, immune cells containing them, and their use for controllable adoptive cell therapy are disclosed. TIFF2024537170000016.tif79128

Description

[Technical field]

[0001] Related Applications CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 252,850, filed October 6, 2021, which is incorporated by reference herein in its entirety.

[0002] Government License This disclosure was made with Government support under Grant No. 1DP1 DK105602-01 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on Aug. 27, 2022, is named 52095-631001WO_ST.xml and is 69 kilobytes in size. [Background technology]

[0004] Background of the disclosure Immunotherapy using adoptive cell transfer (ACT) aims to stimulate or suppress immunity. One of the most promising approaches of ACT is the administration of antigen-specific T cells or chimeric antigen receptor-T (CAR T) cells. CAR T cells targeting the B cell antigen CD19 have had remarkable clinical responses in patients with B cell malignancies (Davila and Brentjens, Clin. Adv. Hematol. Oncol. 14(10):802-808(2016) (Non-Patent Document 1); Halim and Maher, Ther. Adv. Vaccines Immunother. 8:1-17(2020) (Non-Patent Document 2)). Despite success in hematological cancers, the efficacy of CAR-T cells against solid tumors has been limited in part due to the limited persistence, survival, proliferation, and efficacy of CAR-T cells after infusion (Kosti, et al., Front. Immunol. 9:1104-9 (2018) (Non-Patent Document 3); Jafarzadeh, et al., Front. Immunol. 11, 702-17 (2020 (Non-Patent Document 4)).

[0005] The generation of memory T cells is essential for the long-term persistence of CAR T cell therapy. Their formation requires three signals: antigen, co-stimulation, and pro-inflammatory cytokines. Cytokines play an important role in the development, proliferation, survival, and differentiation of various immune cells (Foster, Int. J. Exp. Pathol. 82(3):171,192(2001) (Non-Patent Document 5)). Cytokine-targeted immunotherapy can modulate immune responses by promoting or inhibiting specific immune cell functions. Interleukin-2 (IL-2) stimulates the development and survival of T cells. High-dose IL-2 therapy has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic melanoma and renal cancer (Jiang, et al., Oncoimmunology 5(6):e1163462-10(2016) (Non-Patent Document 6)). However, systemic administration of cytokines has been largely unsuccessful due to uncontrolled cytokine release syndrome and intolerable toxicity. Therefore, controllable activation of cytokine signaling in specific cells is necessary to prevent systemic toxicity and improve engineered immune cell-driven efficacy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Davila and Brentjens, Clin.Adv.Hematol.Oncol.14(10):802-808(2016) [Non-Patent Document 2] Halim and Maher,Ther.Adv.Vaccines Immunother.8:1-17(2020) [Non-Patent Document 3] Kosti,et al.,Front.Immunol.9:1104-9(2018) [Non-Patent Document 4] Jafarzadeh,et al.,Front.Immunol.11,702-17(2020 [Non-Patent Document 5] Foster, Int. J. Exp. Pathol. 82(3):171,192(2001) [Non-Patent Document 6] Jiang, et al., Oncoimmunology 5(6):e1163462-10(2016) Summary of the Invention

[0007] Disclosure Summary A first aspect of the present disclosure relates to a cytokine receptor switch comprising a signal peptide, a single chain antibody fragment (scFv) that specifically binds to a synthetic, substantially non-immunogenic small molecule (hereinafter "synthetic small molecule"), a hinge domain, a transmembrane domain, and an intracellular domain of a cytokine receptor.

[0008] In some embodiments, the signal peptide is native or derived from a cytokine receptor, where the signal peptide and the intracellular domain may be native or derived from different cytokine receptors. In some embodiments, the signal peptide is native or derived from interleukin-2 receptor alpha chain (IL-2RA), IL-2RB, IL-2RG, IL-4RA, IL-7RA, IL-9R, IL-15RA, or IL-21R. In other embodiments, the signal peptide is native or derived from cluster of differentiation 8 (CD8).

[0009] In some embodiments, the synthetic small molecule that specifically binds to an scFv is fluorescein, e.g., fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylate (AQ), tetraxetane (DOTA), or a polyhistidine tag (His tag).

[0010] In some embodiments, the hinge domain is native to or derived from CD8.

[0011] In some embodiments, the transmembrane domain and the intracellular domain are each independently native to or derived from IL-2RA, IL-2RB, IL-2RG, IL-4RA, IL-7RA, IL-9R, IL-15RA, or IL-21R.

[0012] Another aspect of the present disclosure pertains to nucleic acids encoding the cytokine receptor switches described herein.

[0013] A further aspect relates to a composition comprising an immune cell comprising an exogenous nucleic acid encoding a cytokine receptor switch. In some embodiments, the immune cell is a CD8 + and CD4 + In another embodiment, the immune cell is a NK cell.

[0014] In some embodiments, the immune cells are Different cytokine receptor switches, comprising different signal peptides from different cytokine receptors, different transmembrane domains, and / or different intracellular domains of the cytokine receptor. The nucleic acid sequence includes at least two nucleic acids encoding the

[0015] In some embodiments, the immune cell contains an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) for a cell surface antigen. In some embodiments, the cell surface antigen is CD19, B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), mucin 1 (MUC1), or TNF receptor superfamily member 13B (TNFRSF13B).

[0016] In some embodiments, the immune cells comprise a nucleic acid encoding a binary activated chimeric antigen receptor (BAT-CAR).

[0017] In some aspects, the disclosure is directed to a method for stimulating an immune cell that comprises an exogenous nucleic acid encoding a cytokine receptor switch, comprising contacting the immune cell with a sufficient concentration of a synthetic small molecule, where the contacting promotes proliferation of the immune cell, which may also promote a change in the phenotype (e.g., memory, cytotoxic, and regulatory phenotype) of the immune cell.

[0018] In some embodiments, the contacting is performed ex vivo. Immune cells containing exogenous nucleic acid encoding a cytokine receptor switch are placed in a suitable medium and contacted with an effective concentration of the synthetic small molecule, for example, 0.1-1000 μg / mL, based on the total volume of the medium. In some embodiments, the concentration of the synthetic small molecule ranges from 0.1-100 μg / mL, based on the total volume of the medium. The duration of the contact (also referred to herein as "treatment" or "treating") can be as long as hours, days, and weeks (e.g., 1, 2, 3, 4 weeks or more). In some embodiments, the contacting can be performed in a high affinity plate, dish, or flask in which the synthetic small molecule is conjugated to a carrier, which may be polymeric in nature, for example, bovine serum albumin (BSA) immobilized on its surface.

[0019] In some other embodiments, the immune cells are contacted with the synthetic small molecule in vivo. In some embodiments, the immune cells are contacted with the synthetic small molecule systemically. In other embodiments, the immune cells are CAR-containing immune cells that are directed to a specific cell surface antigen (e.g., tumor surface antigen) due to the binding specificity of the CAR, thus allowing local stimulation of the immune cells. A further aspect of the disclosure relates to a method of treating cancer. In some embodiments, a therapeutically effective concentration of a composition containing ex vivo stimulated immune cells is administered to a subject. In other embodiments, the method includes administering a therapeutically effective concentration of immune cells to a subject in need thereof, administering an effective concentration of a synthetic small molecule to the subject for a suitable period of time, thereby stimulating the immune cells, and reducing the stimulation of the immune cells by administering to the subject a composition containing a synthetic monomeric or polymeric small molecule. To optimize stimulation, the synthetic small molecule is advantageously administered as a multimer, for example, a multimer in which a monomer of the small molecule is conjugated to a carrier such as BSA.

[0020] In some embodiments, the cancer is a blood cancer, such as leukemia, lymphoma, or multiple myeloma. In some embodiments, the cancer is characterized by the presence of a solid tumor, such as breast cancer, ovarian cancer, lung cancer, or brain cancer, e.g., glioblastoma multiforme.

[0021] Methods of stimulating immune cells that contain nucleic acids encoding cytokine receptor switches are known in the art, for example, as described in U.S. Pat. No. 5,747,292 and WO 2018 / 111834 and WO 2019 / 193197, each of which is incorporated by reference in its entirety. Methods for generating and stimulating immune cells containing nucleic acids encoding cytokine receptor switches are also described in the following publications: Nelson et al., Nature, 369(6478):333-336(1994); Gerhartz et al., J. Biol. Chem., 271:12991-12998(1996); Sockolosky et al., Science, 2018, 359(6379):1037-1042(1996); Chang et al., Nat. Chem. Biol. 14(3):317-324(2018); Leung et al., JCI Insight 4(11):e124430-18(2019); and Yang et al., PNAS 118:e2106612118-12(2021).

[0022] The cytokine receptor switch of the present invention is different from known chimeric cytokine receptors. For example, the chimeric receptor taught in U.S. Pat. No. 5,747,292 is activated by an endogenous protein that cross-reacts with the natural receptor. The chimeric receptor taught in WO2019 / 193197 is activated by a bacterial protein (e.g., GFP and mCherry) that is not substantially non-immunogenic (i.e., the bacterial proteins GFP and mCherry are immunogenic).

[0023] The present disclosure provides compositions and methods for directing controllable cytokine receptor signaling to a desired set of immune cells, independent of their natural ligands. In contrast to the prior art, the present disclosure requires the use of synthetic small molecules that act as artificial ligands and substitutes for natural cytokines and are also substantially non-immunogenic. Thus, the cytokine receptor switch is under the control of an exogenous moiety, i.e., a synthetic small molecule, allowing precise control of cytokine receptor activation or stimulation and subsequent signaling, both ex vivo and in vivo. Essentially, the synthetic small molecule acts as an on / off switch.

[0024] The present disclosure may provide several additional advantages, particularly in relation to CAR-T cells. The combination of different cytokine receptor switches with CARs may precondition T cells with memory, effector, or regulatory phenotypes, thereby significantly improving the persistence and cytotoxicity of CAR T cells. In addition to controlling their activation or stimulation, CAR-T cells containing cytokine receptor switches may exhibit greater resistance to the immunosuppressive environment characteristic of solid tumor microenvironments. As shown in the examples below, T cell subsets expressing memory markers were increased by using the compositions of the present disclosure. The use of synthetic small molecules provides additional advantages, particularly with respect to ex vivo expansion of CAR-T cells containing cytokine receptor switches, i.e., in terms of cost compared to the use of antibodies and recombinant cytokines. [Brief description of the drawings]

