Reversible regulation of chimeric antigen receptor T cells

Abstract

Disclosed are receptor traps that modulate immune cells, which can have tumor cell-derived ectodomains that can bind to a CAR on a chimeric antigen receptor (CAR)-T cell and reversibly inhibit activation of the CAR-T cell.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 376,389, filed September 20, 2022, and U.S. Provisional Patent Application No. 63 / 462,828, filed April 28, 2023, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety, and the disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction of the patent document or patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0004] FIELD OF THE INVENTION Aspects of the present invention relate to compositions and methods for modulating cell surface molecules, including chimeric antigen receptors (CARs) on the surface of CAR-T cells, to reversibly control CAR receptors and receptors on cancer cells to inhibit cancer cell signaling and proliferation.

[0005] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy made at [] is named [] and is [] bytes in size. [Background technology]

[0006] Background of the Invention Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with hematological malignancies. However, widespread use of CAR-based immunotherapies has been limited by potentially life-threatening toxicities, and therapeutic efficacy is limited by diminished persistence. Cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the two most common toxicities observed after CAR T-cell therapy. Furthermore, diminished persistence is often due to immune cell exhaustion, and continued signaling through CARs ultimately leads to reduced or no efficacy, leading to treatment failure or relapse. Therefore, desirable CAR-based immunotherapies would include features to limit toxicities due to undesirable downstream signaling effects and to control or restrict CAR activity to avoid exhaustion. Summary of the Invention

[0007] Disclosed herein are novel reagents and methods for modulating molecules on the surface of cells. In some embodiments, the reagents and methods are used to control the level of CAR molecules on CAR-T cells. In some embodiments, the reagents and methods are used to reversibly control (e.g., inhibit) CAR-T cell activation. In some embodiments, CAR-T cell activation is controlled in vivo. In some embodiments, the reagents and methods are used to control or inhibit cancer cell growth or modulate immune responses.

[0008] In some embodiments, antigen or receptor traps as described herein are disclosed. In some embodiments, the antigen / receptor trap has an ectodomain or ectodomain means to which a chimeric antigen receptor (CAR) can bind (e.g., an extracellular region of a cell membrane protein to which a CAR on a CAR-T immune cell can bind; e.g., an ectodomain-based ligand trap for an immune cell). In some embodiments, the ectodomain means to which a CAR-T cell can bind substantially in the same way as a CAR can bind to an ectodomain, but the ectodomain means can be derived from an ectodomain, can be a variant thereof, etc.

[0009] In some embodiments, the ectodomain means can be fused to a dimerization means. The dimerization means can cause two receptor traps to form dimers. Dimerization can increase the valency of the receptor trap. In some embodiments, the dimerization means can be the Fc portion of an antibody. In some embodiments, binding of the trap molecule by the CAR can block CAR-T cells (e.g., inhibit binding of the CAR to a molecule that can activate CAR-T cells; inhibit CAR-T cell activation). In some embodiments, blocking or inhibition can be reversible. In some embodiments, binding of the trap molecule by the CAR does not activate or does not substantially activate CAR-T cells. In some embodiments, the antigen / receptor trap can be referred to as a CAR-Trap molecule. In some embodiments, the CAR-Trap molecule is multivalent as well.

[0010] As disclosed herein, nucleic acids encoding these molecules, vectors containing the nucleic acids, and cells containing the vectors and / or expressing the receptor traps are disclosed.

[0011] In some embodiments, a method for administering an antigen / receptor trap to a subject is disclosed. [Brief explanation of the drawings]

