Fluorescein-specific CARs display optimal T cell function against tumors labeled with fluorescein-conjugated ether phospholipids (FL-PLE)

Abstract

Embodiments described herein relate to recombinant chimeric antigen receptors (CARs) and compositions thereof that have specificity and affinity for fluorescein-containing ligands displayed on the surface of tumor cells. Additionally, provided herein are compositions comprising CARs that further comprise spacer arms, as well as methods for making and using such compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 627,147, filed February 6, 2018, entitled "Fluorescein-Specific CARs Exhibiting Optimal T Cell Function Against FL-PLE-Labeled Tumors," which is expressly incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which is provided as an approximately 42 kb file created on January 17, 2019, under the file name SCRI163WOSEQLIST. The information set forth in this electronic Sequence Listing is expressly incorporated herein by reference in its entirety.

[0003] Embodiments described herein relate to engineered chimeric antigen receptors (CARs) and compositions thereof that have specificity and affinity for fluorescein-containing ligands displayed on the surface of tumor cells. Accordingly, the present invention provides such compositions and methods for making and using the compositions. [Background technology]

[0004] Adoptive transfer of human T lymphocytes engineered by gene transfer to express chimeric antigen receptors (CARs) specific for surface molecules expressed on tumor cells has the potential to effectively treat cancer. Chimeric receptors are synthetic receptors that contain an extracellular ligand-binding domain, most commonly a single-chain variable fragment (scFv) of a monoclonal antibody linked to an intracellular signaling domain. The intracellular signaling domain is most commonly CD3ζ alone or in combination with one or more costimulatory domains. Much of the research into chimeric antigen receptor design has focused on finding scFvs and other ligand-binding factors that target malignant cells without causing significant toxicity to vital normal tissues, and on determining the optimal configuration of the intracellular signaling module that can activate T cell effector functions. Therefore, there remains a need for CAR T cell-based therapies that are selective for specific targets and minimize adverse side effects. Summary of the Invention [Means for solving the problem]

[0005] In a first embodiment, a complex comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the CAR or TCR is configured to bind to a lipid comprising a targeting moiety, and binding of the CAR to the lipid is via an interaction with the targeting moiety; The CAR or TCR comprises a spacer domain that is 1 to 22 amino acids in length, 23 to 50 amino acids in length, 51 to 100 amino acids in length, 100 to 150 amino acids in length, or 151 to 250 amino acids in length, or a spacer domain with a length within a range defined by any two of these lengths; A composite is provided. In some embodiments, the CAR or TCR comprises a sequence set forth in any of SEQ ID NOs: 1-6. In some embodiments, the spacer domain is an IgG4 hinge linked to a CH2 domain linked to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the spacer domain is an IgG4 hinge linked to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the spacer domain consists solely of an IgG4 hinge. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 9. Some embodiments include a nucleic acid encoding any of SEQ ID NOs: 1-9, which may be incorporated into a vector and / or introduced into a cell (e.g., a T cell). In some embodiments, the spacer is 229 amino acids in length. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group is a choline, a phosphatidylcholine, a sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, HoIn some embodiments, the polar head group comprises phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the hydrophobic group is a fatty acid, such as a fatty chain. In some embodiments, the fatty acid is a saturated or unsaturated fatty acid. In some embodiments, the hydrophobic group comprises an alkyl, alkenyl, or alkynyl group. In some embodiments, the hydrophobic group comprises a steroid or a terpenoid lipid, such as cholesterol. In some embodiments, the hydrophobic group comprises an ether bond between the polar head group and the fatty chain. In some embodiments, the sugar residue is glycerol. In some embodiments, the hydrophobic group comprises an alkyl carbon chain, the alkyl carbon chain comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, or a number of carbon atoms within a range defined by any two of these numbers. In some embodiments, the alkyl carbon chain comprises 18 carbon atoms. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the targeting moiety is biotin, digoxigenin, dinitrophenol, or fluorescein. In some embodiments, the spacer comprises a polyethylene glycol (PEG) spacer, a spacer consisting of (two) haptens, or an alkane chain. In some embodiments, the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules, or a number of PEG molecules within a range defined by any two of these numbers. In some embodiments, the CAR or TCR is expressed in a cell or T cell. In some embodiments, the CAR or TCR is expressed on the surface of a cell or T cell. In some embodiments, the cell is a progenitor T cell. In some embodiments, the progenitor T cell is a hematopoietic stem cell.In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the lipid is inserted into the lipid bilayer of the target cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the target cell is present in a tumor microenvironment.

[0006] In a second aspect, a cell comprising a complex according to any one of the embodiments of the present invention, comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the CAR or TCR binds or is configured to bind to a lipid comprising a targeting moiety, and a cell comprising the CAR binds to the targeting moiety of the lipid, and the CAR or TCR comprises a spacer domain. Provide the cells. In some embodiments, the complex comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the CAR or TCR binds or is configured to bind to a lipid comprising a targeting moiety, wherein the binding of the CAR to the lipid is via an interaction with the targeting moiety, or the CAR is configured to bind to the lipid via an interaction with the targeting moiety; The CAR or TCR comprises a spacer domain that is 1 to 22 amino acids in length, 23 to 50 amino acids in length, 51 to 100 amino acids in length, 100 to 150 amino acids in length, or 151 to 250 amino acids in length, or a spacer domain of a length within a range defined by any two of these lengths. In some embodiments, the CAR or TCR comprises a sequence set forth in any of SEQ ID NOs: 1-6. In some embodiments, the spacer domain is an IgG4 hinge linked to a CH2 domain linked to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the spacer domain is an IgG4 hinge linked to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the spacer domain consists solely of an IgG4 hinge. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the cell comprises a nucleic acid encoding any of SEQ ID NOs: 1-9. In some embodiments, the spacer is 229 amino acids in length. In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group is selected from the group consisting of choline, phosphatidylcholine, sphingomyelin, a phosphoethanolamine group, an oligosaccharide residue, a sugar residue, HoIn some embodiments, the polar head group comprises phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the hydrophobic group is a fatty acid, such as a fatty chain. In some embodiments, the fatty acid is a saturated or unsaturated fatty acid. In some embodiments, the hydrophobic group comprises an alkyl, alkenyl, or alkynyl group. In some embodiments, the hydrophobic group comprises a steroid or a terpenoid lipid, such as cholesterol. In some embodiments, the hydrophobic group comprises an ether bond between the polar head group and the fatty chain. In some embodiments, the sugar residue is glycerol or a sugar alcohol. In some embodiments, the hydrophobic group comprises an alkyl carbon chain, the alkyl carbon chain comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, or a number of carbon atoms within a range defined by any two of these numbers. In some embodiments, the alkyl carbon chain comprises 18 carbon atoms. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the targeting moiety is biotin, digoxigenin, dinitrophenol, or fluorescein. In some embodiments, the spacer comprises a polyethylene glycol (PEG) spacer, a spacer consisting of (two) haptens, or an alkane chain. In some embodiments, the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules, or a number of PEG molecules within a range defined by any two of these numbers. In some embodiments, the CAR or TCR is expressed in a cell or T cell. In some embodiments, the CAR or TCR is expressed on the surface of a cell or T cell. In some embodiments, the cell is a progenitor T cell. In some embodiments, the progenitor T cell is a hematopoietic stem cell.In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the lipid is inserted into the lipid bilayer of the target cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the target cell is present in a tumor microenvironment. In some embodiments, the cell is a progenitor T cell. In some embodiments, the progenitor T cell is a hematopoietic stem cell. In some embodiments, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the lipid is inserted into the lipid bilayer of the target cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the target cell is present in a tumor microenvironment.

[0007] In a third aspect, there is provided a method of treating, alleviating, or inhibiting cancer in a subject, comprising: a) introducing, providing or administering to a subject a composition comprising a lipid comprising a targeting moiety bound to a masking moiety; b) introducing, providing or administering to the subject a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) specific for the targeting moiety from which the masking moiety has been removed and comprising a spacer domain; c) removing the masking moiety from the targeting moiety, thereby binding the targeting moiety to the CAR present on the cell; d) optionally, after steps a) to c), measuring or assessing binding of the cells containing the CAR to the lipid; e) optionally, after steps a) to d), measuring or evaluating the treatment, alleviation or inhibition of cancer; and / or f) before steps a) to c), optionally, a step of identifying a subject in need of cancer treatment The present invention provides a method comprising: In some embodiments, the complex or the cells are provided to the subject simultaneously with administration of the composition, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, or 48 hours before administration of the composition, or within a range of time defined by any two of these numbers, prior to administration of the composition, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, or 48 hours after administration of the composition, or after a period of time within a range defined by any two of these numbers has elapsed since administration of the composition. In some embodiments, the cells are provided to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, or 48 hours prior to administration of the composition, or within a range defined by any two of these numbers, prior to administration of the composition. In some embodiments, the cells are provided to the subject within seconds or minutes, e.g., before the end of an hour, after providing the composition to the subject. In some embodiments, a booster dose of the cells and / or the composition is provided to the subject. In some embodiments, another cancer therapy is provided, such as a small molecule (e.g., a compound), antibody therapy (e.g., a humanized monoclonal antibody conjugated to a radionuclide, toxin, or drug, or an unconjugated humanized monoclonal antibody), surgery, and / or radiation therapy. In some embodiments, the cancer is a solid tumor such as colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, or liver cancer; or a non-solid tumor such as leukemia or multiple myeloma.In some embodiments, the spacer domain is an IgG4 hinge linked to a CH2 domain linked to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the spacer domain is an IgG4 hinge linked to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the spacer domain consists solely of an IgG4 hinge. In some such embodiments, the spacer domain comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, binding of the targeting moiety to a CAR present on the cell induces production of at least one cytokine. In some embodiments, the at least one cytokine comprises IL-2, TNF-α, and / or INF-α.

[0008] In a fourth aspect, a kit is provided that includes pharmaceutical-grade fluorescein-conjugated ether phospholipid (FL-PLE). In some embodiments, the pharmaceutical-grade FL-PLE includes a hapten. In some embodiments, the pharmaceutical-grade FL-PLE includes fluorescein (e.g., FITC). [Brief explanation of the drawings]

[0009] [Figure 1A-1B]Schematic diagrams showing various CAR T cells with different spacers. Figure 1A shows (i) an example of a second-generation CAR. The antigen-recognition moiety (the top part of the molecule shown in the figure) is displayed at a desired distance from the cell surface via a short spacer domain. This spacer is linked to a transmembrane domain, which is linked to two signaling domains. (ii) In CARs with a medium-length spacer, an additional domain is added to the short spacer. (iii) In CARs with a long spacer, an additional domain is added to the medium-length spacer. Figure 1B shows an example of various spacers exposed on a CAR T cell. In some embodiments, the long spacer may include the sequence set forth in SEQ ID NO: 7, the medium-length spacer may include the sequence set forth in SEQ ID NO: 8, and the short spacer may include the sequence set forth in SEQ ID NO: 9.

[0010] [Figure 2]Schematic diagrams showing examples of spacer length and spacer orientation relative to the surface of tumor cells. Tumor cells are shown surface-labeled with a tumor-specific CAR T cell targeting agent (CTCT). The tumor surface contains proteins and other cellular debris that protrude from the tumor surface, which can affect the ability of a CTCT-specific CAR to recognize the tumor-specific CAR T cell targeting agent (CTCT). Panels A-C show an example of an embodiment of a PLE-CTCT embedded in a lipid membrane, and Panels D-E show an example of an embodiment of a tumor-specific CAR T cell targeting agent (CTCT) bound to a surface protein. In Panel A, a CAR with a long spacer reaches and is activated by the PLE-CTCT. In Panel B, a CAR with a short spacer cannot reach and is not activated by the tumor-specific CAR T cell targeting agent (CTCT). In Panel C, a CAR with a long spacer, in which the recognition domain is oriented in a different direction, cannot recognize the tumor-specific CAR T cell targeting agent (CTCT). Panels D and E show that both the CAR with the long spacer shown in Panel C and the CAR with the short spacer shown in Panel B can recognize and be activated by a tumor-directed CAR T cell targeting agent (CTCT) bound to a surface protein.