[0025] [Figure 1A] 1A-1D are images showing the structure and primary sequence of the cytokine receptor switch of the present invention. Figure 1A is an image showing the components of the cytokine receptor switch. A synthetic small molecule acts as an artificial ligand in place of the natural cytokine for the cytokine receptor. [Figure 1B]Figures 1A-1D are images showing the structure and primary sequence of the cytokine receptor switch of the present invention. Figure 1B is an image showing systemic and local activation of the cytokine receptor switch by small molecules conjugated to non-immunogenic scaffolds or antibodies. The cytokine receptor switch can be combined with a chimeric antigen receptor (CAR). Either synthetic small molecules (scFv) directed against the same small molecule (homotargeted) or different scFvs directed against different small molecules (heterotargeted) were fused to the cytokine receptor switch or CAR. [Figure 1C] Figures 1A-1D are images showing the structure and primary sequence of a cytokine receptor switch of the present invention, and Figure 1C is an image showing a representative primary sequence of a cytokine receptor switch of the present invention. [Figure 1D] Figures 1A to 1D are images showing the structure and primary sequence of the cytokine receptor switch of the present invention. Figure 1D is an image showing the primary sequence of the cytokine receptor switch of the present invention. [Diagram 2] FIG. 1 is an image showing a cytokine receptor switch expressed on primary human T cells stimulated with fluorescein isothiocyanate (FITC) bound to bovine serum albumin (BSA) coated on a culture plate. [Figure 3A] Figures 3A-3D are a series of graphs showing that a cytokine receptor switch of the invention increased effector and central memory markers on T cells with CD3 / CD28 costimulation. Figure 3A is a series of graphs showing that an IL2RA and IL15RA- cytokine receptor switch of the invention increased effector memory markers on CD4+ T cells with CD3 / CD28 costimulation, and an IL2RA-, IL2RB-, IL2RG-, IL7RA-, and IL15RA- cytokine receptor switch increased effector memory markers on CD8+ T cells with CD3 / CD28 costimulation. [Figure 3B]Figures 3A-3D are a series of graphs showing that a cytokine receptor switch of the invention increased effector and central memory markers on T cells with CD3 / CD28 costimulation. Figure 3B is a series of graphs showing that an IL2RA and IL7RA-cytokine receptor switch of the invention increased central memory markers on CD8+ T cells with CD3 / CD28 costimulation. [Figure 3C] Figures 3A-3D are a series of graphs showing that the cytokine receptor switch of the present invention increased effector and central memory markers on T cells with CD3 / CD28 costimulation. Figure 3C is a series of graphs showing that the cytokine receptor switch of the present invention had little effect on effector memory markers CD4+ and CD8+ T cells without CD3 / CD28 costimulation. [Figure 3D] Figures 3A-3D are a series of graphs showing that a cytokine receptor switch of the invention increased effector and central memory markers on T cells with CD3 / CD28 costimulation, and Figure 3D is a series of graphs showing that an IL7RA-cytokine receptor switch of the invention increased central memory markers on CD4+ T cells without CD3 / CD28 costimulation. [Figure 4] FIG. 1 is an image showing the use of the cytokine receptor switch of the present invention by a CAR or by a binary activated T cell comprising a nucleic acid encoding a chimeric antigen receptor (BAT-CAR). [Figure 5A] Figures 5A-5C are a series of graphs showing that BSA-FITC bound on high affinity plates efficiently stimulated fluorescein-specific BAT-CAR-T cells. Figure 5A is a graph showing the expression of interleukin-2 in BAT-CAR-CD8+ T cells stimulated with BSA-FITC bound on regular tissue culture plates (plate bound (normal)), BSA-FITC bound on high affinity plates (plate bound high affinity), and BSA-FITC solution added directly to the cells (free). [Figure 5B]Figures 5A-5C are a series of graphs showing that BSA-FITC bound on high affinity plates efficiently stimulated fluorescein-specific BAT-CAR-T cells. Figure 5B is a graph showing expression of interferon gamma (IFNg)+ in BAT-CAR-CD8+ T cells stimulated by BSA-FITC bound on regular tissue culture plates, BSA-FITC bound on high affinity plates, and BSA-FITC solution added directly to the cells. [Figure 5C] Figures 5A-5C are a series of graphs showing that BSA-FITC bound on high affinity plates efficiently stimulated fluorescein-specific BAT-CAR-T cells. Figure 5C is a graph showing the expression of CD69 on BAT-CAR-CD8+ T cells stimulated by BSA-FITC bound on regular tissue culture plates, BSA-FITC bound on high affinity plates, and BSA-FITC solution added directly to the cells. [Figure 6A] Figures 6A-6B are a series of graphs showing that the cytokine receptors of the present invention switch activated NK cells. Figure 6A is a line graph showing that the IL15RA-, IL2RB- and IL2RG-cytokine receptor switch combination of the present invention promoted cell proliferation of human natural killer (NK) cells, NK92. [Figure 6B] Figures 6A-6B are a series of graphs showing that the cytokine receptor switch of the invention activates NK cells. Figure 6B is a bar graph showing that the combination of the IL7RA and IL2RG cytokine receptor switch of the invention increased the expression of the activation marker CD69 on NK92 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Disclosure Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. As used in this specification and the appended claims, unless otherwise specified, the following terms have the meanings provided to facilitate understanding of this disclosure.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an inhibitor" includes mixtures of two or more such inhibitors, and so forth.

[0028] Unless otherwise specified, the term "about" means within 10% (eg, within 5%, 2%, or 1%) of the particular value that is modified by the term "about."

[0029] The transitional phrase "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to certain materials or steps of the claimed disclosure "and that do not materially affect the basic and novel characteristics."

[0030] As used herein, "immune cells" refers to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. Representative examples of immune cells include T cells and natural killer (NK) cells. The term "immune cells" as used herein also refers to cells derived from stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.

[0031] In some embodiments, the immune cells are CD8 + In some embodiments, the immune cells are CD4 T cells. + In some embodiments, the immune cells are CD8 + T cells and CD4 + In some embodiments, the immune cells are NK cells.

[0032] The term "memory phenotype" as used herein refers to immune cells that contain an exogenous nucleic acid encoding a cytokine receptor switch of the present invention being ready to respond to an antigen more quickly than a naive immune cell (that does not contain exogenous nucleic acid).

[0033] The term "cytotoxic phenotype" as used herein refers to immune cells that are toxic (i.e., immune cells that induce the death of other cells, such as tumor cells, infected cells, or cells that are otherwise damaged or dysfunctional). For example, cytotoxic T cells mediate the lysis of target cells (e.g., cancer cells) that bear their cognate antigen. Cytotoxic T cells are generally antigen-specific and major histocompatibility complex (MHC)-restricted, in that they recognize antigenic peptides only when associated with MHC molecules on the surface of the target cell. NK cells target and kill abnormal cells, including stressed, virally or microbially infected cells, or malignant cells.

[0034] The term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, fragment antigen binding (Fab) fragments, F(ab') fragments, and the like. 2The term "scFv" includes fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments containing a variable heavy chain (VH) region capable of specifically binding to an antigen, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. An scFv contains the variable regions of the VH and light chain (VL) of an antibody and typically contains up to about 50 (e.g., about 10) amino acid residues. The linker can either connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. An scFv can be prepared according to methods known in the art (see, e.g., Bird et al., Science 242:423-426 (1988) and Hustone et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). In some embodiments, the linker sequence comprises the amino acids glycine and serine, and in some cases a set of glycine and serine repeats, such as (Gly4Ser)n, where n is an integer equal to or greater than 1. The length and amino acid composition of the linker can be varied, for example, to achieve optimal folding and interaction between the VH and VL to create a functional epitope. See, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448 (1993). The term "antibody" includes 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, nanobodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, and the like. Unless otherwise specified, the term "antibody" should be understood to include functional antibody fragments thereof. The term also encompasses intact or full length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0035] As used herein, a "synthetic, substantially non-immunogenic small molecule" refers to an organic molecule or compound that can bind to an scFv on a cytokine receptor switch of the present invention without eliciting a significant immune response in a subject, that is monofunctional and ranges in size from about 50 daltons to about 10,000 daltons, usually from about 50 daltons to about 5000 daltons, more usually from about 100 daltons to about 1000 daltons.

[0036] Examples of substantially non-immunogenic small molecules as used herein include fluorescein and fluorescein derivatives (e.g., fluorescein isothiocyanate (FITC)), 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylate (AQ), tetraxane (DOTA), or polyhistidine-tag (His-tag). A "significant immune" response is any immune response that limits or restricts the in vivo usefulness of a synthetic small molecule used in accordance with the teachings of the present disclosure. A detectable immune response is not necessarily a "significant immune response." That is, "substantially non-immunogenic" encompasses a detectable but not significant immune response. In some embodiments, the substantially non-immunogenic small molecule is conjugated to a carrier.

[0037] To the extent that the following terms are used herein with respect to and to further describe the synthetic small molecules described herein, the following definitions apply.

[0038] The term "specific binding", when it comes to interactions between synthetic small molecules and single chain antibody fragments (scFv), refers to intermolecular interactions that are substantially specific in that binding of the synthetic small molecule to other endogenous entities (proteinaceous and non-proteinaceous, etc.) may be detectable but not functionally significant.

[0039] As used herein in the context of cytokine receptor switch, CAR and BAT-CAR components, the term "derived from" (also referred to as "derived from, e.g., native to") encompasses components that have at least a portion of a sequence identical to the sequence of that component in a native gene, such as a signal peptide naturally associated with an IL-2RA receptor (e.g., a signal peptide "native" to the IL-2RA receptor), and components that have a non-native sequence that differs from the native sequence in terms of at least one modification, such as an amino acid substitution, addition (e.g., at either or both termini) or deletion (e.g., "derived from"), provided that the modification does not impair the function that the component performs as part of the cytokine receptor switch. In some embodiments, cytokine receptor switch, CAR and BAT-CAR elements that are derived from proteins include the sequence of a portion of a native gene with one or more modifications from the native gene, or can be identical to the native gene for a range of amino acids or nucleic acids, but with modifications, substitutions, or deletions outside of the range of identical sequences.

[0040] As used herein, the term "sufficient concentration" refers to the concentration of a synthetic small molecule necessary to activate a cytokine switch in the context in which the synthetic small molecule is used (e.g., in vitro or in vivo).

[0041] Cytokine Receptor Switch An important aspect of the present disclosure is directed to a cytokine receptor switch that includes a signal peptide native to or derived from a cytokine receptor, e.g., a single chain antibody fragment (scFv) that specifically binds a synthetic small molecule, a hinge domain, a transmembrane domain, and an intracellular domain of a cytokine receptor (Figure 1A, Figure 1C, and Figure 1D). The DNA and amino acid sequences of representative components of the cytokine receptor switch described herein are listed in Table 1.

[0042] (Table 1) TIFF2024537170000002.tif178170TIFF2024537170000003.tif175170TIFF2024537170000004.tif239170 TIFF2024537170000005.tif182170TIFF2024537170000006.tif179170TIFF2024537170000007.tif162170

[0043] The signal peptide targets the nascent protein to the endoplasmic reticulum. In some embodiments, the signal peptide included in the cytokine receptor switch is or is derived from a native form of interleukin-2 receptor alpha chain (IL-2RA), IL-2RB, IL-2RG, IL-4RA, IL-7RA, IL-9R, IL-15RA, or IL-21R. In some embodiments, the signal peptide is a native form of CD8, i.e., a CD8 signal peptide. In some embodiments, the signal peptide and the intracellular domain are native forms of the same cytokine receptor. In some embodiments, the signal peptide and the intracellular domain are native forms of different cytokine receptors.

[0044] In some embodiments, the signal peptide is that of a native form of IL-2RA and has the nucleic acid sequence of SEQ ID NO:1 and the amino acid sequence of SEQ ID NO:2.

[0045] In some embodiments, the signal peptide is that of the native form of IL-2RB and has the nucleic acid sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:4.

[0046] In some embodiments, the signal peptide is that of the native form of IL-2RG and has the nucleic acid sequence of SEQ ID NO:5 and the amino acid sequence of SEQ ID NO:6.

[0047] In some embodiments, the signal peptide is that of a native form of IL-4RA and has the nucleic acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:8.

[0048] In some embodiments, the signal peptide is that of a native form of IL-7RA and has the nucleic acid sequence of SEQ ID NO:9 and the amino acid sequence of SEQ ID NO:10.

[0049] In some embodiments, the signal peptide is that of the native form of IL-9R and has the nucleic acid sequence of SEQ ID NO:11 and the amino acid sequence of SEQ ID NO:12.

[0050] In some embodiments, the signal peptide is that of a native form of IL-15RA and has the nucleic acid sequence of SEQ ID NO:13 and the amino acid sequence of SEQ ID NO:14.

[0051] In some embodiments, the signal peptide is that of the native form of IL-21R and has the nucleic acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:16.

[0052] The scFv is conjugated to a synthetic small molecule. As used herein, the term "synthetic small molecule" refers to an organic molecule or compound that is monofunctional and ranges in size from about 50 to about 10,000 daltons, usually from about 50 to about 5000 daltons, more usually from about 100 to about 1000 daltons.

[0053] Representative examples of synthetic small molecules include fluorescein and fluorescein derivatives (e.g., FITC, 5-carboxyfluorescein, 6-carboxyfluorescein, 5 / 6-carboxyfluorescein, NHS-fluorescein (5(6)-carboxyfluorescein N-hydroxysuccinimide ester), 5-(iodoacetamido)fluorescein, 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride, 5-(bromomethyl)fluorescein, and fluorescein 5-carbamoylmethylthiopropanoate), 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylate (AQ), and tetraxetane (DOTA). In some embodiments, the synthetic small molecule is a polymer, and the polymer is a monopolymer, a heteropolymer, or a branched polymer. A representative example of a polymeric synthetic small molecule is a polyhistidine tag (His tag) having about 6 to about 9 histidine (His) residues. An exemplary 6His tag has a molecular weight of about 800 daltons.

[0054] In some embodiments, the scFv binds to synthetic small molecules that are fluorescein and fluorescein derivatives, 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylate (AQ), tetraxetane (DOTA), polyhistidine tag (His tag).

[0055] The synthetic small molecule is substantially non-immunogenic. In some embodiments, the synthetic small molecule is non-immunogenic such that when injected alone into an animal, it does not cause the animal to produce antibodies or T cells reactive thereto. In some embodiments, the synthetic small molecule generates IgM antibodies in the animal but does not cause antibody class switching. In some embodiments, the synthetic small molecule generates low levels of antibodies in the animal such that the synthetic small molecule can still bind to one or more cytokine receptor switches without being neutralized. In some embodiments, the synthetic small molecule does not generate a significant immune response. A "significant immune" response is any immune response that limits or restricts the in vivo usefulness of a synthetic small molecule used in accordance with the teachings of the present disclosure.