[0012] Certain diagrams, charts, or flow charts are provided to facilitate a better understanding of the invention. It should be noted, however, that the drawings depict only selected embodiments of the invention and are therefore not intended to limit the scope. Additional and equally effective embodiments and applications of the invention exist. [Figure 1] FIG. 1 is a schematic diagram showing CAR-T cell therapy in patients. [Figure 2] We present an example approach to treat toxicities observed after CAR-T cell therapy with immunosuppressants. [Figure 3] 10A-10C illustrate exemplary approaches to treat toxicity observed after CAR-T cell therapy using suicide genes or exclusion markers. [Figure 4] We present an example approach to treat toxicity observed after CAR-T cell therapy using a reversible gene switch. [Figure 5] We demonstrate a reversible CAR-T modulation mechanism as a strategy to enhance CAR-T cell efficacy. [Figure 6] FIG. 1 shows an example of an approach for controlling CAR-T cell activation using receptor traps as disclosed herein. [Figure 7] FIG. 1 shows examples of CD19 receptor trap monomer and dimer expression. [Figure 8] FIG. 1 shows an example of grafting of the CD19 extracellular domain onto human IgG1 FC and expression of the molecule. [Figure 9] FIG. 1 shows an example of an anti-CD19 Jurkat NFAT GFP / CD19-K562 co-culture assay. [Figure 10] FIG. 1 shows the results of CD19 receptor trap monomer blocking anti-CD19 Jurkat CAR activation in a dose-dependent manner. [Figure 11]FIG. 1 shows the results of the CD19-Fc dimeric receptor trap molecule being more effective than the monomer in blocking anti-CD19 Jurkat CAR activation. [Figure 12] FIG. 1 shows the results of improved expression yield and reduced aggregation of engineered CD19 receptor trap variants. [Figure 13] FIG. 1 shows an example of improved grafting of CD19 extracellular domain into human IgG1 FC and expression of the molecule. [Figure 14] FIG. 1 shows the results of engineered CD19 receptor trap variants that block anti-CD19 Jurkat CAR activation with improved IC50. [Figure 15] FIG. 1 shows results demonstrating that receptor trap inhibition of Jurkat cell activation is reversible. [Figure 16] FIG. 10 shows further results demonstrating that receptor trap inhibition of Jurkat cell activation is reversible. [Figure 17] FIG. 10 shows further results demonstrating that receptor trap inhibition of Jurkat cell activation is reversible. [Figure 18] FIG. 1 shows the results of antigen / receptor trap inhibition of Jurkat cell activation in the presence of K562 and antigen / receptor trap minimally affecting Jurkat CAR-T cells in the absence of activating tumor cells. [Figure 19] FIG. 10 shows further results of antigen / receptor trapping minimally affecting Jurkat CAR-T cells in the absence of activating tumor cells. [Figure 20] FIG. 10 shows further results of antigen / receptor trapping minimally affecting Jurkat CAR-T cells in the absence of activating tumor cells. [Figure 21] FIG. 10 shows further results of antigen / receptor trapping minimally affecting Jurkat CAR-T cells in the absence of activating tumor cells. [Figure 22]FIG. 10 shows further results of antigen / receptor trapping minimally affecting Jurkat CAR-T cells in the absence of activating tumor cells. [Figure 23] FIG. 1 shows the expression of antigen / receptor traps containing the HER2 extracellular domain, the epitope derived from the HER2 protein to which the Herceptin antibody binds (Herceptin BD-FC), or the extracellular domain of BCMA. [Figure 24] FIG. 1 shows the results of expression of CARs specific for CD19, HER2 or BCMA in primary T cells. [Figure 25] FIG. 1 shows a schematic representation of the retroviral vectors used to express various CARs. [Figure 26A] Figures 26A, 26B, and 26C show the activity of the antigen / receptor traps shown in Figure 32 against BMCA, HER2, and HER2, respectively. Each panel shows the results without (left) and with (right) target cells. [Figure 26B] See legend to Figure 26A. [Figure 26C] See legend to Figure 26A. [Figure 27A] Figures 27A, 27B, and 27C show schematic diagrams of the study used to detect the effect of CD19-specific CARTrap 9 on primary cells (A), cytokine production (B), and tumor cell killing (C). The data show that CARTrap inhibited / abolished interferon gamma production (B). The data show that CARTrap abolished tumor killing, and that removal of CARTrap resumed tumor killing. [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 28]Figures 28A-E. Characterization of CD19 ectodomain variants for inhibiting CAR-T cell activity. (A) Schematic representation of CD19wt, NT.1, 19.1, C6.2, and CT.2 fused to IgG1 Fc to form CAR-Trap. (B) Dose-dependent inhibition of CAR-Jurkat cell activation by K562 cells with various CD19 CAR-Trap molecules and corresponding IC50 values ​​are shown. CAR-Jurkat cell activation levels were measured using NFAT-GFP via flow cytometry. (C) SDS-PAGE gel characterization of five CAR-Trap proteins. (D) Comparison of protein expression levels for CD19 ectodomain variant CAR-Trap with CD19wt CAR-Trap. (E) Illustration of the structure of the CD19 / CAR scFv complex showing that mutations in CD19 NT.1 are primarily located far from the interaction interface. The model was built using PyMOL based on PDB 7URV. [Figure 29] Figure 29A-E. Characterization of multivalent CD19 ectodomain fusions for CAR-T cell activity inhibition. (A) Schematic diagram of CD19 NT.1 monomer, dimer, and tetramer. (B) SDS-PAGE gel confirmation of monomer, dimer, and tetramer fusion protein formation. (C) Dose-dependent inhibition of CAR-Jurkat cell activation by K562 cells with various CAR-Trap molecules, along with corresponding IC50 values. CAR-Jurkat cell activation levels were quantified using NFAT-GFP via flow cytometry. (D) An illustration comparing monomeric versus dimeric CAR-Trap in competition with tumor surface antigens for CAR binding on the T cell membrane. Given the simultaneous engagement of multiple CARs and tumor antigens at the cancer cell-T cell interface, dimeric CAR-Trap may exhibit superior ability to disrupt this interaction. (E) shows the reversibility of CAR-Jurkat activation by CAR-Trap. [Figure 30]Figure 30A-D. (A) Schematic representation of the effect of CAR density on baseline, ligand-independent, tonic signaling and response to both CAR-Trap and tumor cells. (B) Depicts the strength of various CAR-T cell signals. (C) Various expression groups for Jurkat cells expressing the EF1a-CAR construct untreated or exposed to various concentrations of CAR-Trap or tumor cells at a 1:1 effector:T cell ratio. (D) CD69 levels for the various groups included in (C). [Figure 31] Figure 31A-D. CAR-Trap reversibly regulates primary CAR-T cells. (A) Overview of the setup for the primary CAR-T cell co-culture assay. Secreted IFN-γ levels are measured to determine CAR-T cell activation levels in the presence of CD19+ A375 cells and CAR-Trap; live-cell fluorescence microscopy is used to determine anti-tumor efficacy. (B) Measurement of human primary CAR-T cell IFN-γ release in the co-culture assay described in (a) with an IFNγ split luciferase assay (Promega). Data are representative of two independent experiments. (C) Fluorescence microscopy of mCherry-labeled A375 cells showing CAR-T cell-mediated A375 killing reversibly regulated by CAR-Trap. (D) Overlay of bright-field and mCherry channel images showing that CAR-T cell-mediated A375 killing activity resumed over time after CAR-Trap washout. [Figure 32] Figure 32A-C. Engineering of BCMA CAR-Trap. (A) Structure of the BCMA ectodomain from PDB 4ZFO. (B) Schematic representation of BCMA CAR-Trap and its corresponding SDS-PAGE validation. (C) Dose-dependent inhibition of anti-BCMA CAR-Jurkat cell activation by H929 cells with BCMA CAR-Trap and corresponding IC50 values. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description of the Invention Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with hematological malignancies (Figure 1). However, in some cases, CAR-T therapy can cause side effects, such as cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), in patients receiving CAR-T therapy.