[0011] [Figure 3] Figure 1 shows an example schematic diagram demonstrating that CTCT-specific CAR T cells recognize and are activated only by unmasked PLE-CTCT. A prodrug form of PLE-CTCT is incorporated into the tumor. In this example embodiment, the prodrug sterically hampers the CTCT-specific CAR T cells, inhibiting their recognition of Pro-PLE-CTCT. Once the prodrug moiety of Pro-PLE-CTCT is cleaved by the tumor microenvironment, the CTCT-specific CAR can recognize and be activated by the unmasked PLE-CTCT.

[0012] [Figure 4] Figure 4A shows an example of an embodiment of a phospholipid conjugated to fluorescein as a hapten (PLE-CTCT). The structure of an ether phospholipid (PLE) linked to fluorescein as a hapten (FL-PLE) is shown. (i) FL (fluorescein): the target of CAR T cells. (ii) Polyethylene glycol (PEG): a spacer to separate the target from the cell surface at an ideal distance. (iii) and (iv) PLE: (iii) is a polar head group, and (iv) is a hydrophobic tail that integrates into or anchors to the cell membrane. Figure 4B shows an example of a prodrug form of FL-PLE. A pro-drug moiety is attached to the hapten (fluorescein) via a cleavable bond indicated by the arrow. The masked FL-PLE is substantially non-fluorescent. This bond can be cleaved in the presence of reactive oxygen species (ROS), such as those found in the tumor microenvironment. Once unmasked, the FL-PLE structure emerges, providing a fluorescent signal and allowing binding to an anti-FL CAR.

[0013] [Figure 5] The top panel shows examples of various anti-FL scFvs selected to generate the anti-FL CAR library. The bottom panel shows examples of CAR structures with long (L), medium (M), or short (S) spacers. These structures contain a leader sequence, scFv, hinge / spacer, CD28tm domain, 41BB domain, CD3ζ, T2A sequence, and EGFTt domain. A total of 18 different plasmids were used to generate various anti-FL CAR T cells.

[0014] [Figure 6]FACS analysis of CD8+ T cells containing anti-FL CARs is shown. Panel (a) shows staining of the EGFRt selection marker in T cells containing anti-FL(FITC-E2) CARs containing SEQ ID NO:5 (96% positive), and panel (b) shows staining of the EGFRt selection marker in T cells containing anti-FL(4M5.3) CARs containing SEQ ID NO:2 (80% positive). Panels (c) and (d) show the results of incubating each T cell with mouse CD19-FITC antibody and then staining with anti-mouse Fc-Alexa647 antibody to detect bound FITC antibody. Panel (c) shows T cells containing anti-FL(FITC-E2) CARs (97% positive), and panel (d) shows T cells containing anti-FL(4M5.3) CARs (79% positive).

[0015] [Figure 7A] FACS analysis of cells treated with FL-PLE is shown.

[0016] [Figure 7B] Specific lysis of K562 cells in the presence or absence of FL-PLE and in the presence or absence of CD8+ T cells containing anti-FL CAR is shown.

[0017] [Figure 7C] Cytokine production by K562 cells in the presence or absence of FL-PLE and in the presence or absence of CD8+ T cells containing anti-FL CAR is shown.

[0018] [Figure 8A] 1 shows FACS analysis of various cell lines treated with FL-PLE.

[0019] [Figure 8B] Specific lysis of cells treated with FL-PLE followed by contact with CD8+ T cells containing an anti-FL(FITC-E2) CAR containing a long spacer is shown.

[0020] [Figure 9]FACS analysis of staining for the surface marker EGFRt of CD8+ T cells containing various anti-FL CARs is shown.

[0021] [Figure 10] FACS analysis of CD4+ T cells stained for the surface marker EGFRt with various anti-FL CARs is shown.

[0022] [Figure 11] FACS analysis of FL-PLE labeled cells is shown.

[0023] [Figure 12] Specific lysis of target cells incubated with FL-PLE followed by contact with CD8+ T cells containing anti-FL CAR is shown.

[0024] [Figure 13] Cytokine production is shown when FL-PLE is added, incubated, and then contacted with CD4+ T cells.

[0025] [Figure 14A] Specific lysis of target cells incubated with FL-PLE followed by contact with CD8+ T cells containing anti-FL CAR is shown.

[0026] [Figure 14B] Cytokine production is shown when FL-PLE is added, incubated, and then contacted with CD4+ T cells.

[0027] [Figure 15A] FACS analysis of various cells with different anti-FL CARs is shown.

[0028] [Figure 15B] FACS analysis of various cells treated with FL-PLE is shown.

[0029] [Figure 15C]1 shows a series of graphs demonstrating the specific lysis of various tumor cells contacted with various anti-FL CARs in the presence or absence of FL-PLE.

[0030] [Figure 15D] 1 shows a series of graphs depicting cytokine production by different anti-FL CAR T cells when contacted with various tumor cells in the presence or absence of FL-PLE.

[0031] [Figure 15E] 1 shows a series of graphs depicting cytokine production by different anti-FL CAR T cells when contacted with various tumor cells in the presence or absence of FL-PLE.

[0032] [Figure 16] The upper left panel shows an image of a glioma xenograft in a subject evaluated 48 hours after intravenous administration of FL-PLE. The lower left panel shows an image of the contralateral hemisphere of the same subject. Furthermore, the orthotopic glioma xenograft and the contralateral hemisphere of the same subject were stained with anti-fluorescein antibody and evaluated. The amount of FL-PLE retained in the orthotopic glioma xenograft and the contralateral hemisphere of the same subject was quantified over time (right panel).

[0033] [Figure 17] 1 shows a graph demonstrating the retention of FL-PLE in various in vivo tumors.

[0034] [Figure 18A] FACS analysis of cells treated with FL-PLE or ProFL-PLE is shown.

[0035] [Figure 18B] Specific lysis of cells treated with FL-PLE or ProFL-PLE followed by contact with CD8+ T cells containing anti-FL CAR is shown.

[0036]

[0037] [Figure 18C] 1 shows a series of graphs showing cytokine production from T cells in the presence or absence of masked ProFL-PLE, unmasked ProFL-PLE, or FL-PLE, and in the presence or absence of CD4+ T cells containing anti-FL(FITCE2) CAR with a long spacer.

[0038] [Figure 19A] FACS analysis of cell-bound FL-PLE or ProFL-PLE is shown.

[0039] [Figure 19B] A series of graphs showing cytokine production from T cells in the presence or absence of ProFL-PLE or FL-PLE, and in the presence or absence of CD4+ T cells containing an anti-FL(FITCE2) CAR with a long spacer are shown.

[0040] [Figure 19C] FACS analysis of cells treated with FL-PLE or ProFL-PLE is shown.

[0041] [Figure 20] 1 shows a graph depicting the relative survival rate of mice bearing neuroblastoma (Be2) tumors treated with ProFL-PLE, FL-PLE, or a control, and administered T cells containing an anti-FL (FITCE2) CAR with a long spacer and comprising SEQ ID NO: 5, or a control.

[0042] [Figure 21A] Graphs showing the mean time to tumor progression (upper panel) and survival rate (lower panel) in mice administered T cells containing an anti-FL(FITCE2) CAR with a long spacer after intratumoral injection of ProFL-PLE.

[0043] [Figure 21B] Graphs showing the mean time to tumor progression (upper panel) and survival rate (lower panel) in mice administered T cells containing anti-FL(FITCE2) CAR with a long spacer after intravenous injection of ProFL-PLE. DETAILED DESCRIPTION OF THE INVENTION

[0044] Described herein are engineered chimeric antigen receptors (CARs) that exhibit specificity and selected or engineered affinity for fluorescein molecules when presented on the surface of tumor cells loaded with exogenous fluorescein-conjugated ether phospholipids (FL-PLE), thereby eliciting targeted anti-tumor T cell reactivity. In some embodiments, FL-PLE is a synthetic molecular structure designed to be incorporated into tumor cell membranes, thereby presenting the fluorescein in the FL-PLE molecule adjacent to the outer layer of the cell membrane in the extracellular space. In some embodiments, the FL-PLE structure possesses therapeutically important properties. In some embodiments, CARs that target the fluorescein (FL) moiety of FL-PLE are provided, and methods for producing and using the CARs are also contemplated. These CARs can be constitutively expressed or can be placed under regulated conditions.

[0045] Although adoptive transfer of transgene-modified T cells has been successful under selected conditions (e.g., CD19 B-cell malignancies), the lack of a single target antigen found in all forms of cancer but not in normal, healthy cells makes it difficult to generalize T cell adoptive transfer therapy for use in other cancer types. Development of CAR T cell therapy for the treatment of each type of human cancer involves the tedious task of identifying and characterizing thousands of antigens (e.g., CAR targets), hindering its development. Previous research has developed fluorescein-conjugated ether phospholipids (FL-PLEs), which can be incorporated into any tumor and present a single synthetic target molecule for recognition by CAR T cells. The development of FL-PLEs eliminates the need to test thousands of CARs and allows for the use of a single fluorescein-specific CAR.

[0046] The present invention discloses the identification, generation, and engineering of CAR T cells suitable for conjugation of fluorescein-conjugated ether phospholipids (FL-PLEs) to a fluorescein (FL) moiety. In some embodiments, the FL moiety may be masked and unmasked so that the FL moiety can be activated only when CAR T cells are administered. In some embodiments, the FL-specific CAR may recognize only an unmasked tumor-directed CAR T cell targeting agent (CTCT), but not a masked FL.

[0047] Some embodiments of the methods and compositions provided herein may include aspects disclosed in WO2018 / 148224, entitled "PHOSPHOLIPID ETHER (PLE) CAR T CELL TUMOR TARGETING (CTCT) AGENTS," which is expressly incorporated herein by reference in its entirety.

[0048] Definition of Terms In the following description, with reference to the present specification, each term has its common and ordinary meaning. A person skilled in the art would understand each term as it is used throughout this specification.

[0049] As used herein, "a" or "an" may mean one or more than one.

[0050] As used herein, "about" indicates that the particular value includes the variation of error inherent in the method employed to determine the value or experimental variation.

[0051] As used herein, the term "chimeric receptor" refers to a synthetically engineered receptor containing a ligand-binding domain of an antibody or other protein sequence that binds to a molecule associated with the disease or disorder, linked via a spacer domain to one or more intracellular signaling domains (e.g., costimulatory domains) derived from a T cell receptor or other receptor. Chimeric receptors can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors (CARs). The specificity of a monoclonal antibody or its binding fragment can be transferred to T cells by transferring the coding sequence of the chimeric receptor into T cells using a vector, such as a retroviral or lentiviral vector. CARs are genetically engineered T cell receptors designed to confer targeting to target cells expressing a specific cell surface antigen. T cells are first obtained from a subject using a method called adoptive cell transfer, and then genetically engineered to express a receptor capable of expressing specificity for the antigen. These T cells are then reintroduced into the patient, where they are able to recognize and target the antigen. Such CARs are engineered receptors that can transfer selected specificities into cells expressing immune receptors. Some researchers understand chimeric antigen receptors (CARs) to include an antibody or antibody fragment, a spacer, a signaling domain, and a transmembrane domain. The CARs described herein are engineered with various components or domains (e.g., epitope-binding regions (e.g., antibody fragments, scFvs, or portions thereof), spacers, transmembrane domains, and / or signaling domains), which have produced surprising effects, and when focusing on each element, the components of the CAR can often be clearly distinguished as separate entities throughout the disclosure herein.

[0052] The term "CAR T cell targeting agent (CTCT)" has its common and ordinary meaning in the context of this specification, and can be described, for example, as a composition capable of incorporating into the cell membrane of a target cell. In embodiments of the present invention, the CTCT comprises a lipid, which comprises a targeting moiety and a masking moiety. The masking moiety may be removed, for example, by low pH or the presence of ROS species, or by being present in the tumor microenvironment. In some embodiments, the masking moiety inhibits specific binding of the CAR to the targeting moiety. The targeting moiety may also be recognized and bound by a chimeric antigen receptor specific for the targeting moiety. In some embodiments of the present invention, the masking moiety is removed by a pH of 4, 5, 6, or 6.5, or a pH within a range defined by any two of these values.