[0056] A representative example of a fluorescein-binding scFv has the nucleic acid sequence of SEQ ID NO:51 and the amino acid sequence of SEQ ID NO:52.

[0057] A representative example of an scFv that binds to MPOB has the nucleic acid sequence of SEQ ID NO:53 and the amino acid sequence of SEQ ID NO:54.

[0058] A representative example of an scFv that binds to AQ has the nucleic acid sequence of SEQ ID NO:55 and the amino acid sequence of SEQ ID NO:56.

[0059] A representative example of a scFv that binds to DOTA has the nucleic acid sequence of SEQ ID NO:57 and the amino acid sequence of SEQ ID NO:58.

[0060] The transmembrane (TM) domain allows the cytokine receptor switch to be stably anchored in the cell membrane of the immune cell. The transmembrane domain can be derived from the same protein from which the other domains of the cytokine receptor switch are derived or from a different protein. The transmembrane domain can be of natural or recombinant origin. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0061] In some embodiments, the transmembrane domain is derived from IL-2RA, IL-2RB, IL-2RG, IL-4RA, IL-7RA, IL-9RA, IL-15RA, or IL-21R.

[0062] In some embodiments, the transmembrane domain peptide is derived from IL-2RA and has the nucleic acid sequence of SEQ ID NO:17 and the amino acid sequence of SEQ ID NO:18.

[0063] In some embodiments, the transmembrane domain peptide is derived from IL-2RB and has the nucleic acid sequence of SEQ ID NO:19 and the amino acid sequence of SEQ ID NO:20.

[0064] In some embodiments, the transmembrane domain peptide is derived from IL-2RG and has the nucleic acid sequence of SEQ ID NO:21 and the amino acid sequence of SEQ ID NO:22.

[0065] In some embodiments, the transmembrane domain peptide is derived from IL-4RA and has the nucleic acid sequence of SEQ ID NO:23 and the amino acid sequence of SEQ ID NO:24.

[0066] In some embodiments, the transmembrane domain peptide is derived from IL-7RA and has the nucleic acid sequence of SEQ ID NO:25 and the amino acid sequence of SEQ ID NO:26.

[0067] In some embodiments, the transmembrane domain peptide is derived from IL-9R and has the nucleic acid sequence of SEQ ID NO:27 and the amino acid sequence of SEQ ID NO:28.

[0068] In some embodiments, the transmembrane domain peptide is derived from IL-15RA and has the nucleic acid sequence of SEQ ID NO:29 and the amino acid sequence of SEQ ID NO:30.

[0069] In some embodiments, the transmembrane domain peptide is derived from IL-21R and has the nucleic acid sequence of SEQ ID NO:31 and the amino acid sequence of SEQ ID NO:32.

[0070] The cytokine receptor switch can be designed to include a transmembrane domain indirectly linked to the scFv. In such embodiments, the transmembrane domain is attached to the scFv via a hinge domain. As used herein, the term "hinge domain" refers to a domain that links the extracellular binding domain to the transmembrane domain and may confer flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular domain close to the plasma membrane of an immune cell to minimize the possibility of recognition by an antibody or a binding fragment thereof. The hinge domain can be natural (e.g., a hinge from a human protein) or synthetic. Sources of hinge domains include human Ig (immunoglobulin) hinges (e.g., IgG4 hinge, IgD hinge), and CD8 (e.g., CD8α hinge).

[0071] In some embodiments, the hinge domain is derived from cluster of differentiation 8 (CD8).

[0072] In some embodiments, the hinge domain peptide has the nucleic acid sequence of SEQ ID NO:49 and the amino acid sequence of SEQ ID NO:50.

[0073] As used herein, the term "intracellular domain" refers to a signaling moiety that provides a signal to an immune cell, such as a T cell, that mediates a cellular response, such as, for example, activation, proliferation, differentiation, and / or cytokine secretion. In some embodiments, the intracellular domain is of or is derived from a native form of IL-2RA, IL-2RB, IL-2RG, IL-4RA, IL-7RA, IL-9RA, IL-15RA, or IL-21R.

[0074] In some embodiments, the intracellular domain is derived from IL-2RA and has the nucleic acid sequence of SEQ ID NO:33 and the amino acid sequence of SEQ ID NO:34.

[0075] In some embodiments, the intracellular domain is derived from IL-2RB and has the nucleic acid sequence of SEQ ID NO:35 and the amino acid sequence of SEQ ID NO:36.

[0076] In some embodiments, the transmembrane domain peptide is derived from IL-2RG and has the nucleic acid sequence of SEQ ID NO:37 and the amino acid sequence of SEQ ID NO:38.

[0077] In some embodiments, the transmembrane domain peptide is derived from IL-4RA and has the nucleic acid sequence of SEQ ID NO:39 and the amino acid sequence of SEQ ID NO:40.

[0078] In some embodiments, the transmembrane domain peptide is derived from IL-7RA and has the nucleic acid sequence of SEQ ID NO:41 and the amino acid sequence of SEQ ID NO:42.

[0079] In some embodiments, the transmembrane domain peptide is derived from IL-9R and has the nucleic acid sequence of SEQ ID NO:43 and the amino acid sequence of SEQ ID NO:44.

[0080] In some embodiments, the transmembrane domain peptide is derived from IL-15RA and has the nucleic acid sequence of SEQ ID NO:45 and the amino acid sequence of SEQ ID NO:46.

[0081] In some embodiments, the transmembrane domain peptide is derived from IL-21R and has the nucleic acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:48.

[0082] Representative cytokine receptor switches include combinations of the sequences of the signal peptide, scFv, transmembrane domain, hinge domain and intracellular domain disclosed above.

[0083] In some embodiments, the cytokine receptor switch comprises an IL-2RB signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RB transmembrane domain, and an IL-2RB intracellular domain. In some embodiments, the cytokine receptor switch comprises an IL-2RG signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain. In some embodiments, the cytokine receptor switch comprises an IL-7RA signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-7RA transmembrane domain, and an IL-7RA intracellular domain. In some embodiments, the cytokine receptor switch comprises an IL-15RA signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-15RA transmembrane domain, and an IL-15RA intracellular domain.

[0084] In some embodiments, the cytokine receptor switch of the disclosure is an anti-fluorescein-IL2-RA cytokine receptor switch and has the following amino acid sequence (SEQ ID NO:59): TIFF2024537170000008.tif24140.

[0085] Immune cells comprising a nucleic acid encoding the cytokine receptor switch of the present invention In some aspects, the present disclosure relates to compositions comprising immune cells comprising at least one nucleic acid encoding a cytokine receptor switch. Immune cells useful in the present disclosure are immune cells from mammals, preferably primates, such as monkeys and humans. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are allogeneic to the recipient subject (from the same species but from a different donor), in some embodiments, the immune cells are autologous (donor and recipient are the same), and in some embodiments, the immune cells are syngeneic (donor and recipient are different but identical twins). Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, in some embodiments, NK cells useful in the present disclosure are allogeneic, autologous, or syngeneic. For example, in some embodiments, the T cells are CD8 + or CD4 + In some embodiments, the NK cells are CD56 dim CD16 + In some embodiments, the NK cells are CD56 bright CD16 - In some embodiments, the NK cells are primary NK cells, memory-like NK cells, or induced memory-like NK cells. The composition may include a combination of two or more types of immune cells that contain the same or different cytokine receptor switches encoded by nucleic acids.

[0086] In some embodiments, the composition comprises an immune cell comprising a nucleic acid encoding an anti-fluorescein-IL2-RA cytokine receptor switch having the amino acid sequence of SEQ ID NO:59.

[0087] In some embodiments, the immune cell comprises at least two nucleic acids encoding at least two cytokine receptor switches, each of which differs in at least one of its signal peptide, transmembrane domain, and intracellular domain.

[0088] For example, in some embodiments, at least two cytokine receptor switches comprise different scFvs.

[0089] In some embodiments, the immune cell comprises at least two nucleic acids encoding at least two cytokine receptor switches, the nucleic acids comprising: a first cytokine switch comprising an IL-2RG signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain; a second cytokine switch comprising the IL-7RA signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-7RA transmembrane domain, and an IL-7RA intracellular domain; Code the following:

[0090] In some embodiments, the immune cell comprises at least three nucleic acids encoding at least three cytokine receptor switches, each of which differs in at least one of its signal peptide, transmembrane domain, and intracellular domain.

[0091] In some embodiments, the immune cells are a first cytokine switch comprising an IL-2RB signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RB transmembrane domain, and an IL-2RB intracellular domain; a second cytokine switch comprising an IL-2RG signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain; and A third cytokine switch comprising the IL-15RA signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-15RA transmembrane domain, and an IL-15RA intracellular domain. The present invention comprises at least three nucleic acids encoding at least three cytokine receptor switches comprising:

[0092] In some embodiments, the immune cells are derived from induced pluripotent stem cells (iPSCs), umbilical cord blood, or peripheral blood mononuclear cells (PBMCs). Progenitor harvesting, generation, and maintenance of iPSCs are known in the art. See, for example, U.S. Patent Nos. 9,260,696, 10,214,722, 10,428,309, 10,844,356, and 11,193,108. Methods for harvesting, generating, and maintaining stem cells from umbilical cord blood, as well as iPSCs, are also known in the art. See, U.S. Patent Nos. 6,338,942, 7,311,905, 8,889,411, and 9,260,696. Similarly, methods for harvesting, generating, and maintaining immune cells from PBMCs are known in the art, see U.S. Patent Nos. 9,476,028, 11,162,072, 11,229,689, and U.S. Patent Publication No. 2017 / 0051252. Methods for differentiating and isolating T cells and NK cells into desired cell subsets from progenitor, pluripotent, or stem cells are known in the art.

[0093] Chimeric Antigen Receptor (CAR)-Immune Cells In some embodiments, the immune cells are chimeric antigen receptor (CAR)-immune cells (e.g., CAR-T cells) and also contain a CAR against a cell surface antigen (Figure 1B, Figure 4).

[0094] As used herein, the term "chimeric antigen receptor" (CAR) refers to a synthetically engineered receptor that comprises an extracellular binding domain comprising an antibody or binding fragment thereof, nanobody or other protein sequence that binds to a cellular antigen ("cell-associated antigen") associated with a disease or disorder and is linked to an intracellular domain of a T cell via a spacer domain. The spacer domain comprises a transmembrane domain and, in some embodiments, a hinge domain.

[0095] Extracellular binding domain As used herein, an extracellular binding domain is a moiety that specifically binds to a target antigen, i.e. a cell surface antigen, such as a tumor-associated antigen. The extracellular binding domain may comprise a protein, a polypeptide, an oligopeptide, or a peptide. The extracellular binding domain may be naturally occurring, synthetic, semi-synthetic, or recombinantly produced.

[0096] In some embodiments, the extracellular binding domain binds to a cell-associated antigen on a tumor cell (a "tumor associated antigen" (TAA)).

[0097] In some embodiments, the extracellular binding domain of the CAR is a single chain variable fragment (scFv) of an antibody (as defined above).

[0098] Other types of antibody fragments with specificity for cell-associated antigens that may be useful as components of CARs include Fv, Fab, and (Fab) 2 See, e.g., U.S. Patent No. 4,946,788.

[0099] Representative examples of such extracellular binding domains are shown in Table 2.

[0100] (Table 2) TIFF2024537170000009.tif219170TIFF2024537170000010.tif214170TIFF2024537170000011.tif220170TIFF2024537170000012.tif194170

[0101] In some embodiments involving the treatment of brain cancer, for example, the targeting ligand binds to a brain tumor associated antigen. For example, tumor associated antigens present on GBM cells include ACVR1, EGFRvIII, IL13Rα2, and HER2. For example, a multiplexed approach that simultaneously targets EGFRvIII, IL13Rα2, and HER2 can be used to treat brain cancer. Other proteins that are involved in brain cancer and can be targeted by the bifunctional compounds of the present disclosure include EphA2, CSPG4, GD2, PDGFRα, and GRP78. Antibodies and / or functional fragments thereof that bind to brain tumor associated antigens are known in the art. For example, see Table 1 above, which describes antibodies and / or fragments thereof that bind to ACVR1, PDGFRα, GD2, and EphA2, among others. The targeting moiety that binds to PDGFRα can include a scFv based on olaratumab (and its binding variants).