[0014] To address these adverse events, several regulatory mechanisms have been developed to control CAR-T cells in vivo, but these strategies do not adequately meet current needs, as toxicity and mortality continue to be reported in CAR-T clinical trials.

[0015] Disclosed herein are novel, modular, and reversible strategies for modulating CAR-T cell activity. Generally, these methods do not require additional genetic manipulation of CAR-T cells. In some embodiments, these strategies can modulate CAR-T toxicity. In some embodiments, these strategies can enhance the efficacy of CAR-T cell therapy. In some embodiments, these strategies are based on reversible blockade or internalization of the CAR receptor.

[0016] Disclosed herein are recombinant protein switches based on the ectodomain of a cell surface molecule to which a chimeric antigen receptor (CAR) can bind. These protein switches can be referred to as "CAR-Trap." In some embodiments, the ectodomain can be a tumor cell surface antigen. CAR-Trap can reversibly and quantitatively "fine-tune" CAR-T cell activity.

[0017] In some embodiments, the antigen trap, including the receptor trap, can comprise an antigen or ligand to which a CAR receptor can bind. The receptor trap can function as a CAR OFF switch by binding to the CAR receptor and blocking its interaction with an antigen or ligand that would normally be found on tumor cells to which the CAR-T cell binds and triggers CAR-T cell activation.

[0018] For example, the antigen / receptor trap molecule can be a molecule to which the CAR of a CAR-T cell specifically binds. In some embodiments, binding of the antigen / receptor trap molecule by the CAR inhibits the CAR from binding to the same or similar molecule on the surface of a tumor cell. In some embodiments, binding of the antigen / receptor trap by the CAR prevents or reduces activation of the CAR-T cell. In some embodiments, binding of the antigen / receptor trap by the CAR can activate the CAR-T cell.

[0019] In some embodiments, receptor trapping does not require engineering of the CAR-T receptor or CAR-T cells and can be applied to CAR-T therapies already approved or in clinical development.