[0053] A "T cell receptor" or "TCR" is a molecule present on the surface of a T lymphocyte or T cell, which is responsible for recognizing antigen fragments bound to major histocompatibility complex molecules.

[0054] As used herein, the term "targeting moiety" refers to a specific group or site on a specific molecule or chemical that serves as a binding target for another chemical or protein of interest. In some embodiments, a complex is provided that includes a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR is configured to bind to a lipid that includes a targeting moiety, and the binding of the CAR to the lipid is mediated by interaction with the targeting moiety. In some embodiments, the targeting moiety is biotin, digoxigenin, dinitrophenol, or fluorescein.

[0055] A "single-chain variable fragment (scFv)" is a fusion protein that may contain an immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) linked by a short linker peptide consisting of 10 to 25 amino acids. The short linker peptide may contain 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, or a number of amino acids within a range defined by any two of these numbers. The linker is typically glycine-rich for flexibility and serine- or threonine-rich for solubility, and can link the N-terminus of VH to the C-terminus of VL, or the C-terminus of VH to the N-terminus of VL. Despite the removal of the constant regions and the introduction of a linker, scFvs retain the specificity of the original immunoglobulin. The scFv can be specific for an antigen. As used herein, "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunocompetent cells, or both. Antigens can be recombinantly produced, synthesized, and manufactured, or obtained from biological samples. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids (e.g., blood, plasma, or ascites). In some embodiments described herein, compositions are provided that include cells produced by any of the methods according to the embodiments described herein. In some embodiments, the cells comprise a chimeric antigen receptor comprising an scFv specific for a particular antigen.

[0056] Some embodiments provided herein relate to an scFv (SEQ ID NO: 1) referred to herein as anti-FL(FITCE2 TyrH133Ala) (also referred to as anti-FL(FITC-E2 Mut2) or anti-FL(Mut2)), which can be incorporated into a CAR in accordance with the present disclosure and has the amino acid sequence SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGGGGGSGGGGSGGGGSQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFASYMDVWGQGTLVTVS.

[0057] Some of the embodiments provided herein relate to an scFv (SEQ ID NO: 2) referred to herein as anti-FL (4M5.3), which can be incorporated into a CAR in accordance with the present disclosure and has the amino acid sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVS.

[0058] Some of the embodiments provided herein relate to an scFv (SEQ ID NO: 3) referred to herein as anti-FL(4420), which can be incorporated into a CAR in accordance with the present disclosure and has the amino acid sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSQGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIGGGGSGGGGSGGGGSEVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS.

[0059] Some of the embodiments provided herein relate to an scFv (SEQ ID NO: 4) referred to herein as anti-FL (4D5Flu), which can be incorporated into a CAR in accordance with the present disclosure and has the amino acid sequence: DYKDIQMTQSPSSLSASVGDRVTITCRASQSLVHSQGNTYLRWYQQKPGKAPKVLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTHVPWTFGQGTKVELKRAGGGGSGGGGSGGGGSSGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMNWVRQAPGKGLEWVAQIRNKPYNYETYYADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCTGSYYGMDYWGQGTLVTVSS.

[0060] Some of the embodiments provided herein relate to an scFv (SEQ ID NO: 5) referred to herein as anti-FL (FITCE2), which can be incorporated into a CAR in accordance with the present disclosure and has the amino acid sequence SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGGGGGSGGGGSGGGGSQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGHFYSYMDVWGQGTLVTVS.

[0061] Some of the embodiments provided herein relate to an scFv (SEQ ID NO: 6) referred to herein as anti-FL (FITCE2 HisH131Ala), which can be incorporated into a CAR in accordance with the present disclosure and has the amino acid sequence SVLTQPSSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGGGGGSGGGGSGGGGSQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYWGAFYSYMDVWGQGTLVTVS.

[0062] An "antigen-specific binding domain" may comprise a protein or protein domain capable of specifically binding to an epitope on a protein with low or high binding affinity (fM to mM avidity). In some embodiments, the fusion protein comprises a protein or portion thereof capable of modulating an immune response. In some embodiments, the protein comprises an antigen-specific binding domain.

[0063] As used herein, "T cells" or "T lymphocytes" may be derived from any mammalian species, such as monkeys, dogs, humans, etc., and preferably from primates. In some embodiments, the T cells are allogeneic (from the same species but from a different donor) to the recipient subject. In some embodiments, the T cells are autologous (donor and recipient are the same). In some embodiments, the T cells are syngeneic (donor and recipient are different but identical twins).

[0064] As used herein, the term "combination therapy" refers to the use of two or more drugs or therapies. Combination therapy can also refer to the use of multiple therapies, for example, to treat a single disease, often involving the use of multiple pharmaceutical preparations. Combination therapy also includes the administration of two or more active ingredients by formulating and administering separate drugs. In some embodiments, a combination therapy is provided that includes administering genetically modified immune cells to modify the tumor microenvironment. In some embodiments, the combination therapy includes administering genetically modified immune cells to modulate the suppression of immune responses in the tumor microenvironment. In some embodiments, the combination therapy includes administering genetically modified immune cells to minimize the proliferation of tumor cells and immunosuppressive cells to a subject (e.g., a human) in need thereof. In some embodiments, the combination therapy includes administering genetically modified immune cells to improve the efficacy of anti-cancer, anti-infective, anti-bacterial, anti-viral, or anti-tumor therapy to a subject (e.g., a human) in need thereof. In some embodiments, the combination therapy further includes administering an inhibitor. In some embodiments, the inhibitor is not an enzyme inhibitor. In some embodiments, the inhibitor is an enzyme inhibitor. In some embodiments, the combination therapy comprises administering a therapeutic dose of an inhibitor or an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof may be humanized. In some embodiments, the combination therapy further comprises administering CAR-expressing T cells to a subject (e.g., a human) in need thereof.

[0065] A "chemotherapeutic agent" is a class of anti-cancer medications that may be used, including chemicals (e.g., anti-cancer drugs (chemotherapeutics)) that can be administered as part of a standardized chemotherapy regimen. Chemotherapeutics can be administered with the intent of cure, prolongation of survival, or relief of symptoms (palliative chemotherapy). Chemotherapy may also include hormonal therapy and targeted therapy, which are major categories of medical oncology (cancer chemotherapy). These chemotherapies are often combined with other cancer therapies, such as radiation therapy, surgery, and / or hyperthermia. Surgery is known to cause cancer spread in some cases. In some embodiments, genetically modified immune cells are administered to the tumor site before or after the surgical procedure.

[0066] Chemotherapy involves the administration of chemotherapeutic agents, and one type of agent may be used in a single chemotherapy session (single-agent chemotherapy) or multiple agents may be used simultaneously (combination chemotherapy or polychemotherapy). Chemotherapy combined with radiation therapy is called chemoradiotherapy. Chemotherapy using agents that are converted into cytotoxic substances upon exposure to light is called photochemotherapy or photodynamic therapy. In some embodiments of the methods comprising administering genetically modified immune cells described herein, the methods can further include administering the genetically modified immune cells or genetically modified macrophages (GEMs) of the present invention to a subject with cancer, followed by administering photochemotherapy or photodynamic therapy to the subject.

[0067] Chemotherapeutic agents include, but are not limited to, antibody-drug conjugates (e.g., antibodies attached to a drug via a linker), nanoparticles (which may be, for example, nanoparticles of 1-1000 nm in size, which improve tumor selectivity and aid in the delivery of low-solubility drugs), electrochemotherapy, alkylating agents, antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, or thioguanine), antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators, or checkpoint inhibitors (e.g., checkpoint kinases CHK1 or CHK2). In some embodiments of the methods described herein, the genetically modified immune cells, or compositions comprising the genetically modified immune cells, are administered in combination with one or more anti-cancer agents, such as one or more of the compounds or therapies described above. In some embodiments, the one or more anti-cancer agents co-administered or administered in combination with the genetically modified immune cells of the invention include antibody-drug conjugates, nanoparticles, electrochemotherapy, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalators, or checkpoint inhibitors. In some embodiments, the antimetabolites include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cladribine, clofarabine, cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, pemetrexed (Alimta®), pentostatin, and thioguanine.

[0068] As used herein, a "subject" or "patient" refers to any living organism to which an embodiment described herein may be used or administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. A subject or patient includes, for example, an animal. In some embodiments, the subject is a mouse, rat, rabbit, non-human primate, or human. In some embodiments, the subject is a cow, sheep, pig, horse, dog, cat, primate, or human.

[0069] Some embodiments relate to tumor-directed CAR T cell targeting agents (CTCTs). Some embodiments provided herein relate to ether phospholipids (PLEs) linked to fluorescein (FL-PLEs) as a hapten. As used herein, "fluorescein" is a synthetic organic compound soluble in water and alcohol. Fluorescein is widely used as a fluorescent tracer in a variety of applications. In embodiments of the present invention, fluorescein is a targeting moiety on a lipid that is specifically recognized by a chimeric antigen receptor designed and / or selected to have the ability to bind to or interact with fluorescein. In some embodiments, the lipid is an ether phospholipid.

[0070] As used herein, a "lipid" is a type of organic compound containing a carbon chain, a fatty acid, or a fatty acid derivative. It is generally insoluble in water but can be miscible or mixed in a hydrophobic or organic solvent. Examples of lipids include, but are not limited to, fats, waxes, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, sphingolipids, cerebrosides, ceramides, or phospholipids. As used herein, amphipathic lipids are described, which may have a polar head group and a hydrophobic moiety or portion thereof. As used herein, a "hydrophobic group" or portion thereof refers to a molecule or portion of a molecule that tends to repel water and is non-polar. The hydrophobic portion may include an alkane, oil, or fat. Lipids may also include, but are not limited to, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some embodiments, a complex comprising a lipid is provided. In some embodiments, the lipid comprises a polar head group and a hydrophobic moiety. In some embodiments, the hydrophobic moiety is a hydrophobic carbon chain tail. In some embodiments, the hydrophobic carbon chain tail is a saturated or unsaturated carbon chain tail. In some embodiments, the hydrophobic carbon chain tail contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms, or a number of carbon atoms within a range defined by any of these numbers. In some embodiments, the hydrophobic moiety is a steroid or cholesterol. In some embodiments, the lipid comprises a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, or a polyketide. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the lipid comprises a branched alkyl tail.

[0071] In some embodiments, the lipid may be a sphingolipid. Sphingolipids may contain a backbone consisting of a sphingoid base, such as a series of aliphatic amino alcohols, including sphingosine. A sphingolipid in which the R group consists solely of hydrogen atoms is ceramide. Other common R groups include phosphocholine (when the R group is phosphocholine, sphingomyelin is obtained) and various sugar monomers or sugar dimers (when the R group is a sugar monomer, cerebrosides are obtained, and when the R group is a sugar dimer, globosides are obtained). Cerebrosides and globosides are known collectively as glycosphingolipids. In some embodiments, the lipid is a glycosphingolipid.

[0072] According to the present specification, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the hydrophobic group comprises a fatty acid, such as a fatty chain. The fatty acid may be saturated or unsaturated. In some embodiments, the hydrophobic group comprises an alkyl, alkenyl, or alkynyl group. In some embodiments, the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol. In some embodiments, the hydrophobic group comprises an ether linkage between the polar head group and the fatty chain. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the polar head group is selected from the group consisting of choline, phosphatidylcholine, sphingomyelin, phosphoethanolamine, oligosaccharide residues, sugar residues, Ho In some embodiments, the sugar is glycerol or a sugar alcohol.

[0073] In some embodiments, the lipid is a short-chain alkyl phospholipid.

[0074] In some embodiments, the lipid comprises a synthetic alkylphospholipid structure, such as edelfosine, perifosine, or erucylphosphocholine. In some embodiments, the lipid is lysophosphatidylcholine, edelfosine, erucylphosphocholine, D-21805, or perifosine. Such lipids have been reported, for example, by van der Lui et al. ("A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells," Mol Cancer Ther 2007; 6(8), 2007, which is expressly incorporated herein by reference in its entirety). In some embodiments of the lipids described herein, the choline in the polar head group can be replaced with a piperidine moiety. In some embodiments, the lipid is an anticancer alkylphospholipid. Anticancer phospholipids have also been reported by van der Lui et al. (“A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells” Mol Cancer Ther 2007; 6(8), 2007, which is expressly incorporated herein by reference in its entirety.