[0102] In some embodiments, the targeting ligand is a HER2 targeting ligand for cancers such as breast cancer, lung cancer, colorectal cancer, brain cancer, ovarian cancer, and pancreatic cancer. + Binds to HER2 on malignant tumors. Exemplary targeting ligands that bind to HER and may be useful in the present disclosure include trastuzumab and pertuzumab, which bind to the extracellular domains IV and II of HER, respectively, and HER-binding fragments thereof (e.g., scFv).

[0103] Antibody fragments that bind EGFRvIII are described in O'Rourke, et al., Sci. Transl. Med. 9(399):eaaa0984-30 (2017). Other antibodies or fragments thereof that bind EGFRvIII are commercially available siltuximab and mAb DH8.3 (Novus Biologicals). Further representative examples of amino acid or gene sequences encoding scFvs targeting EGFRvIII that may be useful in the present disclosure are described in U.S. Patent Application Publication No. 2015 / 0259423.

[0104] Antibody fragments that bind to IL13Rα2 are described in Brown, et al., N. Engl. J. Med. 375(26):2561-2569 (2016). Other antibodies or fragments thereof that bind to IL13Rα2 are commercially available from Abnova and Millipore.

[0105] Antibody fragments that bind to HER2 are described in Ahmed, et al., JAMA Oncol. 3(8):1049-1101 (2017). Other antibodies or fragments thereof that bind to HER2 are commercially available, including trastuzumab and FRP5. Further representative examples of amino acid or gene sequences encoding scFvs that target HER2 that may be useful in the present disclosure are described in U.S. Patent Application Publication No. 2011 / 0313137.

[0106] Another example of an antibody fragment that binds EphA2 is described in Chow, et al., Mol. Ther. 21(3):629-637 (2013). Still other antibodies or fragments thereof that bind EphA2 are commercially available from Thermo Fisher (mAb4H5 and mAb 1C11A12) and RND Systems. Further representative examples of amino acid or gene sequences encoding scFvs that target EphA2 that may be useful in the present disclosure are described in U.S. Patent Application Publication No. 2010 / 436783.

[0107] Antibody fragments that bind CSPG4 are described in Pellegatta, et al., Sci. Transl. Med., 10:eaao2731-33 (2018). Another antibody that binds CSPG4 is described in Fenton et al., Oncol. Res. 22(2):117-21 (2015). Other antibodies or fragments thereof that bind CSPG4 are commercially available bevacizumab and Creative Biolabs mAb225.28. Still other antibodies or fragments thereof that bind CSPG4 are commercially available from Aviva Systems Biology. Further representative examples of amino acid or gene sequences encoding scFvs that target CSPG4 that may be useful in the present disclosure are described in U.S. Pat. No. 9,801,928 and U.S. Patent Application Publication No. 2019 / 0008940.

[0108] Another example of an antibody fragment that binds to GD2 is described in Mount et al., Nat. Med. 24, 572-579 (2018). Other antibodies or fragments thereof that bind to GD2 include dinutuximab, mAb 3F8, mAb 14g2a, and mAb 14.18. Further representative examples of amino acid or gene sequences encoding scFvs that target GD2 and may be useful in the present disclosure are described in U.S. Patent No. 4,675,287.

[0109] Another example of an antibody fragment that binds PDGFRα is described in Brennan et al., PLoS One, 4(11):e7752-10 (2009). Other antibodies or fragments thereof that bind PDGFRα are commercially available from Abcam, LifeSpan Bio, Santa Cruz (sc-338), and Thermo Fisher (mAb APA5). Further representative examples of amino acid or gene sequences encoding scFvs targeting PDGFRα that may be useful in the present disclosure are described in U.S. Patent Application Publication No. 2012 / 0027767.

[0110] Antibody fragments that bind to GRP78 are described in Kang et al., Sci. Rep. 6, 34922-7 (2016). Other antibodies or fragments thereof that bind to GRP78 are commercially available from Thermo Fisher (PA1-014A) and Abcam (N-20). Further representative examples of amino acid or gene sequences encoding scFvs that target GRP78 and may be useful in the present disclosure are described in U.S. Patent No. 10,259,884.

[0111] Other proteins that have been implicated in brain cancer and can be targeted by the bifunctional compounds of the present disclosure include neural cell adhesion molecule (NCAM), cluster of differentiation 276 (CD276), and the neuroectodermal stem cell marker (nestin).

[0112] Antibodies that bind to NCAM are described in Modak et al., Cancer Res. 61, 4048-4054 ​​(2001). Other antibodies or fragments thereof that bind to NCAM are mAb UJ13A and mAb ERIC-1. Further representative examples of amino acid or gene sequences encoding scFvs that target NCAM and may be useful in the present disclosure are described in U.S. Patent No. 7,402,560.

[0113] Antibodies or fragments thereof that bind nestin are commercially available from Abcam (ab6142) and Novus Biologicals (NB100-1604). Antibodies or fragments thereof that bind βIII-tubulin are commercially available from Abcam (2G10) and RND Systems (mAB1195).

[0114] Another example of an antibody fragment that binds CS-1 is described in Chu et al., Blood, 122:14 (2013). Other antibodies or fragments thereof that bind CS-1 are commercially available, including REA150 (Miltenyi) or 162.1 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CS-1 that may be useful in the present disclosure are described in WO 2004 / 100898(A2).

[0115] Another example of an antibody fragment that binds BCMA is described in Raje et al., N. Engl. J. Med. 380(18):1726-1737 (2019). Other antibodies or fragments thereof that bind BCMA are commercially available and include REA315 (Miltenyi), J6M0 (Abeomics) (anti-BCMA included in the belantamab conjugation composition), and 19F2 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target BCMA that may be useful in the present disclosure are described in WO 2010 / 104949 (A2) and WO 2003 / 014294 (A2).

[0116] In some embodiments, the cell surface antigen is CD19, B cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CD38, CS1 (SLAM family member 7, SLAMF7), G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), or TNFRSF13B (TACI).

[0117] The transmembrane (TM) domain allows the CAR to be stably anchored to the cell membrane of immune cells. The transmembrane domain may be derived from the same protein or from a different protein from which the other domains of the CAR are derived. The transmembrane domain may be derived from natural or recombinant sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Representative examples of transmembrane domains that may be useful in the present disclosure include the transmembrane regions of CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0118] CARs can be designed to include a transmembrane domain that is indirectly linked to an extracellular binding domain. In such embodiments, the transmembrane domain is linked to the extracellular region of the CAR via a hinge domain. As used herein, the term "hinge domain" refers to a domain that links the extracellular binding domain to the transmembrane domain and may confer flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular domain close to the plasma membrane of immune cells to minimize the possibility of recognition by antibodies or binding fragments thereof. The hinge domain can be natural (e.g., hinges from human proteins) or synthetic. Sources of hinge domains include human Ig (immunoglobulin) hinges (e.g., IgG4 hinges, IgD hinges), and CD8 (e.g., CD8α hinges).

[0119] In some embodiments, the hinge domain is derived from cluster of differentiation 8 (CD8), e.g., SEQ ID NO:50.

[0120] The intracellular signaling domain assists in immune cell activation upon binding of the CAR (e.g., second generation, third generation, engineered T cell receptor (TCR)) to a cell-associated antigen on a target cell. Such domains are known in the art and are generally referred to as second, third and fourth generation CARs and engineered T cell receptors (TCRs). The intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. Examples of intracellular signaling domains include the cytoplasmic sequence of the TCR and co-receptors that act in concert to initiate signaling after antigen receptor binding. As is known in the art, signals generated through the TCR alone are insufficient for full activation of T cells, and therefore secondary or costimulatory signals are also required. Thus, T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences, namely, those that initiate antigen-dependent primary activation via the TCR (i.e., primary intracellular signaling domains) and those that act antigen-independently to provide secondary or costimulatory signals (i.e., secondary cytoplasmic or costimulatory domains). The primary signaling domain regulates the primary activation of the TCR complex, either stimulatory or inhibitory. Primary intracellular signaling domains that act stimulatory may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Representative examples of ITAM-containing primary intracellular signaling domains that may be suitable for use in the present disclosure include those of CD3zeta, common FcRgamma (FCER1G), Fc-gammaRIIa, FcR-beta (Fc-εR1b), CD3gamma, CD3δ, and CD3ε. In some embodiments, the CAR comprises an intracellular signaling domain that contains the primary signaling domain of CD3zeta.

[0121] The intracellular signaling domain of the CAR may also include at least one other intracellular signaling or costimulatory domain. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for efficient lymphocyte response to antigen. Representative examples of costimulatory domains that may be useful in the CARs of the present disclosure include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, HVEM (LIGHTR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. For example, CD27 costimulation has been demonstrated to enhance the proliferation, effector function, and survival of human CAR T cells in vitro, and to increase the persistence and antitumor activity of human T cells in vivo (see Song, et al., Blood 119(3):696-706 (2012)).

[0122] The intracellular signaling domain can be designed to include one or more, e.g., 1, 2, 3, 4, 5, or more, costimulatory signaling domains, which can be linked together in a specified or random order, optionally via a linker molecule. A polypeptide linker about 1-10 amino acids in length can link consecutive intracellular signaling sequences. Examples of such linkers include doublets such as Gly-Ser, as well as single amino acids such as Ala and Gly. Combinations that can constitute a T cell activation domain can be based on the cytoplasmic regions of CD28, CD137 (4-1BB), OX40, and HVEM, which act to enhance T cell survival and proliferation, and CD3, CD3ζ, and FcRε, which induce T cell activation. For example, CD3ζ, which contains three ITAMs, is the most commonly used intracellular domain component of CARs and transmits activation signals to T cells after antigen binding. However, to provide additional costimulatory signaling, CD28 and OX40 domains can be used together with CD3ζ, allowing the CAR immune cells to transmit proliferation / survival signals.

[0123] CARs that may be used in the present disclosure and methods for making them are known in the art and are described, for example, in U.S. Patent Application Publication No. 2018 / 0169109, each of which is incorporated herein by reference in its entirety. Additional CARs that may be useful have been approved by the FDA and include tisagenlecleucel (Kymriah™), axicabtagene ciloleucel (Yescarta™), idecabtagene biculeucel (Avekuma™), and lysocabtagene maraleucel (Breyangi™), brexcabtagene autoleucel (Tecartas), and siltacabtagene autoleucel (Carvikti).

[0124] Exemplary cytokine receptor switches and CAR constructs are shown in Table 3 below.

[0125] Table 3: Cytokine receptor switches and CAR constructs TIFF2024537170000013.tif141168TIFF2024537170000014.tif211168 * CARs including CD19-, BCMA-, HER2-, and EGFR-CARs.

[0126] Table 3 illustrates exemplary combinations of CARs with the same or different scFvs and cytokine receptor switches that can be present in immune cells.

[0127] BAT-CAR In some embodiments, the immune cells are binary activated T cells that contain a nucleic acid encoding a chimeric antigen receptor (BAT-CAR). BAT-CARs are substantially identical to CARs in terms of design and each spacer domain (e.g., transmembrane domain and intracellular domain). However, in contrast to CAR T cells that are engineered to directly bind to cell-associated antigens, such as tumor-associated antigens, BAT-CAR cells bind to synthetic antigens (which may be masked pro-antigens or unmasked) that are not present on normal or cancer cells. The synthetic antigens are delivered and attached to the cell-associated antigens in the form of a conjugate with an antibody or fragment thereof that binds to the cell-associated antigen. BAT-CAR cells can be administered to a subject with a single conjugate or multiple conjugates that contain antibodies or fragments that bind to different cell-associated antigens. By separating tumor cell targeting and tumor cell killing, CAR T cells with a single specificity (for synthetic antigens or unmasked pro-antigens) can target multiple tumor-associated antigens simultaneously. Thus, the design of BAT-CAR immune cells differs from CAR immune cells primarily, but not exclusively, in terms of the extracellular binding domain.

[0128] The extracellular domain of the BAT-CAR is typically present at the amino terminus and is presented on the surface of immune cells. Apart from its specificity, the extracellular domain of the BAT-CAR is typically an antibody or an antigen-binding fragment thereof, such as an scFv.

[0129] Representative examples of synthetic antigens include fluorescein and fluorescein derivatives (e.g., FITC). Representative examples of extracellular binding domains that bind fluorescein and FITC are described above (in relation to the cytokine receptor switch itself). Representative examples of other binding moieties that may be useful as extracellular binding domains in BAT-CARs are, for example, 4M5.3 ScFv (disclosed in Midelfort et al. J. Mol. Biol. 343:685-701 (2004)) and 2D12.5, 2D12.5ds, or C8.2.5 (disclosed in Orcutt et al. Nucl. Med. 38(2):223-233 (2011)). Representative examples of masked pro-antigens are known in the art, e.g., WO 2017 / 143094, WO 2018 / 200713, WO 2019 / 236522, and WO 2020 / 006312, each of which is incorporated herein by reference.