[0020] In other embodiments, the receptor trap can be reversible, allowing for fine tuning of CAR-T cell activity, such as rejuvenating cells for toxicity management and / or continued therapy.

[0021] In some embodiments, receptor traps can be tailored to target CAR-T cells to different tumor antigens, for example, by replacing the components used in their design (i.e., the traps can be modularized). These antigen traps can be designed by manipulating the ectodomain of tumor surface antigens. Antigen traps can disrupt the interaction between CAR-T cells and tumor cells.

[0022] Also disclosed are approaches to enhance CAR-T efficacy. Temporary "resting" of CAR-T cells can reverse CAR-T cell exhaustion. In some embodiments, the disclosed reversible CAR modulators can increase CAR-T cell efficacy by alternating CAR-T cells between an "active" and a "resting" state.

[0023] Detailed descriptions of one or more embodiments are provided herein. However, it should be understood that the present invention can be embodied in various forms. Accordingly, the specific details disclosed herein should not be construed as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art how to employ the present invention in any suitable manner.

[0024] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In the claims and / or specification, the use of the words "a" or "an" when used in conjunction with the word "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0025] As used herein, when phrases such as "for example," "such as," "including," and the like are used, they are understood to be followed by the phrase "and without limitation," unless expressly stated otherwise. Similarly, "an example," "exemplary," and the like are understood to be non-limiting.

[0026] The term "substantially" permits deviations from the description so long as the intended purpose is not adversely affected. Descriptive terms are understood to be modified by the word "substantially," even if the word "substantially" is not explicitly stated.

[0027] The terms "comprising," "including," "having," "involving" (and likewise "comprises," "includes," "has," "involving"), etc. are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is to be interpreted as open-ended, meaning "at least" and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. When the terms "a" or "an" are used, they are to be understood as "one or more," unless the context makes such an interpretation meaningless.

[0028] As used herein, the term "about" can refer to approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. As used herein, the term "about" is used to vary a numerical value above and below the stated numerical value by a variance of 20% (higher or lower).

[0029] CAR-T cells, toxicity and toxicity control Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers (Figure 1). However, poor control of transfused CAR T cells can limit the widespread application of this therapy due to potentially life-threatening toxicity. Toxicity has been an obstacle to the development of CAR T therapy for both hematological and solid tumors. Several deaths from CAR T therapy have been reported in recent years (Neelapu, Sattva S., et al. "Toxicity management after chimeric antigen receptor T cell therapy: one size does not fit 'all'." Nature reviews Clinical oncology 15.4(2018):218-218).

[0030] Cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the two most common toxicities observed after CAR-T cell therapy.

[0031] CRS, characterized by hyperthermia, hypoxia, hypotension, and multisystem organ toxicity, occurs in 37% to 93% of patients with lymphoma and 77% to 93% of patients with leukemia. ICANS, characterized by confusion, delirium, seizures, or cerebral edema, occurs in 23% to 67% of patients with lymphoma and 40% to 62% of patients with leukemia. Severe CRS and ICANS require intensive care unit monitoring and treatment, and multiple deaths have been reported due to uncontrollable CRS or ICANS toxicity.

[0032] Three types of treatment are currently used to address these toxicities.

[0033] In some instances, patients are treated with systemic immunosuppressants (Figure 2), including corticosteroids, IL-6 receptor antibodies (e.g., tocilizumab), lymphocytotoxic anti-CD52 antibodies (e.g., alemtuzumab), and tyrosine kinase inhibitors (e.g., dasatinib) (LCK inhibitors do not inhibit already activated T cells). However, these treatments have limitations. For example, high-dose steroid treatment limits the duration of CAR T cell function and can induce hematologic hypoplasia and toxicity. Anti-IL-6 receptor antibodies have diverse biological activities and can nonspecifically inhibit the immune system (Bonifant, Challice L., et al. “Toxicity and management in CAR T-cell therapy.” Molecular Therapy-Oncolytics 3(2016):16011).

[0034] In some embodiments, patients are treated with suicide genes or exclusion markers (Figure 3), including iCasp9, anti-CD20 (e.g., rituximab), anti-EGFR (e.g., cetuximab), and the like. However, these treatments have limitations. For example, they can irreversibly and / or permanently eliminate CAR T cells from the body (Brandt, Laerke JB, et al. "Emerging approaches for regulation and control of CAR T cells: a mini review." Frontiers in Immunology 11 (2020): 326).