[0075] In some embodiments, the lipids provided herein are structurally related synthetic antitumor agents characterized by their ability to interact with cell membranes. These synthetic lipids are alkylphospholipids, as described, for example, by van Blitterswijk et al. ("Anticancer mechanisms and clinical application of alkylphopholipids," Biochimica et Biophysica Acta 1831 (2013) 663-674, which is incorporated herein by reference in its entirety). Such synthetic alkylphospholipids include, but are not limited to, edelfosine, miltefosine, perifosine, erucylphosphocholine, or erufosine. In some embodiments, the lipid is edelfosine, miltefosine, perifosine, erucylphosphocholine, or erufosine. In some embodiments, the lipid is a stable analog of lysophosphatidylcholine. In some embodiments, the lipid is a thioether-linked variant of edelfosine or 1-hexadecylthio-2-methoxymethyl-rac-glycero-3-phosphocholine, hi some embodiments, the lipid is LysoPC, edelfosine, irmofosine, miltefosine, perifosine, erucylphosphocholine, or erufosine.

[0076] As used herein, a "polar head group" refers to the hydrophilic group of a lipid (e.g., a phospholipid). As used herein, a "phospholipid" is a special type of lipid that has amphiphilic properties and can form lipid bilayers. A phospholipid molecule comprises at least one hydrophobic fatty acid "tail" and a hydrophilic "head" or "polar head group." In embodiments herein, a phospholipid or ether phospholipid comprises a polar head group. In some embodiments, the polar head group comprises a phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the lipid comprises a targeting moiety, and a CAR binds to or is configured to bind to the lipid through interaction with the targeting moiety. In some embodiments, the lipid comprises a polar head group (e.g., a polar head group comprising an aromatic ring) and an alkyl carbon chain. In embodiments of the present invention, a conjugate comprising one or more of the above lipids is provided. In some embodiments, the lipid comprises a polar head group. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the ether phospholipid comprises a targeting moiety, and the CAR binds or is configured to bind to the ether phospholipid through interaction and / or binding with the targeting moiety. In some embodiments, the ether phospholipid comprises a polar head group and an alkyl carbon chain. In some embodiments, the polar head group is selected from the group consisting of choline, phosphatidylcholine, sphingomyelin, phosphoethanolamine groups, oligosaccharide residues, sugar residues, HoIn some embodiments, the polar head group comprises sphatidylserine or phosphatidylinositol. In some embodiments, the polar head group comprises a phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the sugar is glycerol or a sugar alcohol. In some embodiments, the polar head group comprises a saccharide group. In some embodiments, the lipid comprises a head group comprising mannose. In some embodiments, the polar head group comprises sphingosine. In some embodiments, the polar head group comprises glucose. In some embodiments, the polar head group comprises a disaccharide, trisaccharide, or tetrasaccharide. In some embodiments, the lipid is a glucosylcerebroside. In some embodiments, the lipid is lactosylceramide. In some embodiments, the lipid is a glycolipid. In some embodiments, the glycolipid comprises a sugar unit such as n-glucose, n-galactose, or N-acetyl-n-galactosamine. In some embodiments, the lipid comprises a hydrocarbon ring such as a sterol.

[0077] In some embodiments, the polar head group of the lipid comprises glycerol or a sugar alcohol. In some embodiments, the polar head group of the lipid comprises a phosphate group. In some embodiments, the polar head group of the lipid comprises a choline. In some embodiments, the lipid is phosphatidylethanolamine. In some embodiments, the lipid is phosphatidylinositol. In some embodiments, the lipid comprises a backbone consisting of a sphingoid base. In some embodiments, the lipid comprises a sterol lipid, such as cholesterol or a derivative thereof. In some embodiments, the lipid comprises a saccharolipid. In some embodiments, the polar head group comprises choline, phosphate, and / or glycerol.

[0078] In some embodiments, the lipid is a glycolipid. In some embodiments, the lipid comprises a sugar. In some embodiments, the lipid is derived from sphingosine. In some embodiments, the lipid is a glyceroglycolipid or a glycosphingolipid.

[0079] In some embodiments, the lipid is an ether lipid having a hydrophobic branched chain.

[0080] As used herein, "saturated" refers to a fatty acid molecule that does not have a double bond between carbon atoms. As used herein, "unsaturated" refers to the presence of one or more double bonds within the fatty acid chain. In some embodiments of the present invention, a complex comprising a lipid is provided. In some embodiments, the lipid comprises a saturated or unsaturated fatty acid chain.

[0081] As used herein, an "alkyl" is an alkyl substituent having one hydrogen atom removed.

[0082] An "alkenyl" group is an unsaturated hydrocarbon containing at least one carbon-carbon double bond.

[0083] An "alkynyl" group is an unsaturated hydrocarbon containing at least one carbon-carbon triple bond.

[0084] As used herein, "terpenoids" are molecules derived from five-carbon isoprene units. Steroids and sterols can be produced from terpenoid precursors. For example, steroids and cholesterol can be biosynthesized from terpenoid precursors.

[0085] As used herein, "ether phospholipids" are lipids in which one or more carbon atoms of the polar head group are attached to an alkyl chain via an ether bond, rather than the more common ester bond. In some embodiments, the polar head group is glycerol.

[0086] Several types of "spacers" are described herein. A chimeric antigen receptor spacer refers to a polypeptide spacer, the length of which is configured or selected to enhance binding or interaction with the chimeric antigen receptor or reduce or minimize adverse side effects associated with CAR T cell therapy. In some embodiments, the short spacer domain of a CAR has 2 to about 12 amino acids and comprises the entire IgG4 hinge region sequence, or a portion thereof, or a variant thereof. In some embodiments, the medium-length spacer domain of a CAR has 2 to about 119 amino acids and comprises the entire IgG4 hinge region sequence and CH3 region sequence, or a variant thereof. In some embodiments, the long spacer domain of a CAR has 2 to about 229 amino acids and comprises the entire IgG4 hinge region sequence, CH2 region, and CH3 region sequence, or a variant thereof. The lipid may include a spacer to distance the targeting moiety from the lipid, and this spacer is attached to the polar head group of the lipid. The lipid spacer may include a PEG spacer, a hapten spacer, a small peptide, or an alkane chain. In some embodiments, the hapten spacer includes two haptens (hapten (2x) spacer). In some embodiments, the lipid includes a hydrophobic group such as an alkane chain. In some embodiments, the alkane chain may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms, or a number of carbon atoms within a range defined by any two of these numbers. In some embodiments, the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules, or a number of PEG molecules within a range defined by any two of these numbers.

[0087] As used herein, a "hapten" is a small molecule that can induce an immune response only when conjugated to a larger carrier, such as a protein. The carrier may not be capable of inducing an immune response by itself. In vivo, if an antibody is produced against the hapten-carrier adduct, the small hapten may also bind to the antibody, but the hapten itself typically does not initiate an immune response; typically, only the hapten-carrier adduct is capable of initiating an immune response. In some embodiments, the hapten is a small molecule binding moiety to which an scFv or antibody can bind, or which may have specificity for an scFv or antibody.

[0088] In some embodiments of the present invention, the cells provided are cytotoxic T lymphocytes. As used herein, "cytotoxic T lymphocytes (CTLs)" refer to T lymphocytes that express CD8 on their cell surface (e.g., CD8 + In some embodiments, such cells refer to antigen-experienced "memory" T cells (T M Preferably, the cells are CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

[0089] As used herein, a "masking moiety" refers to a portion of an ether lipid attached to a targeting moiety. The masking moiety functions as a protecting group that prevents recognition of the targeting moiety on the lipid by inhibiting binding and recognition by a chimeric antigen receptor specific for the targeting moiety. When the lipid is incorporated into cells present in the tumor environment or at a site where reactive oxygen species are generated, the masking moiety self-cleaves, allowing the chimeric antigen receptor to bind, interact with, and / or recognize the targeting moiety. In some embodiments, the lipid is an ether phospholipid. In some embodiments, the masking moiety comprises a phenolic hydroxyl group or PEG. In some embodiments, the phenolic hydroxyl group is attached to the hydroxyl group of the xanthene moiety of fluorescein. In some embodiments, the masking moiety is attached to the targeting moiety via a cleavable moiety that may be configured to be specifically cleaved in the tumor microenvironment. In some embodiments, the cleavable moiety configured to be cleavable in the tumor microenvironment is cleaved by reaction with reactive oxygen species, acidic pH, hypoxia, or nitrosylation. In some embodiments, the ether phospholipid comprises a targeting moiety, and a CAR is attached to the ether phospholipid via interaction and / or binding with the targeting moiety. In some embodiments, the ether phospholipid comprises a polar head group and an alkyl carbon chain.

[0090] As used herein, "cancer" refers to a group of diseases involving the proliferation of abnormal cells that can invade or spread to other parts of the body. Subjects that can be treated using the methods described herein include those identified or selected to have cancer, including, but not limited to, colon cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, leukemia, multiple myeloma, or brain cancer. Identification and / or selection of cancer patients can be performed by clinical or diagnostic evaluation. In some embodiments, tumor-associated antigens or tumor-associated molecules are known, and the tumors include, but are not limited to, melanoma, breast cancer, brain tumor, squamous cell carcinoma, colon cancer, leukemia, myeloma, or prostate cancer. The cancers include, but are not limited to, B-cell lymphoma, breast cancer, brain tumor, prostate cancer, and / or leukemia. In some embodiments, the one or more tumorigenic polypeptides are associated with kidney cancer, uterine cancer, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer (including melanoma), bone cancer, brain cancer, adenocarcinoma, pancreatic cancer, chronic myeloid leukemia, or leukemia. In some embodiments, methods are provided for treating, alleviating, or inhibiting cancer in a subject. In some embodiments, the cancer is breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, kidney cancer, pancreatic cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver cancer, colon cancer, skin cancer (including melanoma), bone cancer, or brain cancer. In some embodiments, a subject receiving any of the therapies described herein is further selected to receive another cancer therapy, which may include a cancer therapeutic agent, radiation therapy, chemotherapy, or a cancer therapeutic agent.In some embodiments, provided cancer therapies include abiraterone, alemtuzumab, anastrozole, aprepitant, arsenic trioxide, atezolizumab, azacitidine, bevacizumab, bleomycin, bortezomib, cabazitaxel, capecitabine, carboplatin, cetuximab, chemotherapy combinations, cisplatin, crizotinib, cyclophosphamide, cytarabine, denosumab, docetaxel, doxorubicin, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fluorouracil, flucloxone, fluoxetine ... These include vestrant, gemcitabine, imatinib, imiquimod, ipilimumab, ixabepilone, lapatinib, lenalidomide, letrozole, leuprolide, mesna, methotrexate, nivolumab, oxaliplatin, paclitaxel, palonosetron, pembrolizumab, pemetrexed, prednisone, radium-223, rituximab, sipuleucel-T, sorafenib, sunitinib, talc intrapleural suspension, tamoxifen, temozolomide, temsirolimus, thalidomide, trastuzumab, vinorelbine, or zoledronic acid.

[0091] As used herein, the "tumor microenvironment" refers to the cellular environment in which a tumor resides. The tumor microenvironment may include, but is not limited to, surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, or the extracellular matrix (ECM).