[0130] Representative examples of masked pro-antigens and BAT-CARs are masked fluorescein pro-antigens and BAT-CARs with specificity for fluorescein. A stimulus (e.g., UV light) unmasks the masked fluorescein molecule, thereby activating cells expressing the fluorescein-specific BAT-CAR. See, e.g., Kobayashi et al., ChemMedChem17:e202100722-5 (2022). In some embodiments, the synthetic antigen is masked by the addition of one or more 5-carboxymethoxy-2-nitrobenzyl (CMNB) caging groups.

[0131] BAT-CARs that may be used in the present disclosure and methods for making them are known in the art and are described, for example, in WO 2017 / 143094, WO 2018 / 200713, WO 2019 / 236522, and WO 2020 / 006312, each of which is incorporated by reference in its entirety.

[0132] A representative example of a polynucleotide encoding BAT-CAR anti-FL CAR-CD28-4-1BB-CD3ζ has the sequence designated as SEQ ID NO: 60: TIFF2024537170000015.tif107141.

[0133] Introduction of polynucleotides encoding cytokine receptor switches into immune cells Immune cells, such as T cells, can be engineered to contain a nucleic acid encoding an expressed cytokine receptor switch according to known techniques. In general, a polynucleotide vector encoding the cytokine receptor switch is constructed and the vector is introduced (e.g., transfected or transduced) into an immune cell population. The cells are then grown under conditions that promote expression of the polynucleotide encoding the cytokine receptor switch. Successful transfection (or transduction, which refers to viral-mediated gene integration) and presentation of the cytokine receptor switch can be performed via standard techniques. In some embodiments, immune cells can be engineered to produce a cytokine receptor switch by first constructing a retroviral vector encoding a selected cytokine receptor switch. Retroviral transduction can be performed using known techniques (e.g., Johnson, et al., Blood 114:535-546 (2009)). Surface expression of the cytokine receptor switch on the transfected immune cells can be measured, for example, by flow cytometry.

[0134] The expression vector encoding the cytokine receptor switch may be introduced as one or more DNA molecules or constructs, optionally containing at least one marker that allows for selection of host cells containing the construct.

[0135] A DNA construct is an artificially constructed segment of nucleic acid for introduction (i.e., transfection or transduction) into a target cell or tissue. As used herein, the term "nucleic acid" refers to a polymer of nucleotides, each of which is an organic molecule consisting of a nucleoside (a nucleic acid base and a pentose sugar) and a phosphate. The term nucleotide includes nucleosides with a ribose sugar (i.e., ribonucleic acid, ribonucleotides that form RNA) or a 2'-deoxyribose sugar (i.e., deoxyribonucleic acid, deoxyribonucleotides that form DNA), unless otherwise specified or clear from the context. Nucleotides serve as monomeric units of nucleic acid polymers or polynucleotides. The four nucleic acid bases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleic acid bases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). A nucleic acid is a linear chain of nucleotides (e.g., at least three nucleotides) chemically joined by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2'-deoxyribose) in the adjacent nucleotide.

[0136] The nucleic acid encodes at least a cytokine receptor switch protein. The terms "protein" and "polypeptide" as used herein refer to a series of amino acids (typically at least 10 amino acids long) linked by amide bonds. A protein is usually derived from an organism, but is not limited thereto, and may be composed of, for example, an artificially designed sequence. Also, the protein may be any of naturally occurring proteins, synthetic proteins, and recombinant proteins.

[0137] Constructs can be prepared in a conventional manner in which the individual components of the cytokine receptor switch can be ligated in the desired order, cloned into a suitable cloning host, and analyzed by restriction enzymes or sequencing or other convenient means. In particular, PCR can be used to isolate individual fragments containing all or part of the functional unit, and one or more mutations can be introduced using "primer repair", ligation, in vitro mutagenesis, etc., as appropriate. Once complete and demonstrated to have the proper sequence, the construct can then be packaged into a suitable vector, which is then introduced into immune cells (i.e., T cells) by any convenient means. Vectors that can be used to prepare construct DNA stocks and to perform transfections are well known in the art and many are commercially available, containing useful elements such as bacterial or yeast origins of replication, selectable and / or amplifiable markers (e.g., hypoxanthine-guanine phosphoribosyltransferase (hprt), neomycin resistance, thymidine kinase, hygromycin resistance, etc.), promoter / enhancer elements for expression in prokaryotes or eukaryotes, one or more suitable sites for insertion of nucleic acid sequences, such as a multiple cloning site (MCS), etc.

[0138] The construct may be incorporated and packaged into a non-replicating defective viral genome such as Adenovirus, Adeno-associated virus (AAV), Herpes simplex virus (HSV), or others including retroviral and lentiviral vectors for infection and transduction of cells. The construct may optionally include viral sequences for transfection. Alternatively, the construct may be introduced by fusion, electroporation, biolistic methods, transfection, lipofection, etc. The host immune cells may be grown and expanded in culture prior to introduction of the construct, and then the construct may be introduced and treated appropriately to integrate the construct. The cells are then expanded and screened for markers present in the construct.

[0139] In some instances, when it is desired that the construct be integrated into a particular locus, the construct can be engineered to have a target site for homologous recombination. For example, an endogenous gene can be knocked out and replaced with a gene encoded by the construct (at the same locus or elsewhere) using materials and methods known in the art for homologous recombination. For homologous recombination, either OMEGA or O-vectors can be used. See, for example, Thomas and Capecchi, Cell 51:503-512 (1987), Mansour et al., Nature 336:348-352 (1988), and Joyner et al., Nature 338:153-156 (1989).

[0140] In some embodiments, the vector is a lentiviral vector or a recombinant lentiviral vector. In some embodiments, the expression vector is a non-integrating and non-replicating recombinant lentiviral vector. Exemplary lentiviral vectors include, for example, LentiVector and LentiStable from Oxford BioMedica, LV-Max from Gibco, and the like. The construction of lentiviral vectors is described, for example, in U.S. Patent Nos. 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530.

[0141] Formulations and Methods of Use Preparations containing cytokine receptor switch-containing immune cells The immune cells of the present invention may be formulated in a pharma- ceutically acceptable vehicle or carrier, the choice and amount of which may be determined depending on the mode of administration. The therapeutically effective amount of the formulation may depend on the concentration of cells in the total volume of the formulation. The number of immune cells of the present invention administered to a subject may vary over a wide range, depending on a variety of factors, including, for example, the location, type, and severity of the cancer, and the age and condition of the individual being treated, and is within the skill level of the treating physician. In general, the formulation contains approximately 1×10 4 ~Approx. 1×10 10 In some embodiments, the preparation contains about 1×10 immune cells of the invention. 5 ~Approx. 1×10 9 5×10 immune cells of the present invention 5 ~Approx. 5×10 8 of the immune cells of the present invention, or about 1×10 6 ~Approx. 1×10 7 The immune cells of the present invention are contained in the host. See, for example, International Publication No. WO 2020 / 006312, the entire contents of which are incorporated herein by reference.

[0142] The immune cell preparations of the present invention can be administered to a subject in need thereof according to accepted medical practice. An exemplary mode of administration is intravenous injection. Other modes of administration may include intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial (including convection-enhanced delivery), intraspinal, and intrathecal (spinal fluid). Any known device useful for parenteral injection or infusion of the preparation can be used to affect such modes of administration. Exemplary vehicles and carriers include buffers such as neutral buffered saline, phosphate buffered saline, and the like. The composition may further comprise one or more pharma- ceutically acceptable excipients. Examples of such excipients include carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants such as aluminum hydroxide, and preservatives.

[0143] Formulations Containing Synthetic Small Molecules To stimulate the immune cells of the present invention in vivo, a therapeutically effective amount of synthetic small molecules can be administered to a subject in need thereof in accordance with accepted medical practice. Synthetic small molecules can be formulated in a pharma- ceutically acceptable vehicle or carrier, the selection and amount of which can be determined according to the mode of administration. An exemplary mode of administration is intravenous injection. Other modes include intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), im, ip, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, and intrathecal. Administration of synthetic small molecules can be performed using any known device useful for parenteral injection or infusion of formulations. Representative examples of pharma-ceutically acceptable vehicles or carriers are serum albumin (e.g., human serum albumin), dextran, and antibodies.

[0144] In general, the amount of synthetic small molecule is in the range of 0.1-1000 μg / mL based on the total volume of the composition. In some embodiments, the amount of synthetic small molecule is in the range of about 0.1-100 μg / mL based on the total volume of the composition. The total amount of small molecule may vary depending on the vehicle or carrier. For example, in the case of in vivo injection, the effective dose may be higher than ex vivo because not all of the injected small molecule may be delivered to the tumor.

[0145] Methods for stimulating immune cells expressing the cytokine receptor switch of the present invention Broadly speaking, the methods of the invention involve treating or contacting immune cells with a sufficient concentration of a synthetic small molecule.

[0146] In some embodiments, the method is performed ex vivo. Immune cells containing an exogenous nucleic acid encoding a cytokine receptor switch are placed in a suitable container, suitable medium, and contacted with a synthetic small molecule, for example, in an amount of 0.1-1000 μg / mL based on the total volume of the medium. In some embodiments, the amount of synthetic small molecule ranges from about 0.1-100 μg / mL based on the total volume of the medium. Representative examples of suitable media that can be used in the practice of the method include RPMI-1640 (Gibco™) and X-VIVO™ 15 (BioWhittaker™).

[0147] The duration of contact (also referred to herein as "treatment" or "treating" or "stimulating") can be as long as hours, days, and weeks (e.g., 1, 2, 3, 4 weeks or more). In some embodiments, the contact can be performed in a high affinity plate, dish, or flask, and the small molecule is conjugated to a carrier immobilized on the surface of the vessel. Representative examples of carriers include bovine or human serum albumin, dextran, and antibodies (e.g., anti-HER2 antibodies, anti-EGFR antibodies, anti-BCMA antibodies, and anti-CD19 antibodies). In some embodiments, pertuzumab, cetuximab, belentamab, J6M0, or daratuzumab.

[0148] In some embodiments, the stimulation promotes immune cell proliferation or phenotypic change, for example, the stimulation may promote an increase in the population of stimulated immune cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more.

[0149] In some embodiments, immune cell stimulation promotes a change in immune cell phenotype. Exemplary phenotypes include a memory phenotype, a cytotoxic phenotype, and a regulatory phenotype.

[0150] In some embodiments, immune cells containing nucleic acids encoding a cytokine receptor switch are stimulated with increasing doses of fluorescein isothiocyanate (FITC) (0, 0.1, 1, 10, 100, and 1000 μg / mL) on FITC-conjugated bovine serum albumin (BSA) coated plates for up to 2 weeks in the presence of CD3 / CD28 costimulation. + and CD8 + Central memory on T cells (CD45RA-, CC chemokine receptor type 7 (CCR7) + ) markers, and effector memory (CD45RA - CCR7 - ) Markers can be assessed by flow cytometry.

[0151] In some embodiments, immune cells containing nucleic acids encoding a cytokine receptor switch are stimulated with increasing doses of FITC (0, 0.1, 1, 10, 100, and 1000 μg / mL) on FITC-conjugated BSA-coated plates for up to 2 weeks in the presence or absence of CD3 / CD28 costimulation. + and CD8 + Effector memory on T cells (CD45RA - CCR7 - ) markers, central memory (CD45RA-, CCR7 + ) markers, and activation (CD69) markers can be assessed by flow cytometry.

[0152] In some embodiments, immune cells containing nucleic acids encoding a cytokine receptor switch are stimulated with increasing doses of carboxyfluorescein (0, 0.1, 1, 10, 100, and 1000 μg / mL) on carboxyfluorescein-conjugated BSA-coated plates for up to 2 weeks in the presence of CD3 / CD28 costimulation.

[0153] In some embodiments, immune cells containing a nucleic acid encoding a CAR with or without one or a combination of different cytokine receptor switches are stimulated on small molecule conjugate antibody coated plates with increasing doses (0, 0.1, 1, 10, and 100 μg / mL) of small molecule conjugate in the presence or absence of CD3 / CD28 costimulation. + and CD8 + Expression of IL-2, IFN-γ and CD69 on T cells can be assessed by flow cytometry.

[0154] The stimulated immune cells can be isolated from the culture medium and then formulated for delivery to a subject, hi some embodiments, the immune cells are stimulated in vivo.