[0092] In some embodiments, the method comprises administering to a patient a combination of an infusion of fluorescein-conjugated ether phospholipid (FL-PLE) or a derivative thereof and an infusion of CAR T cells specific for fluorescein (FL). In this case, the system of the present invention can be used as a generic target antigen in combination with generic CARs and / or anti-tumor effector cells expressing generic CARs. In some embodiments, fluorescein (FL)-appended ether phospholipid (PLE) (C) with a CAR recognition site can be used to treat human tumors. 18The CAR recognition site is attached to the polar head group choline via a three PEG repeat spacer. Anti-FL CAR libraries may be generated using a variety of single-chain variable fragments (scFvs) (e.g., six or more scFvs) with dissociation constants between the spacer element of the CAR and the scFv ranging from 200 fM to 10 nM, e.g., 200 fM, 210 fM, 220 fM, 230 fM, 240 fM, 250 fM, 260 fM, 270 fM, 280 fM, 290 fM, and the like. , 300 fM, 400 fM, 500 fM, 600 fM, 700 fM, 800 fM, 900 fM, 1 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nM, or a value within a range defined by any two of these values. Binding of each scFv to FL occurs slightly differently for each scFv. Without wishing to be bound by a particular theory, binding of scFv to FL is influenced in part by mutations in the binding pocket that result in differences in binding affinity and by the fact that some scFvs are derived from phage display rather than yeast display. In some embodiments, three types of spacers (all derived from antibody components) may be used, including, for example, a long spacer consisting of an IgG4 hinge linked to a CH2 domain linked to a CH3 domain; a medium-length spacer consisting of an IgG4 hinge linked to a CH3 domain; and a short spacer consisting of only an IgG4 hinge. In some embodiments, only FL-specific scFv CAR T cells with appropriate orientation and appropriate spacer length exert targeted anti-tumor function against in vitro and in vivo tumors loaded with FL PLE-CTCT. Furthermore, the shape and size of the scFv binding pocket may also play a role in the successful activation of CARs.

[0093] Some embodiments provided herein relate to FL-specific CAR T cells that contain targeting and recognition domains presented in an appropriate orientation and with an appropriate spacer length, and that have a binding affinity selected to elicit a desired T cell response. In some embodiments, the spacer length is selected to induce a T cell response in specific cells of interest. In some embodiments, the binding affinity is selected to induce a T cell response in specific cells of interest. In some embodiments, varying the orientation, spacer length, and affinity of CAR T cells to induce a desired CAR T cell response can be advantageous for different tumors or different cells. Thus, some embodiments provided herein relate to tailoring a combination of orientation, spacer length, and binding affinity to a selected therapy, or selecting a combination of orientation, spacer length, and binding affinity to a selected therapy.

[0094] In some embodiments, the indicators or measures of the success of in vitro CAR T cell generation include cytolysis and cytokine production.CAR with short spacer usually does not produce cytokine, while CAR with long spacer produces a large amount of cytokine.This will be described in more detail herein.

[0095] In some embodiments, an anti-FL(4D5Flu) CAR comprising SEQ ID NO: 4 is provided. This anti-FL(4D5Flu) CAR is unable to produce cytokines when combined with FL-PLE, but can produce cytokines when cells are labeled with FL by other methods. This result highlights that the orientation of the scFv relative to the FL portion of the FL-PLE is an important variable in T cell activity, which has not been previously reported. In some embodiments, decreasing the dissociation constant, such as to improve binding affinity, does not necessarily improve CAR activation. In some embodiments, heterogeneous anti-tumor reactivity of T cells is not correlated with the KD of the scFv. In some embodiments, heterogeneous anti-tumor reactivity of T cells may be spacer length dependent. In some embodiments, an anti-FL(FITC-E2 Mut2) CAR comprising SEQ ID NO: 1 is a robust CAR, which was unpredictable based on previous knowledge of CAR structure functionality or previous knowledge of scFv research. Contrary to the findings of the present invention, the art teaches that the highest affinity scFv results in the best CAR.

[0096] In some embodiments, when FL-PLE is administered to a tumor-bearing mammal, the administered FL-PLE is retained on the cell membrane of tumor cells and functions as a versatile tumor-targeting agent recognized by FL-specific CAR T cells. Also provided herein is the identification of a FL-specific CAR that, when associated with FL-PLE-loaded tumor cells, can induce robust signaling in T cells that was unpredictable based on previous knowledge. Furthermore, in some embodiments, the present disclosure relates to the development of a final form (e.g., a kit) of pharmaceutical-grade FL-PLE to be used in combination with a CAR T cell formulation. Alternatively, other haptens designed to react with the PLE can be used in combination with a CAR instead of a FL.

[0097] Table 1 provides an example of a sequence useful in the embodiments provided herein. [Table 1] TIFF0007774961000002.tif232167TIFF0007774961000003.tif235167TIFF00077749610 00004.tif232166TIFF0007774961000005.tif232167TIFF0007774961000006.tif108166

[0098] Treatment method Some embodiments of the methods and compositions provided herein include methods of treating, alleviating, or inhibiting cancer in a subject. Some such embodiments include: administering to a subject an effective amount of a composition comprising a lipid having a targeting moiety attached thereto, the targeting moiety comprising a masking moiety; and administering to the subject a cell (e.g., a population of cells) that comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR) that specifically binds to the targeting moiety in the absence of the masking moiety. Including, the CAR or TCR comprises an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOs: 1-6; and / or The spacer domain is an IgG4 hinge linked to a CH2 domain linked to a CH3 domain (e.g., having an amino acid sequence with at least 95% identity to SEQ ID NO: 7, or a long spacer having the amino acid sequence of SEQ ID NO: 7); an IgG4 hinge linked to a CH3 domain (e.g., having an amino acid sequence with at least 95% identity to SEQ ID NO: 8, or a medium-length spacer having the amino acid sequence of SEQ ID NO: 8); or IgG4 hinge (e.g., having an amino acid sequence having at least 95% identity to SEQ ID NO: 9, or a short spacer having the amino acid sequence of SEQ ID NO: 9) The present invention relates to a method for producing a pharmaceutical composition comprising the steps of:

[0099] In some embodiments, the CAR or TCR comprises an amino acid sequence selected from SEQ ID NOs: 1 to 6. In some embodiments, the spacer is 229 amino acids in length. In some embodiments, the CAR or TCR comprises an scFv domain (FITC-E2 Mut2) having the amino acid sequence of SEQ ID NO: 1 and a spacer domain (an example of a long spacer) having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the CAR or TCR comprises an scFv domain (4M5.3) having the amino acid sequence of SEQ ID NO: 2 and a spacer domain (an example of a long spacer) having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the CAR or TCR comprises an scFv domain (FITC-E2) having the amino acid sequence of SEQ ID NO: 5 and a spacer domain (an example of a long spacer) having the amino acid sequence of SEQ ID NO: 7.

[0100] Some embodiments of the methods and compositions provided herein include a method of treating, alleviating, or inhibiting cancer in a subject, the method comprising: (a) introducing, providing, or administering to a subject a composition comprising a lipid comprising a targeting moiety bound to a masking moiety; (b) introducing, providing, or administering to the subject a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) specific for the targeting moiety from which the masking moiety has been removed; (c) removing the masking moiety from the targeting moiety, thereby binding the targeting moiety to the CAR present on the cell; (d) optionally, after steps (a)-(c), measuring or assessing binding of the cells containing the CAR to the lipid; (e) optionally, after steps (a) to (d), measuring or evaluating the treatment, alleviation, or inhibition of cancer; and / or (f) optionally, prior to steps (a) to (c), identifying a subject in need of cancer treatment; Including, the CAR or TCR comprises a spacer domain of 1 to 22 amino acids, 23 to 50 amino acids, 51 to 100 amino acids, 100 to 150 amino acids, or 151 to 250 amino acids in length; the CAR or TCR comprises a sequence selected from SEQ ID NOs: 1 to 6, and / or The spacer domain is an IgG4 hinge linked to a CH2 domain linked to a CH3 domain (e.g., having an amino acid sequence with at least 95% identity to SEQ ID NO: 7, or a long spacer having the amino acid sequence of SEQ ID NO: 7); an IgG4 hinge linked to a CH3 domain (e.g., having an amino acid sequence with at least 95% identity to SEQ ID NO: 8, or a medium-length spacer having the amino acid sequence of SEQ ID NO: 8); or IgG4 hinge (e.g., having an amino acid sequence with at least 95% identity to SEQ ID NO: 9, or a short spacer having the amino acid sequence of SEQ ID NO: 9) The present invention relates to a method for producing a pharmaceutical composition comprising the steps of:

[0101] In some embodiments, the cells are provided to the subject simultaneously with administration of the composition, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, or 48 hours before administration of the composition, or within a range of hours defined by any two of these numbers prior to administration of the composition, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, or 48 hours after administration of the composition, or within a range of hours defined by any two of these numbers after administration of the composition. In some embodiments, the cells are provided to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 20 hours, 24 hours, 36 hours, or 48 hours before administration of the composition, or within a range of hours defined by any two of these numbers before administration of the composition. In some embodiments, the cells are provided to the subject within seconds or minutes, e.g., before the elapse of an hour, after providing the composition to the subject.

[0102] In some embodiments, a booster dose of the cells and / or the composition is provided to the subject.

[0103] In some embodiments, the subject is provided with another cancer therapy, such as a small molecule (e.g., a chemical compound), antibody therapy (e.g., a humanized monoclonal antibody conjugated to a radionuclide, toxin, or drug, or an unconjugated humanized monoclonal antibody), surgery, and / or radiation therapy.

[0104] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colorectal cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, bladder cancer, head and neck cancer, or liver cancer. In some embodiments, the cancer is a non-solid tumor such as leukemia or multiple myeloma. Examples of solid tumors include sarcoma, carcinoma, and lymphoma. Further examples of cancers, such as solid tumors and non-solid tumors, are described in Amin, MB, et al. (Eds.). AJCC Cancer Staging Manual (8th edition). Springer International Publishing: American Joint Commission on Cancer; 2017, which is incorporated herein by reference in its entirety.

[0105] In some embodiments, binding of the targeting moiety to a CAR present on the cell induces production of at least one cytokine, hi some embodiments, the at least one cytokine comprises IL-2, TNF-α, and / or INF-α.

[0106] In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group is selected from the group consisting of choline, phosphatidylcholine, sphingomyelin, phosphoethanolamine groups, oligosaccharide residues, sugar residues, HoIn some embodiments, the polar head group comprises phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the hydrophobic group is a fatty acid, such as a fatty chain. In some embodiments, the fatty acid is a saturated or unsaturated fatty acid. In some embodiments, the hydrophobic group comprises an alkyl, alkenyl, or alkynyl group. In some embodiments, the hydrophobic group comprises a steroid or a terpenoid lipid, such as cholesterol. In some embodiments, the hydrophobic group comprises an ether bond between the polar head group and the fatty chain. In some embodiments, the sugar residue is glycerol or a sugar alcohol. In some embodiments, the hydrophobic group comprises an alkyl carbon chain, the alkyl carbon chain comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, or a number of carbon atoms within a range defined by any two of these numbers. In some embodiments, the alkyl carbon chain comprises 18 carbon atoms. In some embodiments, the lipid is an ether phospholipid.

[0107] In some embodiments, the targeting moiety is biotin, digoxigenin, dinitrophenol, fluorescein, or a derivative thereof. In some embodiments, the targeting moiety is fluorescein or a derivative thereof.

[0108] In some embodiments, the spacer comprises a polyethylene glycol (PEG) spacer, a spacer consisting of (two) haptens, or an alkane chain. In some embodiments, the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules, or a number of PEG molecules within a range defined by any two of these numbers.

[0109] In some embodiments, the cells are progenitor T cells. In some embodiments, the progenitor T cells are hematopoietic stem cells. In some embodiments, the cells are naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cells are CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

[0110] kit Some embodiments of the methods and compositions provided herein include kits, which in some embodiments may include a pharmaceutical grade lipid conjugated to a targeting moiety.

[0111] In some embodiments, the lipid comprises a polar head group and a hydrophobic group. In some embodiments, the polar head group is selected from the group consisting of choline, phosphatidylcholine, sphingomyelin, phosphoethanolamine groups, oligosaccharide residues, sugar residues, HoIn some embodiments, the polar head group comprises phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety. In some embodiments, the hydrophobic group is a fatty acid, such as a fatty chain. In some embodiments, the fatty acid is a saturated or unsaturated fatty acid. In some embodiments, the hydrophobic group comprises an alkyl, alkenyl, or alkynyl group. In some embodiments, the hydrophobic group comprises a steroid or a terpenoid lipid, such as cholesterol. In some embodiments, the hydrophobic group comprises an ether bond between the polar head group and the fatty chain. In some embodiments, the sugar residue is glycerol or a sugar alcohol. In some embodiments, the hydrophobic group comprises an alkyl carbon chain, the alkyl carbon chain comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, or a number of carbon atoms within a range defined by any two of these numbers. In some embodiments, the alkyl carbon chain comprises 18 carbon atoms. In some embodiments, the lipid is an ether phospholipid.

[0112] In some embodiments, the targeting moiety is biotin, digoxigenin, dinitrophenol, fluorescein, or a derivative thereof. In some embodiments, the targeting moiety is fluorescein, or a derivative thereof.

[0113] In some embodiments, the spacer comprises a polyethylene glycol (PEG) spacer, a spacer consisting of (two) haptens, or an alkane chain. In some embodiments, the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules, or a number of PEG molecules within a range defined by any two of these numbers. [Example]

[0114] Example 1 - Preparation of CAR T cells T cells were isolated from leukocyte reduction "cones" (conical filters) that are typically discarded after plateletpheresis. Specifically, a peripheral blood mononuclear cell (PBMC) fraction containing T cells was obtained by Ficoll density gradient centrifugation, followed by sequential purification of CD8+ and CD4+ T cells using an appropriate magnetic enrichment kit. All obtained T cells were immediately used for CAR production on the day of isolation. On day 0, 0.5 to 5 million T cells were stimulated with anti-human CD3 / CD28 activating beads at a 1:1 ratio. In some embodiments, the concentration of T cells used for stimulation with anti-human CD3 / CD28 activating beads may be 0.5 million, 1 million, 2 million, 3 million, 4 million, or 5 million.

[0115] On days 1–3 of stimulation, T cells were transduced with CAR-containing lentivirus at a multiplicity of infection (MOI) of 1–6 in the presence of protamine sulfate using the spinoculation method. Cell cultures were maintained at an appropriate cell density by replacing half or all of the medium every 2–3 days and, if necessary, transferred to larger culture vessels for expansion. Typically, cells were transferred to larger culture vessels when the cell concentration reached 1.5–2 million cells / mL, or when the culture was visually determined to be confluent and the medium turned yellow. Gene reconstitution of CD4+ T cells with fresh rhIL-7 at a final concentration of 50 ng / mL and fresh rhIL-15 at a final concentration of 0.5 ng / mL, and CD8+ T cells with fresh rhIL-2 at a final concentration of 50 U / mL and fresh rhIL-15 at a final concentration of 0.5 ng / mL, respectively, was initiated on day 0. These cytokines were added at each cell nutrient supply. On days 9–11 of stimulation, the activation beads were magnetically removed.

[0116] DHFRdm-free CAR T cells for selection of CAR expression using methotrexate (e.g., CAR T cells in the examples shown in Figures 8B, 15A, 15C, 15D, 15D, 15E, 18C, 19b, 19C, 21A, and 21B) were magnetically sorted using a biotinylated antibody and anti-biotin microbeads based on expression of the reporter EGFRt on days 10-21 of culture. For CAR T cells containing DHFRdm for enrichment of CAR expression using methotrexate (e.g., the CAR T cells in the examples shown in Figures 9, 10, 12, 13, 14A, and 14B), the cells were first treated with 50 nM methotrexate on days 7 to 14 of culture, the methotrexate concentration was increased to 100 nM on days 14 to 19 of culture, and then the methotrexate concentration was returned to 50 nM on days 19 to 21 of culture. To avoid a decrease in cell viability due to methotrexate selection, which could lead to culture failure, the cells were separated with Ficol on day 12 of culture to remove dead cells and improve the viability of the cultured cells.

[0117] On days 14 or 21 of culture, 0.5 million to 2 million CAR T cells were cultured using the rapid endothelial plate (REP) method with irradiated PBMCs and TM-LCL cells as feeder cells. When fresh, unfrozen PBMCs were used, 25 million PBMCs and 5 million TM-LCL cells were used in each rapid culture. When frozen PBMCs were used, the number of feeder cells was doubled. PBMCs were irradiated with 3500 rads and TM-LCL cells with 8000 rads using a cesium-based radiation irradiator. On days 0–5 of rapid culture, 30 ng / μL OKT3 antibody was added to the cells in addition to the usual cytokines described above to rapidly stimulate TCR expression. On day 5 of rapid culture, the irradiated feeder cells were removed, and CAR T cells were maintained as described above. CAR T cells containing DHFRdm for enrichment of CAR expression using methotrexate (e.g., CAR T cells in the examples shown in Figures 9, 10, 12, 13, 14A, and 14B) were supplemented with 100 nM methotrexate from day 5 of rapid culture onwards and maintained until day 12 of rapid culture to further enrich for CAR-positive cells. On day 14 of rapid culture, all CAR T cells obtained in this experiment were assessed in a functional chromium release cytotoxicity assay (CD8+ T cells only) and a functional 3-plex cytokine release assay (CD4+ and CD8+ T cells).

[0118] Example 2 - Cytotoxicity and Chromium Release Assays to target cells 51 Cr was added and incubated overnight. For target cells treated with ether phospholipid linked to a tumor-directed CAR T cell targeting agent (CTCT-PLE), CTCT-PLE was further added to the medium. 51The target cells were incubated overnight in the presence of Cr. The next day, they were washed and seeded into 96-well plates at a concentration of 5000 cells per well. CD8+ anti-fluorescein (FL) effector T cells and mock effector T cells were washed (usually on days 8–16 of the rapid culture) and seeded in triplicate with target cells at various E:T ratios (30:1, 10:1, 3:1, and 1:1) and co-cultured for 4 hours at 37°C. Control T cells were also used. 51 To assess Cr release, each target cell line was seeded in medium alone. 51 To assess the maximum release of Cr, each target cell line was seeded and lysed with 2% SDS. A control group was run in sextuplicate. After co-culture, the supernatant was collected, seeded onto LUMA plates, and allowed to dry overnight. The following day, samples were measured using a Top Count scintillation counter. Specific lysis (%) was calculated using the following formula:

number

[0119] See, e.g., Gonzalez, S., Naranjo, A., Serrano, LM, Chang, W.-C., Wright, CL, & Jensen, MC (2004). Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma. The Journal of Gene Medicine, 6(6), 704-711, which is expressly incorporated herein by reference in its entirety.

[0120] Example 3 - Cytokine Release Assay For target cells treated with CTCT-PLE, CTCT-PLE was added to the medium and incubated overnight. The next day, all target cells were collected, washed, and plated at 5 × 10 cells per well. 4CD8+ anti-FL effector T cells and mock effector T cells were washed (usually on days 8–16 of rapid culture) and each T cell (1 × 10) was cultured in a 96-well plate at a concentration of 1 × 10. 5 Cells (cells / well) were seeded with target cells and co-cultured for 24 hours at 37°C. After 24 hours, the supernatant was collected and the concentrations of IFN-γ, TNF-α, and IL-2 in the supernatant were measured using the Bio-Plex® 200 system (Bio-Rad).

[0121] Example 4 - FACS analysis of EGFRt and anti-FL CAR expression CD8+ T cells containing anti-FL CARs were stained to analyze the binding of EGFRt or anti-FL. Figure 6 shows the positive rates of CD8+ anti-FL CAR T cells analyzed by staining for the EGFRt selection marker. Figure 6(a) shows the anti-FL (FITC-E2) CAR containing SEQ ID NO: 5 [positive rate: 96%]. Figure 6(b) shows the anti-FL (4M5.3) CAR containing SEQ ID NO: 2 [positive rate: 80%]. To test the binding ability of the anti-FL CARs to FL, each T cell was incubated with mouse CD19-FITC antibody and washed. The bound FITC antibody was then stained using anti-mouse Fc-Alexa647 antibody. Figure 6(c) shows the anti-FL (FITC-E2) CAR [positive rate: 97%], and Figure 6(d) shows the anti-FL (4M5.3) CAR [positive rate: 79%]. The same positive rate was obtained by the two staining methods, confirming that there is a one-to-one relationship between CAR and surface markers and that anti-FL CAR can bind to FL. The CAR binding domain may contain the sequences shown in SEQ ID NOS: 1 to 6, and the spacer domain that may be included as part of CAR may contain the sequences shown in SEQ ID NOS: 7 to 9.

[0122] Example 5 - In vitro FL-PLE-mediated CAR T cell recognition and activation We tested the binding of anti-FL CAR-containing T cells to FL-PLE and their activation. Figures 7A, 7B, and 7C show in vitro FL-PLE-mediated recognition and activation by CAR T cells. K562 (leukemia) cells were incubated overnight with FL-PLE. FL-PLE uptake into cells was analyzed by flow cytometry (Figure 7A). A clear shift was observed between control K562 parental cells and those incubated with 5 μM FL-PLE, whereas only a very slight shift was observed in those incubated with 0.5 μM FL-PLE. This slight shift indicated a difference in the amount of FL exposed on the cell surface recognized by CAR T cells. Furthermore, K562 OKT3+ cells (a cell line developed to test intrinsic TCR-mediated activation of T cells) showed identical results to those of the parental K562 cells. Furthermore, we performed chromium release assays (Figure 7B) and cytokine release assays (Figure 7C) using these cells to compare the activation of CD8+ anti-FL(FITC-E2) CAR T cells with that of the CD8+ mock T cells shown in Figure 6. These experiments confirmed that anti-FL(FITC-E2) CAR T cells recognized the FL moiety of FL-PLE incorporated into the cell membrane and were activated. The degree of activation was related to the amount of FL exposed on the cell surface.

[0123] Figures 8A and 8B demonstrate the versatility of FL-PLE loading on tumor cells and the recognition of the fluorescein moiety by anti-FL CAR T cells. FL-PLE was added to K562 (leukemia), U87 (glioblastoma), 251T (glioma), Be2 (neuroblastoma), MDA-MB-231 (adenocarcinoma), NIH OVCAR (adenocarcinoma), and 143B (osteosarcoma) cells and incubated overnight. The uptake of FL-PLE into cells was analyzed by flow cytometry (Figure 8A). A clear shift was observed from the control parental cell line to the parental cell line incubated in the presence of 5 μM FL-PLE, reflecting the amount of FL exposed on the cell surface recognized by CAR T cells. As expected, K562 OKT3+ cells (a cell line generated to test intrinsic T cell activation via the TCR) showed identical results to the parental K562 cells. Furthermore, we performed a chromium release assay (Figure 8B) using these cells to compare the cytotoxic response of CD8+ anti-FL(FITC-E2) CAR T cells with that of CD8+ mock T cells. These experiments demonstrated that anti-FL(FITC-E2) CAR T cells can recognize the FL moiety of FL-PLE incorporated into the cell membrane of various types of cancer cells and lyse target cells.

[0124] Example 6 - Selection of CAR T cells with methotrexate CAR T cells were selected with methotrexate. Figure 9 shows various CD8+ anti-FL CAR T cells selected with the chemotherapy drug methotrexate. Specifically, we used anti-FL CAR cells containing a CAR with an FITC-E2 scFv domain and a long spacer, anti-FL CAR cells containing a CAR with an FITC-E2 scFv domain and a medium-length spacer, anti-FL CAR cells containing a CAR with an FITC-E2 scFv domain and a short spacer, and anti-FL CAR cells containing other CARs with the anti-FL scFv domain and a long spacer as shown in the figure. Furthermore, we stained each CD8+ anti-FL CAR T cell for the surface marker EGFRt to detect positive CARs. These anti-FL CARs harbor a double-mutated dihydrofolate reductase (DHFRdm) gene, which confers methotrexate resistance, allowing for the enrichment of CAR-positive cells using methotrexate. Except for the cell line with the lowest percentage of EGFRt+ cells, each cell line was diluted with mock T cells to create a stock of identical cells for use in functional assays. Target value: 18.1% EGFRt+. Actual value: approximately 13-20% EGFRt+.