[0155] These embodiments involve administering a therapeutically effective amount of a composition described herein to a subject in need thereof, and administering a therapeutically effective amount of a synthetic small molecule to the subject. The synthetic small molecule and immune cells may be administered substantially simultaneously or sequentially, via the same or different formulations. The method may further include administering to the subject a formulation of a synthetic monomeric or polymeric small molecule that serves to reduce irritation.

[0156] The administration of ex vivo activated immune cells and in vivo immune cell activation is typically performed in the context of treating a disease or disorder, namely cancer.

[0157] The term "subject" (or "patient") as used herein includes all members of the animal kingdom susceptible to or afflicted with the indicated disease or disorder. In some embodiments, the subject is a mammal, e.g., a human or non-human mammal. The method is also applicable to pet animals such as dogs and cats, as well as livestock such as cows, horses, sheep, goats, pigs, and other domesticated and wild animals. A subject "in need" of treatment according to the present disclosure may be a subject "suffering or suspected of suffering from" a particular disease or disorder, may have been positively diagnosed, or may otherwise exhibit a sufficient number of risk factors, or a sufficient number or combination of signs or symptoms, such that a medical professional may diagnose or suspect that the subject suffers from the disease or disorder. Thus, subjects suffering from and suspected of suffering from a particular disease or disorder are not necessarily two distinct groups.

[0158] Broadly, the method may be effective in treating carcinomas (solid tumors, including both primary and metastatic tumors), sarcomas, melanomas, and hematological cancers (cancers affecting the blood, including lymphocytes, bone marrow, and / or lymph nodes), such as leukemia, lymphoma, and multiple myeloma. Adult tumors / cancers and pediatric tumors / cancers are also included. The cancer may be a tumor that is vascularized or not yet substantially vascularized or not vascularized.

[0159] Representative examples of cancer include adrenal cortical carcinoma, AIDS-related cancer (e.g., Kaposi's and AIDS-related lymphoma), appendix cancer, childhood cancer (e.g., childhood cerebellar astrocytoma, childhood brain astrocytoma), basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, brain cancer (e.g., brain stem glioma, gestational trophoblastic tumor glioma, cerebellar astrocytoma, brain astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, and other gliomas and glioblastomas. ), breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, cancer of the nervous system (e.g., central nervous system cancer, central nervous system lymphoma), cervical cancer, chronic myeloproliferative disorder, colorectal cancer (e.g., colon cancer, rectal cancer), lymphatic neoplasms, mycoses, Sézary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, extramaxillary germ cell tumors, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal cancer (stomach cancer, small intestine cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors) stromal tumor, GIST), bile duct cancer, germ cell tumor, ovarian germ cell tumor, head and neck cancer, neuroendocrine tumor, Hodgkin lymphoma, Ann Arbor stage III and IV childhood non-Hodgkin lymphoma, ROS1-positive refractory non-Hodgkin lymphoma, leukemia, lymphoma, multiple myeloma, hypopharyngeal cancer, intraocular melanoma, eye cancer, pancreatic islet cell tumor (pancreatic endocrine tumor), kidney cancer (e.g., Wilm's tumor, renal cell carcinoma), liver cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), ALK-positive anaplastic large cell lymphoma, ALK-positive advanced malignant solid neoplasm, Waldenstrom giant cell tumor, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell cervical cancer of unknown primary site, multiple endocrine neoplasm neoplasia, MEN), myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer (e.g., mouth cancer, lip cancer, oralcancer of the tongue, oropharynx, throat, and larynx; ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumors, and ovarian low malignant potential tumors); pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma, metastatic anaplastic thyroid cancer, anaplastic thyroid cancer, papillary thyroid cancer, pituitary tumors, plasma cell These include: alveolar neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine cancer (e.g., endometrial carcinoma, uterine sarcoma, endometrial carcinoma), squamous cell carcinoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, juvenile xanthogranuloma, transitional cell carcinoma of the renal pelvis, ureter, and other urinary tract, urethral cancer, gestational trophoblastic neoplasm, vaginal cancer, vulvar cancer, hepatoblastoma, rhabdominal tumor, and Wilms' tumor.

[0160] Sarcomas that may be treatable with the methods of the present disclosure include both soft tissue and bone sarcomas as well, representative examples of which include osteosarcoma or osteogenic sarcoma (bone) (e.g., Ewing's sarcoma), chondrosarcoma (cartilage tissue), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membranous lining of a body cavity), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (fatty tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), mesenchymal or mixed mesodermal tumor (mixed connective tissue type), and histiocytic sarcoma (immune cancer).

[0161] In some embodiments, the methods of the present disclosure involve treatment of a subject having a cell proliferative disease or disorder of the blood system, liver, brain, lung, colon, pancreas, prostate, ovaries, breast, skin, and endometrium.

[0162] As used herein, "cell proliferative diseases or disorders of the blood system" include lymphoma, leukemia, myeloid neoplasm, mast cell neoplasm, myelodysplasia, benign monoclonal gammopathy, lymphomatoid papulosis, polycythemia vera, myeloid metaplasia of unknown etiology, and essential thrombocythemia. Thus, representative examples of blood cancer include multiple myeloma, lymphoma (T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, DLBCL), follicular lymphoma, mantle cell lymphoma, leukemia, myelodysplasia ... lymphoma, MCL) and ALK+ anaplastic large cell lymphoma (e.g., diffuse large B-cell lymphoma (e.g., germinal center B-cell-like diffuse large B-cell lymphoma or activated B-cell-like diffuse large B-cell lymphoma), Burkitt lymphoma / leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphocytic plasmacytic lymphoma / Waldenstrom macroglobulinemia, metastatic pancreatic adenocarcinoma, refractory B-cell non-Hodgkin's lymphoma and B-cell non-Hodgkin's lymphoma selected from relapsed B-cell non-Hodgkin's lymphoma, childhood lymphoma, and lymphomas of lymphocytic and cutaneous origin, such as small lymphocytic lymphoma, leukemias including childhood leukemia, hairy cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (e.g. acute monocytic leukemia), chronic lymphocytic leukemia, small lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia, myeloid neoplasms and mast cell neoplasms.

[0163] Multiple myeloma is a cell proliferative disease or disorder of the blood system. Multiple myeloma is a plasma cell neoplasm. Healthy plasma cells produce antibodies that recognize and attack pathogens. In multiple myeloma, cancerous plasma cells accumulate in the bone marrow and eliminate healthy blood cells. Instead of producing antibodies, cancerous myeloma plasma cells produce abnormal proteins that can cause complications. Hyperproliferation of plasma cells also leads to crowding of normal blood-forming cells, resulting in low blood counts and thrombocytopenia and leukopenia. Myeloma plasma cells produce abnormal antibodies known as monoclonal immunoglobulins, monoclonal proteins (M proteins), M spikes, or paraproteins.

[0164] Other plasmacytomas that do not meet the criteria for multiple myeloma include monoclonal gammopathy of uncertain significance (MGUS), smoldering multiple myeloma (SMM), solitary plasmacytoma, and light chain amyloidosis. Minimal residual disease (MRD) refers to a small number of malignant cells below the detection limit available with traditional morphologic evaluation. In multiple myeloma, MRD refers to myeloma cells that are present in the bone marrow after clinical response (CR) has been measured and the subject is in remission. These residual myeloma cells are clinically significant because they can lead to disease progression and relapse.

[0165] As used herein, "cell proliferative disease or disorder of the liver" includes all forms of cell proliferative disorders that affect the liver.Cell proliferative disorders of the liver can include liver cancer (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma and hepatoblastoma), precancer or precancerous conditions of the liver, benign growths or lesions of the liver, and malignant growths or lesions of the liver, as well as metastatic lesions in tissues and organs of the body other than the liver.Cell proliferative disorders of the liver can include hyperplasia, metaplasia, and dysplasia in the liver.

[0166] As used herein, "brain cell proliferative disease or disorder" includes all forms of cell proliferative disorders affecting the brain. Brain cell proliferative disorders may include brain cancers (e.g., glioma, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, and primitive neuroectodermal tumor (medulloblastoma)), brain precancer or precancerous conditions, brain benign growths or lesions, and brain malignant growths or lesions, as well as metastatic lesions in tissues and organs of the body other than the brain. Brain cell proliferative disorders are also called central nervous system (CNS) tumors, and include astrocytic tumors, oligodendroglial tumors, mixed gliomas, ependymal tumors, medulloblastomas, pineal parenchymal tumors, meningeal tumors, germ cell tumors, and craniopharyngiomas (Grade I). Brain cell proliferative disorders may include brain hyperplasia, metaplasia, and dysplasia. Cancers that have spread to the brain are called metastatic brain tumors. Approximately half of metastatic brain tumors originate from lung tumors. Other tumors that have a tendency to spread to the brain include, for example, melanoma, breast cancer, colon cancer, renal cancer, and nasopharyngeal cancer.

[0167] Glioblastoma (GBM), also called grade IV astrocytoma, is a rapidly growing, aggressive cell proliferative disorder of the brain. GBM is an astrocytic tumor that begins in astrocytes (a type of glial cell) and is sometimes called a glioma. GBM invades nearby brain tissue but does not generally spread to distant organs. GBM can arise de novo in the brain or from low-grade astrocytomas.

[0168] As used herein, "pulmonary cell proliferative diseases or disorders" include all forms of cell proliferative disorders affecting lung cells. Pulmonary cell proliferative disorders include lung cancer, precancer and precancerous conditions of the lung, benign growths or lesions of the lung, hyperplasia, metaplasia, and dysplasia in the lung, and metastatic lesions in tissues and organs of the body other than the lung. Lung cancer includes all forms of lung cancer, such as malignant lung neoplasms, intraepithelial carcinomas, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer includes small cell lung cancer (SLCL), non-small cell lung cancer (NSCLC), adenocarcinoma, small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and mesothelioma. Lung cancer cells can include "scar carcinoma," bronchioloalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer also includes lung neoplasms with histological and ultrastructural heterogeneity (e.g., mixed cell types). In some embodiments, the compounds of the present disclosure can be used to treat non-metastatic or metastatic lung cancer (e.g., NSCLC, ALK-positive NSCLC, NSCLC with ROS1 rearrangements, lung adenocarcinoma, and squamous cell lung cancer).

[0169] As used herein, "cell proliferative disease or disorder of the colon" includes all forms of cell proliferative disorders that affect colon cells, including colon cancer, colon precancer or precancerous conditions, colon adenomatous polyps, and colon metachronous lesions.Colon cancer includes sporadic and hereditary colon cancer, malignant colon neoplasms, intraepithelial carcinomas, typical carcinoid tumors, and atypical carcinoid tumors, adenocarcinomas, squamous cell carcinomas, and squamous cell carcinomas.Colon cancer may be associated with hereditary syndromes, such as hereditary nonpolyposis colorectal cancer, familial adenomatous colorectal cancer, MYH-associated polyposis, Gardner syndrome, Peutz-Jeghers syndrome, Turcot syndrome, and juvenile polyposis.Cell proliferative disorders of the colon may also be characterized by colonic hyperplasia, metaplasia, or dysplasia.

[0170] As used herein, "cell proliferative disease or disorder of the pancreas" includes all forms of cell proliferative disorders affecting pancreatic cells. Cell proliferative disorders of the pancreas may include pancreatic cancer, precancer or precancerous conditions of the pancreas, pancreatic hyperplasia, pancreatic dysplasia, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, as well as metastatic lesions in tissues and organs of the body other than the pancreas. Pancreatic cancer includes all forms of pancreatic cancer, including ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary tumor, mucinous cystadenoma, papillary cystadenoma, and serous cystadenoma, as well as pancreatic neoplasms with histochemical and ultrastructural heterogeneity (e.g., mixed).

[0171] As used herein, "cell proliferative disease or disorder of the prostate" includes all forms of cell proliferative disorders that affect the prostate. Cell proliferative disorders of the prostate may include prostate cancer, precancer or precancerous conditions of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, as well as metastatic lesions in tissues and organs in the body other than the prostate. Cell proliferative disorders of the prostate may include hyperplasia, metaplasia, and dysplasia in the prostate.