[0125] As another example, Figure 10 shows various CD4+ anti-FL CAR T cells selected with the chemotherapy drug methotrexate. Each CD4+ anti-FL CAR T cell was stained for the surface marker EGFRt to detect positive CARs. These anti-FL CARs harbor a double-mutated dihydrofolate reductase (DHFRdm) gene, which confers methotrexate resistance, allowing for methotrexate-mediated enrichment of CAR-positive cells. Except for the cell line with the lowest percentage of EGFRt cells, each cell line was diluted with mock T cells to create stocks under the same conditions and used in functional assays. Target value: EGFRt 18.1%. Actual value: EGFRt approximately 15-24%.

[0126] Example 7 - Labeling of cells with FL-PLE Target cells were labeled with FL-PLE. Figure 11 shows the loading of FL-PLE into cells. Target cells for functional assays were generated using K562 cells and MDA-MB-231 cells. Each target cell group was labeled with FL-PLE. The incorporation of FL-PLE into the cell membrane was analyzed by flow cytometry.

[0127] Example 8 - CAR T cell cytotoxicity assay The cytotoxicity of CAR T cells was tested. Figure 12 shows the cytotoxicity assay of CD8+ anti-FL CAR T cells. MDA-MB-231 cells were incubated with FL-PLE. FL-PLE uptake into MDA-MB-231 cells was analyzed by flow cytometry. Chromium release assays were performed using these MDA-MB-231 cells and CAR T cells. Negative controls (parental K562 cells and MDA-MB-231 cells) showed no killing, while the positive control (K562 OKT3+ cells) showed cytolysis, as expected. For FL-PLE-labeled MDA-MB-231 cells, CARs with long spacers performed slightly better than CARs with medium-length spacers, while CARs with short spacers showed minimal killing. These results demonstrate the importance of spacer length for target cell lysis by CAR T cells and that hapten-based cell labeling is useful for determining spacer length.

[0128] Figure 14A shows the analysis of CD8+ anti-FL CAR T cells with different anti-FL scFvs. MDA-MB-231 cells were incubated with FL-PLE. FL-PLE uptake into MDA-MB-231 cells was analyzed by flow cytometry. A chromium release assay was performed using these MDA-MB-231 cells and CAR T cells. Negative controls (parental K562 cells and MDA-MB-231 cells) showed no killing, whereas the positive control (K562 OKT3+ cells) showed cell lysis, as expected (Figure 14A). Four anti-FL scFvs were tested in this chromium release assay with CARs containing long spacers. The three anti-FL CARs showed comparable lysis of MDA-MB-231 cells labeled with FL-PLE, whereas anti-FL (4D5Flu) containing SEQ ID NO: 4 showed little killing. On the other hand, another experiment using anti-FL(4D5Flu) showed the ability to lyse FL-labeled cells, so anti-FL(4D5Flu) was not suitable for recognizing the FL moiety on FL-PLE incorporated into the cell membrane.

[0129] Example 9 - Cytokine release assay of CAR T cells Cytokine release from CAR T cells was analyzed. Figure 13 shows a cytokine release assay of CD4+ anti-FL CAR T cells. FL-PLE was added to K562 cells and MDA-MB-231 cells, respectively, and the cells were incubated. FL-PLE uptake into each cell was analyzed by flow cytometry. Cytokine release assays were performed using these cells and anti-FL CAR T cells. Negative controls (parental K562 cells and MDA-MB-231 cells) did not produce any cytokines, whereas positive controls (K562 OKT3+ cells) showed production of all three cytokines in all anti-FL CAR T cell lines. This assay was designed to evaluate the relationship between CAR spacer length and FL-PLE. These data indicated that only CARs with long spacers were able to produce all three cytokines when FL-PLE was used.

[0130] Figure 14B shows a cytokine release assay of CD4+ anti-FL CAR T cells using various anti-FL scFvs. FL-PLE was added to K562 cells and MDA-MB-231 cells, respectively, and the uptake of FL-PLE into each cell was analyzed by flow cytometry. A cytokine release assay was performed using these cells and CAR T cells. Negative controls (parental K562 cells and MDA-MB-231 cells) did not produce any cytokines, whereas positive controls (K562 OKT3+ cells) showed production of all three cytokines in all anti-FL CAR T cell lines. In this cytokine release assay, five anti-FL scFvs were tested in CARs with long spacers. Only four anti-FL CARs showed activation in K562 cells labeled with 5 μM FL-PLE. When the amount of FL-PLE was reduced to 500 nM in K562 cells, anti-FL (4M5.3) containing SEQ ID NO:2 and anti-FL (FITC-E2 Mut2) containing SEQ ID NO:1 induced the highest cytokine production, but only anti-FL (FITC-E2 Mut2) was able to produce IL-2. In MDA-MB-231 cells labeled with 5 μM FL-PLE, anti-FL (4M5.3) and anti-FL (FITC-E2 Mut2) also induced the highest cytokine production. With FL-PLE, anti-FL (4M5.3) and anti-FL (FITC-E2 Mut2) showed the best activation, but their dissociation constants were very different: the dissociation constant between FL-PLE and anti-FL (4M5.3) was 270 nM, while the dissociation constant between FL-PLE and anti-FL (FITC-E2 Mut2) was 3.1 nM. These results indicate that simply decreasing the dissociation constant of CARs is not enough to obtain the best CARs. When FL-PLE was used, anti-FL(4D5Flu) containing SEQ ID NO: 4 did not produce CARs. Anti-FL(4D5Flu) did not produce cytokines in either cell line labeled with 5 μM FL-PLE. However, a separate experiment using anti-FL(4D5Flu) demonstrated lytic activity against FL-labeled cells.Therefore, anti-FL(4D5Flu) was not suitable for recognizing the FL moiety on FL-PLE incorporated into the cell membrane.

[0131] Three anti-FL CARs with long spacers (4M5.3, FITC-E2, and FITC-E2 Mut2) that demonstrated high functionality were further tested. CAR T cells were generated using each anti-FL CAR. Figure 15A shows pure CD4+ and pure CD8+ anti-FL CAR T cell populations selected by staining for the EGFRt selection marker. FL-PLE was added to K562 cells (leukemia) and MDA-MB-231 cells (adenocarcinoma), respectively, and incubated overnight. FL-PLE uptake into each cell was analyzed by flow cytometry (Figure 15B). A clear shift was observed from the control parental cell line to the parental cell line incubated in the presence of 5 μM FL-PLE, reflecting the amount of FL exposed on the cell surface recognized by CAR T cells. Additionally, each generated CAR T cell was analyzed by chromium release assay (Figure 15C) and cytokine release assay (Figure 15D and Figure 15E) to test optimal anti-FL CAR T cell activation by comparing each CAR T cell line with each other and with mock T cells. These experiments showed that anti-FL(Mut2) CAR T cells showed the best response in terms of CAR T cell recognition and activation in both leukemia and adenocarcinoma cells, followed by anti-FL(FITC-E2) CAR T cells, and then anti-FL(4M5.3) CAR T cells.

[0132] Example 10 - In vivo targeting and incorporation of FL-PLE We examined in vivo targeting and uptake of FL-PLE. Figure 16 shows the in vivo targeting and uptake of FL-PLE with an available FL moiety for conjugation. After establishing glioblastoma (U87 cells) in one group of mice via intracranial injection, FL-PLE was intravenously injected into the mice. At various time points after FL-PLE injection, the mice were sacrificed and their brains were harvested. Specifically, FL-PLE was administered intravenously to mice bearing orthotopic xenografts of glioma, and the brains were evaluated over a 14-day period. Brain tissue sections were prepared 48 hours later. Nuclei were stained with DAPI. Furthermore, an anti-fluorescein antibody was used to stain the fluorescein molecules of FL-PLE incorporated into the cell membrane, and available fluorescein molecules were examined. Gliomas retained excessive amounts of FL-PLE compared with the contralateral tumor-free brain hemisphere of the same subjects. The upper left panel of Figure 16 shows a 10x fluorescence image, demonstrating that the tumor is significantly brighter than the normal healthy tissue, indicated by N. This indicates that FL-PLE is selectively incorporated into the tumor cell membrane, making the FL moiety available for binding. In the lower left panel, a bright signal is observed when viewing an image of the contralateral brain. To quantify these signals, the MFIs of the tumor and contralateral sites were calculated separately in the right panel. This analysis was repeated at multiple time points. The resulting values ​​were plotted to generate a graph showing the retention time of FL-PLE over several days after FL-PLE administration.

[0133] The results shown in Figure 17 demonstrate the versatility of FL-PLE targeting and uptake in vivo. To demonstrate versatility, we used an adenocarcinoma cell line (MDA-MB-231), an osteosarcoma cell line (143B), and a glioblastoma cell line (U87) in this experiment. Adenocarcinoma and osteosarcoma tumors were inoculated on both flanks in each group, while glioblastoma tumors were inoculated on only one flank. In the fourth group, adenocarcinoma tumors were inoculated on one flank and osteosarcoma tumors on the other flank. After tumor establishment in each mouse group by subcutaneous injection, FL-PLE was injected intravenously into the mice. At various time points after FL-PLE injection, the mice were sacrificed and tumors were harvested. The harvested tumors were immediately subjected to fluorescent imaging to confirm the presence or absence of fluorescein molecules on the FL-PLE. The graph in Figure 17 for three cancer types shows the retention time of FL-PLE over several days until tumor fluorescence reached baseline levels (tumors not inoculated with FL-PLE). In the fourth group, FL-PLE targeting two cancer types simultaneously was shown to be retained for several days.

[0134] Example 11 - Inhibition of CAR T cell binding by ProFL-PLE, a prodrug form of FL-PLE We labeled cells with masked FL-PLE (ProFL-PLE) and tested whether this ProFL-PLE had the ability to inhibit CAR T cell binding to the cells. Figures 18A and 18B show the inhibitory effect of ProFL-PLE on CAR T cell recognition, observed before unmasking. K562 (leukemia) cells were incubated overnight with FL-PLE (high or low dose) or ProFL-PLE. The cellular uptake of FL-PLE or ProFL-PLE was analyzed by flow cytometry (Figure 18A). A clear shift was observed from the control K562 parental cells to those incubated with a high dose of FL-PLE, whereas only a slight shift was observed in those incubated with a low dose of FL-PLE. This slight shift indicates a difference in the amount of FL exposed on the cell surface that was recognized by CAR T cells. ProFL-PLE did not emit fluorescence due to the presence of the phenolic hydroxyl group, which is the masking agent. Thus, flow cytometry revealed that K562 cells in the presence of ProFL-PLE and parental K562 cells had nearly identical fluorescence profiles. Unmasking ProFL-PLE induced fluorescein fluorescence. The amount of FL exposed on the surface of unmasked ProFL-PLE was comparable to that of cells treated with low doses of FL-PLE. Figure 18B shows the cells used in a chromium release assay to test the activation of CD8+ anti-FL CAR T cells by comparing them with mock CD8+ T cells. These experiments demonstrated that anti-FL CAR T cells recognized the FL moiety of FL-PLE incorporated into the cell membrane. ProFL-PLE completely inhibited recognition by anti-FL CAR, whereas removal of the prodrug-protected moiety allowed the FL moiety to be recognized by anti-FL CAR. Lysis of cells with unmasked ProFL-PLE was comparable to that in the presence of low doses of FL-PLE, which correlated with the amount of FL exposed on the surface of K562 cells.

[0135] Figures 18A and 18C show the cells used in a cytokine release assay to evaluate the activation of CD4+ anti-FL CAR T cells by comparison with CD4+ mock T cells. The uptake of FL-PLE or ProFL-PLE into cells was analyzed by flow cytometry (Figure 18A). As shown in Figure 18C, the negative control (K562 parental cells) showed no cytokine production, whereas the positive control (K562 OKT3+ cells) showed the production of all three cytokines in all anti-FL CAR T cell lines. When cells labeled with FL-PLE were used, all three cytokines could be produced, and the amount of these cytokines produced depended on the amount of FL exposed on the cell surface. When cells incorporating ProFL-PLE labeled with a pro-drug moiety were used, no cytokines were produced before unmasking, indicating that the anti-FL CAR T cells were not activated. Unmasking of cells labeled with ProFL-PLE resulted in the production of all three cytokines, indicating that removal of the prodrug moiety activated anti-FL CAR T cells via ProFL-PLE incorporated into the surface of cancer cells. These experimental results demonstrate that tumor cells labeled with a lipid (such as ProFL-PLE) conjugated to a masked hapten can be unmasked from the hapten bound to the lipid incorporated into the tumor cell membrane to form an unmasked hapten, and that contacting this hapten with anti-hapten CAR T cells can activate the CAR T cells, at least in an in vitro environment.

[0136] Figures 19A, 19B, and 19C show that before ProFL-PLE unmasking, CAR T cell recognition was inhibited by ProFL-PLE. After unmasking, the newly exposed fluorescein on the cell surface was recognized by anti-FL CAR T cells, resulting in their activation. K562 cells (leukemia) were incubated overnight with FL-PLE or ProFL-PLE. The cellular uptake of FL-PLE or ProFL-PLE was analyzed by flow cytometry (Figure 19A). A clear shift was observed from the control K562 parental cell line to the K562 parental cell line incubated in the presence of FL-PLE, reflecting the amount of FL exposed on the cell surface recognized by CAR T cells. ProFL-PLE did not emit fluorescence due to the presence of the phenolic hydroxyl group, which acts as a masking agent. Thus, K562 cells incubated in the presence of ProFL-PLE (i.e., ProFL-PLE without the prodrug moiety removed) and parental K562 cells were observed to have nearly identical profiles by flow cytometry. Unmasking ProFL-PLE (overnight incubation in FBS-free medium) resulted in the development of fluorescein fluorescence. Similar amounts of FL were observed exposed on the surface of unmasked ProFL-PLE compared with K562 cells incubated in the presence of FL-PLE. Figure 19B shows the cells used in a cytokine release assay, which subsequently assessed the activation of CD4+ and CD8+ anti-FL CAR T cells by comparing them with CD4+ and CD8+ mock T cells. The negative control (parental K562 cells) showed no cytokine production, whereas the positive control (K562 OKT3+ cells) showed the production of all three cytokines in all anti-FL CAR T cell lines. As expected, all three cytokines were produced when FL-PLE-labeled cells were used.When cells incorporating ProFL-PLE labeled with a prodrug moiety were used, no cytokines were produced before unmasking, indicating that anti-FL CAR T cells were not activated as designed. Unmasking ProFL-PLE-labeled cells produced all three cytokines, indicating that removal of the prodrug moiety activated anti-FL CAR T cells via unmasked ProFL-PLE incorporated on the surface of cancer cells. This demonstrates that the ProFL-PLE design functions in vitro. Figure 19C shows the results of cell staining to examine the upregulation of activation markers (LAG3, 41BB, and PD-1) on CAR T cells after 24 hours of coculture in a cytokine release assay. Live CD8+ anti-FL (FITC-E2) CAR T cells with a long spacer are shown. As expected, the negative control cocultured with K562 parental cells showed no upregulation of activation markers, while the positive control (K562 OKT3+ cells) upregulated all three activation markers. Co-culture with K562 cells containing ProFL-PLE resulted in a slight increase in the expression of activation markers, indicating that the intact prodrug moiety inhibited anti-FL CAR T cell recognition and activation. Co-culture with K562 cells containing FL-PLE or unmasked ProFL-PLE resulted in activation comparable to that of the positive control, indicating that anti-FL CAR T cells were similarly activated by both FL-PLE and unmasked ProFL-PLE.

[0137] Example 12 - Activity of anti-FL CAR T cells in vivo Mice bearing brain tumors were administered masked or unmasked FL-PLE and anti-FL CAR T cells. Figure 20 shows the initial in vivo treatment with FL-PLE or ProFL-PLE. After establishing neuroblastoma (Be2) tumors in three groups of mice by intracranial injection, the mice were intracranially injected with anti-FL(FITCE2) CAR T cells containing a long spacer and SEQ ID NO:5. The control group received only anti-FL(FITCE2) CAR T cells with a long spacer. Tumors in the control group progressed normally (black bars). The second group received a single intravenous injection of FL-PLE before T cell injection. This group's survival time was approximately 20% longer than the control group. The third group received three regular intravenous injections of ProFL-PLE (one before T cell injection and two after T cell injection), and the mice in this group also experienced a survival time of approximately 20% longer. These results also confirmed that re-administration of ProFL-PLE is safe.

[0138] Example 13 - In vivo activity of anti-FL CAR T cells when administered with masked FL-PLE The combined administration of masked FL-PLE (ProFL-PLE) and anti-FL CAR T cells was tested in a mouse breast cancer model. Figures 21A and 21B show in vivo ProFL-PLE therapy in a flank tumor-bearing model. After establishing adenocarcinoma tumors (MDA-MB-231 eGFP:ffLuc IL2+) by subcutaneous injection in three groups of mice (two tumors per mouse), two of the mice were administered ProFL-PLE by intratumoral injection (Figure 21A) or intravenous injection (IV) (Figure 21B), while the control group received no drug injection. After the first drug injection, cells containing anti-FL (FITC-E2) CARs with a long spacer were intravenously injected (IV) into all three groups. The vertical gray dotted lines in the graph indicate the days of injection. The control group died within 16 days due to tumor burden (Figure 21A). In the ProFL-PLE intratumoral injection group, ProFL-PLE was administered 12 times over a 45-day period, and all three mice experienced tumor regression to baseline levels by day 40. These mice survived without tumor development until the end of the study on day 90 (Figure 21B). In the ProFL-PLE intravenous injection group, ProFL-PLE was administered 10 times over a 34-day period, and two mice experienced tumor regression to baseline levels by approximately day 40. These mice survived without tumor development until the end of the study on day 90. One mouse in this group died on day 16, as did the control mouse. These results demonstrate that the combination of ProFL-PLE and anti-FL CAR T cells administered intratumorally or intravenously is a viable treatment.

[0139] All of the following references are expressly incorporated herein by reference in their entirety.

[0140] References 1. Ma, J. S. et al. (2016). Versatile strategy for controlling the specificity and activity of engineered T cells. Versatile strategy for controlling the specificity and activity of engineered T cells. Proceedings of the National Academy of Sciences of the United States of America, 113(4), E450-E458.

[0141] 2. Kim, M. S. et al. (2015). Redirection of Genetically Engineered CAR-T Cells Using Bifunctional Small Molecules. Journal of the American Chemical Society, 137(8), 2832-2835.

[0142] 3. Urbanska, K. et al. (2012). A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor. Cancer Research, 72(7), 1844-1852.

[0143] 4. Wu, C. Y. et al. (2015). Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science, 350(6258), aab4077-aab4077.

[0144] 5. Kranz et al. (1981). Partial elucidation of an anti-hapten repertoire in BALB / c mice: comparative characterization of several monoclonal antiFLuorescyl antibodies. Mol Immunol, 18(10), 889-898.

[0145] 6. Jung et al. (1997). Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Engineering Design & Selection, 10(8), 956-966.

[0146] 7. Schwesinger et al. (2000). Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. PNAS, 97(18), 9972-9977.

[0147] 8. Boder et al. (2000). Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. PNAS, 97(20), 10701-10705.

[0148] 9. Honegger et al. (2005). A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex. Prot Sci, 14(10), 2537-2549.

[0149] The examples and embodiments described herein are for illustrative purposes only, and various modifications and variations of these examples and embodiments are suggested to those skilled in the art, which modifications and variations are within the spirit and scope of this application and the appended claims.

[0150] Substantially all plural and / or singular terms used herein will be interpreted by those skilled in the art as construing the plural terms as singular and / or singular terms as plural, as appropriate to the description and / or application herein. The various singular / plural terms have been intentionally used to clarify the invention.

[0151] Those skilled in the art will understand that the terminology used herein, particularly in the appended claims (e.g., the body of the appended claims), is generally "open-ended" (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," and the term "include" should be interpreted as "including, but not limited to"). Furthermore, those skilled in the art will understand that when a specific number is recited in a claim, such an intention is also clearly recited in the claim, and that when a specific number is not recited, such intention is not present. Specifically, for example, the claims below may use preambles such as "at least one" or "one or more" to define the claim. However, this preface should not be construed to limit a claim reciting an element using the indefinite article "a" or "an" to embodiments containing only one element, nor should it be construed to limit a claim reciting an element using the indefinite article "a" or "an" to embodiments containing only one element, even if the same claim contains the preface "one or more" or "at least one" and the indefinite article "a" or "an" (e.g., "a and / or an" should be interpreted to mean "at least one" or "one or more"). The same applies to claims reciting a definite article. Furthermore, even if a claim explicitly recites a specific number, one of ordinary skill in the art would understand that "at least" refers to the recited number (e.g., "two" without a modifier means "at least two" or "two or more").Furthermore, when idiomatic phrases such as "at least one of A, B, and C" are used, such phrases are typically written in the sense that one of ordinary skill in the art would normally understand the phrase (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Also, when idiomatic phrases such as "at least one of A, B, or C" are used, such phrases are typically written in the sense that one of ordinary skill in the art would normally understand the phrase (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Furthermore, those skilled in the art will understand that disjunctive terms and / or phrases intended to express two or more alternatives, whether in the specification, claims, or drawings, may include one of the stated terms, either of the stated terms, or both of the stated terms. For example, the phrase "A or B" may include "A or B" or "A and B."

Claims

1. 1. A composition for use in treating, inhibiting, or alleviating cancer in a subject, comprising: a lipid having attached thereto a targeting moiety comprising fluorescein or a fluorescein derivative and a masking moiety; the masking moiety is cleavable by reactive oxygen species (ROS) present in the tumor microenvironment of a solid tumor; to be administered to the subject in combination with a cell comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising a ligand binding domain and a spacer domain capable of specifically binding to the target moiety in the absence of the masking moiety; The composition, wherein the ligand-binding domain comprises the amino acid sequence shown in any of SEQ ID NOs: 1 to 3, 5, and 6, and the spacer domain comprises an IgG4 hinge and has a length of 151 to 250 consecutive amino acids.

2. 2. The composition for use according to claim 1, wherein the spacer domain comprises an IgG4 hinge domain, a CH2 domain and a CH3 domain.

3. The composition for use of claim 1 , wherein the spacer domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

7.

4. The composition for use according to claim 1, wherein the ligand binding domain comprises the amino acid sequence shown in SEQ ID NO: 1, 2 or 5, and the spacer domain comprises the amino acid sequence of SEQ ID NO:

7.

5. 2. The composition for use of claim 1, wherein the cells are administered to the subject simultaneously with or after a first administration of the composition, and the subject may receive a second administration of the composition.

6. 2. The composition for use of claim 1, wherein the cells are administered to the subject prior to a first administration of the composition, and the subject may receive a second administration of the composition.

7. 2. The composition for use of claim 1, wherein binding of the targeting moiety to a CAR present on the cell induces production of at least one cytokine.

8. 2. The composition for use of claim 1, wherein the lipid comprises a polar head group, a hydrophobic group, and a polyethylene glycol (PEG) spacer.

9. the polar head group comprises a choline, a phosphatidylcholine, a sphingomyelin, a phosphoethanolamine, a sugar residue, a phosphatidylserine, a phosphatidylinositol, a phosphocholine, a piperidine moiety, or a trimethylarseno-ethyl-phosphate moiety; the hydrophobic group comprises a fatty acid, an alkyl group, an alkenyl group, an alkynyl group, a steroid, a terpenoid lipid, or cholesterol; the hydrophobic group comprises an ether bond between the polar head group and the fatty chain; the hydrophobic group comprises an alkyl carbon chain, which may contain 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, and may even contain 18 carbon atoms; and / or 9. The composition for use of claim 8, wherein the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 PEG molecules.

10. 2. The composition for use according to claim 1, wherein the lipid comprises an ether phospholipid (PLE).

11. 2. The composition for use according to claim 1, wherein the masking moiety comprises a phenolic hydroxyl group attached to a hydroxyl group of a xanthene moiety of the PLE.

12. 2. The composition for use of claim 1, wherein the cells are selected from the group consisting of progenitor T cells; hematopoietic stem cells; CD8+ cytotoxic T lymphocytes selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells; and CD4+ helper T lymphocytes selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.

13. 2. The composition for use of claim 1, wherein the cancer is selected from colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, liver cancer, leukemia, and multiple myeloma.

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