[0172] As used herein, "cell proliferative diseases or disorders of the ovary" includes all forms of cell proliferative disorders affecting cells of the ovary. Cell proliferative disorders of the ovary include proliferation of cells that form in tissues that cover one or both ovaries, the fallopian tubes, or organs in the abdominal cavity (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma). Cell proliferative disorders of the ovary may include precancer or precancerous conditions of the ovary, benign growths or lesions of the ovary, ovarian cancer, and metastatic lesions in tissues and organs of the body other than the ovaries. Cell proliferative disorders of the ovary may include hyperplasia, metaplasia, and dysplasia in the ovary. Cell proliferative disorders of the ovary include ovarian epithelial cancer (epithelial ovarian cancer), germ cell tumors, and stromal cell tumors. Epithelial ovarian cancer is the most common type of ovarian cancer. Approximately 85% to 90% of these cancers involve cells that cover the outer surface of the ovary. They generally spread first to the lining and organs of the pelvis and abdomen, and then to other parts of the body. Nearly 70% of women with this type of ovarian cancer are diagnosed at an advanced stage. Ovarian epithelial cancer, fallopian tube cancer, and primary peritoneal cancer are epithelial ovarian cancers.

[0173] As used herein, "cell proliferative diseases or disorders of the breast" includes all forms of cell proliferative disorders affecting breast cells. Cell proliferative disorders of the breast may include breast cancer, precancer or precancerous conditions of the breast, benign growths or lesions of the breast, and metastatic lesions in tissues and organs of the body other than the breast. Cell proliferative disorders of the breast are a group of disorders in which cells of the breast grow uncontrollably. Cell proliferative disorders of the breast may include hyperplasia, metaplasia, and dysplasia in the breast. Cell proliferative disorders of the breast may begin in different parts of the breast. The breast is composed of three main parts: lobules, ducts, and connective tissue. Lobules are the glands that produce milk. Ducts are the tubes that carry milk to the nipple. Connective tissue, which is composed of fibrous and fatty tissue, surrounds and connects breast tissue. Most breast cancers begin in the ducts or lobules. Infiltrating ductal carcinoma and invasive lobular carcinoma are the two most common types of breast cancer. In invasive ductal carcinoma, cancer cells originate in the ducts and then spread or metastasize outside the ducts to other parts of breast tissue. In invasive lobular carcinoma, cancer cells originate in the lobules and then spread from the lobules to nearby breast tissue. As used herein, "cell proliferative diseases or disorders of the skin" includes all forms of cell proliferative disorders affecting skin cells. Cell proliferative disorders of the skin may include precancer or precancerous conditions of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma or other malignant growths or lesions of the skin, and metastatic lesions in tissues and organs of the body other than the skin. Cell proliferative disorders of the skin may include hyperplasia, metaplasia, and dysplasia in the skin.

[0174] As used herein, "cell proliferative diseases or disorders of the endometrium" include all forms of cell proliferative disorders affecting cells of the endometrium. Cell proliferative disorders of the endometrium may include precancer or precancerous conditions of the endometrium, benign proliferations or lesions of the endometrium, endometrial cancer, and metastatic lesions in tissues and organs of the body other than the endometrium. Cell proliferative disorders of the endometrium may include hyperplasia, metaplasia, and dysplasia in the endometrium.

[0175] In some embodiments, the cancer is breast cancer, ovarian cancer, multiple myeloma, lung cancer, or glioblastoma multiforme. The therapeutic method of the present disclosure can be a "first-line" or initial treatment in patients who have not yet received any anti-cancer treatment, alone or in combination with other treatments. The therapeutic method of the present disclosure can be advantageously used as a "second-line" therapy in that it is administered to patients who have received at least one previous anti-cancer treatment regimen, such as chemotherapy, radioimmunotherapy, toxin therapy, prodrug-activated enzyme therapy, antibody therapy, surgical therapy, immunotherapy, radiation therapy, targeted therapy, or any combination thereof, alone or in combination with other treatments. In some cases, previous therapy may have been unsuccessful or partially successful, but this is when the patient becomes intolerant to a particular treatment, especially when the frontline therapy is no longer effective due to antigen loss / escape.

[0176] Combination therapy In certain embodiments, the methods of the invention for treating cancer can be part of a combination therapy, where the subject is also treated with another agent that exerts an indirect or direct effect. In the case of administration of BAT-CAR immune cells, the other agent is a synthetic antigen conjugated to a binding moiety that binds to a cell surface antigen, such as a tumor-associated antigen. The BAT-CAR immune cells bind to the cell surface antigen indirectly through the extracellular binding domain of the BAT-CAR, which binds to the synthetic antigen. Representative examples of synthetic antigens and their conjugates are described, for example, in WO 2017 / 143094, WO 2018 / 200713, and WO 2020 / 006312, each of which is incorporated herein by reference in its entirety. In some embodiments, the synthetic antigen is masked or caged so that it cannot bind to the extracellular binding domain of the BAT-CAR ("pro-antigen"). These embodiments require administration of an additional agent that unmasks or uncages the antigen so that it can interact with the extracellular binding domain of the BAT-CAR. This feature adds yet another level of control to the methods of the invention. Representative examples of synthetic antigens and conjugates thereof are described, for example, in WO 2017 / 143094, WO 2018 / 200713, and WO 2020 / 006312, which are incorporated by reference in their entireties. In some embodiments, the synthetic antigen is masked by the addition of one or more CMNB caging groups, and the unmasking agent is UV light.

[0177] In some embodiments, the method involves the administration of another anti-cancer drug. An "anti-cancer" drug can adversely affect a subject's cancer, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer. More generally, these other compositions are provided in a combined amount effective to kill or inhibit proliferation of the cells. This process can include contacting the cancer cells with the expression construct and the drug or multiple factors simultaneously. This can be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both drugs, or by contacting the cells with two different compositions or formulations at the same time, one composition that includes the expression construct and another that includes a second drug.

[0178] Tumor cell resistance to chemotherapy and radiotherapy is a major problem in clinical oncology. One goal of current cancer research is to find ways to combine chemotherapy and radiotherapy with other therapies to improve their effectiveness. In the context of the present invention, it is believed that cell therapy can be used in conjunction with chemotherapy, radiotherapy, or immunotherapy interventions, as well as with proapoptotic or cell cycle regulating agents.

[0179] Alternatively, the therapy of the present invention may precede or follow the treatment of the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the present invention are applied separately, one will generally ensure that no significant period of time passes between the respective delivery times so that the agent and the therapy of the present invention can still exert their advantageous combined effect on the cells. In such cases, it is contemplated that the cells may be contacted with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period if several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between the respective administrations.

[0180] It is expected that treatment cycles will be repeated as necessary. It is also contemplated that various standard therapies, as well as surgical interventions, may be applied in combination with the cell therapy of the present invention.

[0181] Suitable myeloma therapeutic agents for combination with the therapies of the invention described herein include belantamab mafodotin-blmf (Brenrep®), bortezomib (Velcade®), carfilzomib (Kyprolis®), carmustine (BiCNU®), siltacabtagene-autoleucel (Carbicty®), cyclophosphamide, daratumumab (Darazalex®), daratumumab and hyaluronidase-fihj (Darazalex-Faspro®), doxorubicin hydrochloride liposomal (Doxil®), elotuzumab (Empliciti®), idecbutagen-bicelucel (Avekuma®). , isatuximab-irfc (Circlisa®), ixazomib citrate (Ninlaro®), lenalidomide (Revlimid), melphalan and melphalan hydrochloride (Alkeran® Tablets, Alkeran® Injection, Evomela®), disodium pamidronate (Aredia®), plerixafor (Mozovir®), pomalidomide (Pomalyst®), selinexor (Xypobio®), thalidomide (Thalomid®), zoledronic acid (Zometa®), and PAD combinations of bortezomib (PS-341), doxorubicin hydrochloride (Adriamycin®) and dexamethasone.

[0182] Breast cancer preventative and therapeutic agents suitable for combination with the inventive therapies described herein also include raloxifene and tamoxifen citrate (Soltamox®), abemaciclib (Verzenio®), paclitaxel (Abraxane®), ado-trastuzumab emtansine (Kadcyla®), everolimus (Afinitor®, Zotres®, Afinitor-Disperz®), alpelisib (Piclay®), anastrozole (Arimidex®), pamidronate disodium (Aredia®), exemestane (Aromasin®), cyclophosphamide, doxorubicin hydrochloride, epirubicin hydrochloride (Ellens®), fam-trastuzumab deluxecan-nxki (Enhertz®), fluorouracil (5-FU;Adolsil®), toremifene (Fareston®), letrozole (Femara®), gemcitabine (Gemzar®, InfuGem®), eribulin mesylate (Halaven®), trastuzumab and hyaluronidase-oysk (Herceptin-Hylecta®), trastuzumab (Herceptin®), palbociclib (Ibrance®), ixabepilone (Ixempra®), pembrolizumab (Keytruda®), ribociclib (Kisqali®), olaparib (Lynparza®), margetuximab-cmkb (Margenza®), neratinib maleate (Ne may include, for example, Lulinx®), pertuzumab (Perjeta®), pertuzumab trastuzumab and hyaluronidase-zzxf (Phesgo®), talazoparib tosylate (Tarzena®), docetaxel (Taxotere®), atezolizumab (Tecentriq®), thiotepa (Tepadyna®), methotrexate sodium (Trexar®), sacituzumab govitecan-hziy (Trodelv®), tucatinib (Tuxa®), lapatinib ditosylate (Tikerub®), vinblastine sulfate, capecitabine (Xeloda®), and goserelin acetate (Zoladex®);

[0183] Suitable ovarian cancer therapeutic agents for combination with the therapies of the present invention described herein include melphalan (Alkeran®), bevacizumab (Alimsys®, Avastin®, Mubashi®, Zirabeb®), cisplatin, cyclophosphamide, doxorubicin hydrochloride, doxorubicin hydrochloride liposomal (Doxyl®), gemcitabine hydrochloride (Gemzar®, Infugem®), topotecan hydrochloride (Hycamtin®), olaparib (Lynparza®), carboplatin (Paraplatin®), rucaparib camsylate (Rubraca®), thiotepa (Tepadyna®), and niraparib tosylate monohydrate (Zedura®). Drugs approved for treating epithelial ovarian cancer may be applied to ovarian germ cell cancer.

[0184] Brain cancer therapeutics suitable for combination with the therapies of the invention described herein include veltutifan (Welireg), bevacizumab (Alimsys, Avastin, Mubashi, Zirabeb), carmustine (BiCNU), carmustine implant (Ziradel Wafer), everolimus (Afinitor, Afinitor Dispertz), lomustine, naxitamab-gqgk (Danielza), and temozolomide (Temodar).

[0185] immunotherapy Checkpoint inhibitors (e.g., pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cemiplimab (LIBTAYO®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), durvalumab (IMFINZI®) or other immunomodulatory antibodies or reagents may also be used in conjunction with the gene therapy of the invention as part of a combination therapy.

[0186] chemotherapy Cancer therapy also includes a variety of combination therapies using both chemical and radiation-based treatments, such as Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, These include plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabine, Navelbine®, farnesyl protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or variants of any of the foregoing analogues or derivatives, and combinations thereof.

[0187] In certain embodiments, chemotherapy for an individual is employed in combination with the present disclosure, for example, before, during, and / or after administration of the present disclosure.

[0188] Radiation therapy Other agents that cause DNA damage and are widely used include gamma radiation, commonly known as X-rays, and / or the guided delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also possible, such as microwave and ultraviolet radiation. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0189] gene In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide (e.g., a therapeutic RNA such as an mRNA or replicon) is administered before, after, or simultaneously with the clinical embodiments of the present disclosure. A variety of expression products are encompassed by the present disclosure, including inducers of cell proliferation, inhibitors of cell proliferation, or regulators of programmed cell death.

[0190] surgery Approximately 60% of cancer patients undergo some type of surgery, including preventative, diagnostic, staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatment of the present disclosure, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0191] Curative surgery includes resection, where all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). It is further contemplated that the present disclosure may be used in combination with the removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0192] When all or part of the cancerous cells, tissues, or tumors are removed, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application to the site with additional anti-cancer therapy. Such treatments may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of various dosages as well.

[0193] Other drugs Additional agents may be used in the methods of the invention. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, or agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers. Immunomodulators include tumor necrosis factor, F42K and other cytokine analogs, or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. Upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL, may enhance the apoptosis-inducing ability of the compositions and methods of the invention by establishing an autocrine or paracrine effect on the hyperproliferative cells. Increasing intercellular signaling by increasing the number of GAP junctions may increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. Thus, in other embodiments, cytostatic or differentiation agents may be used in combination with the present disclosure to further enhance anti-hyperproliferative efficacy. Cell adhesion inhibitors may also enhance the effectiveness of the present disclosure. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Still other agents that increase the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) can be used.

[0194] These and other aspects of the present disclosure will be further understood in view of the following examples, which are intended to illustrate certain specific embodiments of the present disclosure, but not to limit its scope, which is defined by the claims. EXAMPLES

[0195] Example 1: CD4 with and without CD3 / CD28 costimulation + and CD8 + Effect of the cytokine receptor switch of the invention on T cells T cells containing nucleic acids encoding cytokine receptor switches were stimulated with increasing doses of FITC (0, 0.1, 1, 10, 100, and 1000 μg / mL) on FITC-conjugated BSA-coated plates (Figure 2) for up to 2 weeks in the presence or absence of CD3 / CD28 costimulation. + and CD8 + Effector memory on T cells (CD45RA - CCR7 - ) Marker, Central Memory (CD45RA - , CCR7 + ) and activation (CD69) markers were assessed by flow cytometry.

[0196] Figure 3A shows that IL2RA-IL2RB-, IL2RG-, IL7RA- and IL15RA- cytokine receptor switching increased effector memory markers on CD8+ T cells in the presence of CD3 / CD28 costimulation. IL2RA and IL15RA- cytokine receptor switching also increased effector memory markers on CD4+ T cells in the presence of CD3 / CD28 costimulation. + It increased effector memory markers on T cells.

[0197] Figure 3B shows that the IL2RA and IL7RA cytokine receptor switch promotes CD8+ / CD28+ co-stimulation. + This shows that it increased central memory markers on T cells.

[0198] FIG. 3C shows that cytokine receptor switching had little effect on effector memory markers on T cells without CD3 / CD28 costimulation.

[0199] Figure 3D shows that the IL7RA-cytokine receptor switch is conserved in the absence of CD3 / CD28 costimulation by CD4 + This shows that it increased central memory markers on T cells.

[0200] Example 2: BSA-FITC bound on high affinity plates efficiently stimulated chimeric antigen receptor (CAR)-T cells T cells containing nucleic acid encoding an anti-fluorescein CAR were stimulated with increasing doses of FITC (0, 0.1, 1, 10, and 100 μg / mL) on regular (regular) or high affinity (high affinity) plates coated with FITC-conjugated antibodies or antibody-FITC solution (free) in the presence or absence of CD3 / CD28 costimulation. + The expression of IL-2, IFN-γ and CD69 in T cells was assessed by flow cytometry.

[0201] Figure 5A shows that IL-2 expression was increased in CAR T cells in a FITC dose-dependent manner. High affinity plate-bound antibody-FITC stimulated CAR-T cells more effectively than regular plate-bound or solution antibody-FITC.

[0202] Figure 5B shows that IFN-γ expression was increased in CAR T cells in a FITC dose-dependent manner. High affinity plate-bound antibody-FITC stimulated CAR-T cells more effectively than regular plate-bound or solution antibody-FITC.

[0203] Figure 5C shows that CD69 expression was increased in CAR T cells in a FITC dose-dependent manner. High affinity plate-bound antibody-FITC stimulated CAR-T cells more effectively than regular plate-bound or solution antibody-FITC.

[0204] Example 3: Effect of the cytokine receptor switch of the present invention on NK cells Human natural killer (NK) cell line, NK92, expressing different combinations of cytokine receptor switches (IL2RB and IL2RG-cytokine receptor switch, or IL15RA-, IL2RB and IL2RG-cytokine receptor switch) was stimulated with increasing doses of FITC (0, 0,1, 1, 10, 100 μM) on FITC-conjugated BSA coated plates for up to 7 days. The fold increase in viable cells was assessed by cell proliferation assay. The activation marker CD69 on NK92 cells was assessed by flow cytometry. Figure 6A shows that the combination of IL15RA-, IL2RB and IL2RG-cytokine receptor switch of the present invention promoted cell proliferation of NK92 cells. These NK92 cells were a first cytokine switch comprising an IL-2RB signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RB transmembrane domain, and an IL-2RB intracellular domain; a second cytokine switch comprising an IL-2RG signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain; and A third cytokine switch comprising the IL-15RA signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-15RA transmembrane domain, and an IL-15RA intracellular domain. The present invention includes three nucleic acids encoding three cytokine receptor switches comprising:

[0205] NK92 cells expressing different combinations of cytokine receptor switches (IL7RA and IL2RG-cytokine receptor switch or IL2RB and IL2RG-cytokine receptor switch) were stimulated with increasing doses of FITC (0, 0,1,1,10,100,1000 μM) on FITC-conjugated BSA coated plates for up to 7 days. The activation marker CD69 on NK92 cells was assessed by flow cytometry. Figure 6B shows that the combination of the IL7RA and IL2RG-cytokine receptor switch of the present invention increased the expression of the activation marker CD69 on NK92 cells. These NK92 cells were a first cytokine switch comprising an IL-2RG signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-2RG transmembrane domain, and an IL-2RG intracellular domain; A second cytokine switch comprising an IL-7RA signal peptide, an anti-small molecule scFv, a CD8 hinge, an IL-7RA transmembrane domain, and an IL-7RA intracellular domain. The present invention comprises two nucleic acids encoding two cytokine receptor switches comprising:

[0206] All patent and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All such publications, including any specific portions thereof referred to, are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0207] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. It is thus to be understood that numerous modifications can be made to the illustrative embodiments and other arrangements can be devised without departing from the spirit and scope of the disclosure as defined by the appended claims.

Claims

1. Signal peptides and A single-chain antibody fragment (scFv) that specifically binds to synthetic, substantially non-immunogenic small molecules ("synthetic small molecules"), Hinged domain and, Transmembrane domain and The intracellular domain of the first cytokine receptor, which is native to or derived therefrom. Cytokine receptor switches, including

2. The signal peptide is a native type of the second cytokine receptor or derived therefrom. The first and second cytokine receptors are the same or different. The cytokine receptor switch according to claim 1.

3. The cytokine receptor switch according to claim 2, wherein the signal peptide, transmembrane domain, and / or intracellular domain are native forms of or derived from IL-2RA, IL-2RB, IL-2RG, IL-4RA, IL-7RA, IL-9R, IL-15RA, or IL-21R.

4. The cytokine receptor switch according to claim 3, wherein the signal peptide is selected from the group consisting of the following: a) A signal peptide of the native type of IL-2RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 1 or has the amino acid sequence of SEQ ID NO: 2; b) A signal peptide of the native form of IL-2RB, which is encoded by the nucleic acid sequence of SEQ ID NO: 3 or has the amino acid sequence of SEQ ID NO: 4; c) A signal peptide of the native form of IL-2RG, which is encoded by the nucleic acid sequence of SEQ ID NO: 5 or has the amino acid sequence of SEQ ID NO: 6; d) A signal peptide of the native form of IL-4RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 7 or has the amino acid sequence of SEQ ID NO: 8; e) A signal peptide of the native form of IL-7RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 9 or has the amino acid sequence of SEQ ID NO: 10; f) A native signal peptide of IL-9R, which is encoded by the nucleic acid sequence of SEQ ID NO: 11 or has the amino acid sequence of SEQ ID NO: 12; g) A signal peptide of the native form of IL-15RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 13 or has the amino acid sequence of SEQ ID NO: 14; and h) A signal peptide of the native form of IL-21R, which is encoded by the nucleic acid sequence of SEQ ID NO: 15 or has the amino acid sequence of SEQ ID NO:

16.

5. The cytokine receptor switch according to claim 1, wherein the scFv is selected from the group consisting of the following: a) scFv that binds to fluorescein and fluorescein derivatives, and which is encoded by the nucleic acid sequence of SEQ ID NO: 51 or has the amino acid sequence of SEQ ID NO: 52; b) scFv that binds to MPOB and is encoded by the nucleic acid sequence of SEQ ID NO: 53 or has the amino acid sequence of SEQ ID NO: 54; c) scFv that binds to AQ and is encoded by the nucleic acid sequence of SEQ ID NO: 55 or has the amino acid sequence of SEQ ID NO: 56; and d) An scFv that binds to DOTA and is encoded by the nucleic acid sequence of SEQ ID NO: 57 or has the amino acid sequence of SEQ ID NO:

58.

6. The cytokine receptor switch according to claim 1, wherein the hinge domain is derived from surface antigen classification 8 (CD8).

7. The cytokine receptor switch according to claim 3, wherein the transmembrane domain is selected from the group consisting of the following: a) A transmembrane domain derived from IL-2RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 17 or has the amino acid sequence of SEQ ID NO: 18; b) A transmembrane domain derived from IL-2RB, which is encoded by the nucleic acid sequence of SEQ ID NO: 19 or has the amino acid sequence of SEQ ID NO: 20; c) A transmembrane domain derived from IL-2RG, which is encoded by the nucleic acid sequence of SEQ ID NO: 21 or has the amino acid sequence of SEQ ID NO: 22; d) A transmembrane domain derived from IL-4RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 23 or has the amino acid sequence of SEQ ID NO: 24; e) A transmembrane domain derived from IL-7RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 25 or has the amino acid sequence of SEQ ID NO: 26; f) A transmembrane domain derived from IL-9R, which is encoded by the nucleic acid sequence of SEQ ID NO: 27 or has the amino acid sequence of SEQ ID NO: 28; g) A transmembrane domain derived from IL-15RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 29 or has the amino acid sequence of SEQ ID NO: 30; and h) A transmembrane domain derived from IL-21R, which is encoded by the nucleic acid sequence of SEQ ID NO: 31 or has the amino acid sequence of SEQ ID NO:

32.

8. The cytokine receptor switch according to claim 3, wherein the intracellular domain is selected from the group consisting of the following: a) Intracellular domains derived from IL-2RA that are encoded by the nucleic acid sequence of SEQ ID NO: 33 or have the amino acid sequence of SEQ ID NO: 34; b) Intracellular domains derived from IL-2RB, which are encoded by the nucleic acid sequence of SEQ ID NO: 35 or have the amino acid sequence of SEQ ID NO: 36; c) An intracellular domain derived from IL-2RG, which is encoded by the nucleic acid sequence of SEQ ID NO: 37 or has the amino acid sequence of SEQ ID NO: 38; d) Intracellular domains derived from IL-4RA, which are encoded by the nucleic acid sequence of SEQ ID NO: 39 or have the amino acid sequence of SEQ ID NO: 40; e) Intracellular domains derived from IL-7RA, which are encoded by the nucleic acid sequence of SEQ ID NO: 41 or have the amino acid sequence of SEQ ID NO: 42; f) Intracellular domains derived from IL-9R, which are encoded by the nucleic acid sequence of SEQ ID NO: 43 or have the amino acid sequence of SEQ ID NO: 44; g) an intracellular domain derived from IL-15RA, which is encoded by the nucleic acid sequence of SEQ ID NO: 45 or has the amino acid sequence of SEQ ID NO: 46; and h) An intracellular domain derived from IL-21R, which is encoded by the nucleic acid sequence of SEQ ID NO: 47 or has the amino acid sequence of SEQ ID NO:

48.

9. A nucleic acid encoding a cytokine receptor switch according to claim 1.

10. A composition comprising immune cells containing the nucleic acid described in claim 9.

11. The immune cells further comprise at least two of the nucleic acids, The two nucleic acids mentioned above, Different cytokine receptor switches, comprising different signal peptides, different transmembrane domains, and / or different intracellular domains. The composition according to claim 10, which codes for the composition.

12. The composition according to claim 11, wherein at least two of the cytokine receptor switches include different scFvs.

13. The composition according to claim 11, wherein the cytokine receptor switch contains the same scFv.

14. The composition according to claim 10, wherein the immune cells are T cells or NK cells.

15. The T cell is CD8 ＋ or CD4 ＋ The composition according to claim 14.

16. The composition according to claim 10, wherein the immune cells further comprise nucleic acids encoding chimeric antigen receptors (CARs) directed toward cell surface antigens.

17. The composition according to claim 14, wherein the immune cells are binary activated T cells encoding a chimeric antigen receptor (BAT-CAR), or binary activated NK cells containing nucleic acids encoding a chimeric antigen receptor.

18. An ex vivo method for stimulating immune cells containing nucleic acids according to claim 9, comprising contacting the immune cells with a synthetic small molecule in a concentration sufficient to promote proliferation.

19. The method according to claim 18, wherein the contact also promotes a change in the phenotype of the immune cells, which is selected from a memory phenotype, a cytotoxic phenotype, and a regulatory phenotype.

20. A composition for treating a disease or disorder, comprising immune cells containing the nucleic acid described in Claim 9, which is administered in combination with at least one synthetic small molecule conjugated to a carrier in order to stimulate the immune cells.

21. A composition for treating a disease or disorder, comprising immune cells stimulated by the method described in Claim 18 or 19.

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