[0035] In some cases, CAR-T cells with switchable CAR receptors, such as split-CAR, SMaSh-CAR, and CAR PROTAC, can be used in patients (Figure 4). However, these therapies have limitations. For example, they can impair CAR-T activity, the switch can be leaky, and the switch can be immunogenic (Labanieh, Louai, et al. "Enhanced safety and efficacy of protease-regulated CAR-T cell receptors." Cell 185.10(2022):1745-1763).

[0036] However, it is known that the efficacy of CAR-T can be enhanced by using reversible CAR-T regulatory mechanisms (Figure 5). Constitutively expressing CAR-T cells may exhibit elevated levels of exhaustion-associated proteins. However, in some embodiments, transient "rest" can reverse the exhaustion phenotype. In some embodiments, CAR-T cells can be switched between "off" and "on" states by reversibly turning regulated CARs off and on (Weber, Evan W., et al. "Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling." Science 372.6537(2021):eaba1786; Labanieh, Louai, et al. "Enhanced safety and efficacy of protease-regulated CAR-T cell receptors." Cell 185.10(2022):1745-1763). This can be used as a method for treating CAR-T cell exhaustion.

[0037] In some embodiments of the invention disclosed herein, antigen / receptor traps are used to modulate CAR-T cells. In some embodiments, these receptor traps can reversibly modulate CAR-T cells.

[0038] Antigen or receptor traps In some embodiments, the strategies disclosed herein for modulating molecules (e.g., the amount of a molecule) on a cell surface and / or modulating the activity of a molecule on a cell surface can use an antigen trap or receptor trap approach. For example, this strategy can involve a cell surface molecule (e.g., a protein) binding to a "trap" molecule. In some embodiments, the trap molecule can be an ectodomain or ectodomain means to which a chimeric antigen receptor (CAR) on the surface of a CAR-T cell can bind. In some embodiments, the ectodomain means can be derived from an ectodomain, a variant thereof, etc., to which a CAR-T cell can bind in substantially the same way as a CAR can bind to an ectodomain.

[0039] In embodiments, binding of the trap molecule can affect the ability of a general ligand of the cell surface molecule to bind to the cell surface molecule (e.g., CAR) and / or modulate the molecule. In embodiments, the trap molecule can bind to the cell surface molecule such that binding of a regulatory protein (e.g., an activator or repressor of the cell surface molecule; e.g., an epitope to which CAR can bind) to the cell surface molecule is inhibited. In embodiments, binding of the trap molecule itself to the cell surface molecule minimally affects the regulation of the cell surface protein (e.g., binding of the trap has no or minimal effect on the up- and / or down-regulation of cell surface or membrane molecules / proteins). In some embodiments, the trap molecule is not associated with a cell (e.g., is free in solution). In some embodiments, the trap molecule can also have an Fc region of an antibody fused to the trap molecule or its ectodomain.

[0040] In some embodiments, the cell surface molecule or protein can be a CAR molecule. In some embodiments, the CAR molecule can be present on a CAR-T cell. In some embodiments, a strategy for regulating CAR-T activity involves blocking the CAR receptor with an antigen / receptor trap molecule. In some embodiments, blocking the CAR receptor with the trap is reversible. In some embodiments, the receptor trap can function as a CAR OFF switch by binding to the CAR receptor on the T cell and blocking its interaction with molecules (e.g., molecules on the cell) to which the CAR receptor normally binds (Figure 6). In some embodiments, the receptor trap can inhibit activation of CAR-T cells that bind to the receptor trap. In some embodiments, an antigen-receptor trap molecule specific for a CAR can be referred to as a CAR-Trap molecule.

[0041] In some embodiments, the receptor trap can be any antigen or epitope to which a chimeric antigen receptor (CAR) specifically binds, provided that: (i) the receptor trap molecule blocks binding of at least some "conventional" antigens / epitopes that the CAR is designed to bind; and / or (ii) it does not activate or suppress, or only minimally activates or suppresses, CAR-T cells upon binding to the CAR (e.g., the trap molecule does not have the same regulatory effect on the CAR as the antigen / epitope on the cell that the CAR is designed to bind). In some embodiments, the receptor trap can reversibly modulate CAR-T cells.

[0042] In embodiments, the receptor trap can be a variant of the antigen / epitope to which the CAR is designed to bind (e.g., by means of an ectodomain). In some embodiments, the trap molecule can resemble an antigen on the cell surface to which the CAR is designed to bind. In some embodiments, the ectodomain means can have an IC50 for binding to the CAR that is at least 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than the wild-type ectodomain to which the same CAR can bind. In some embodiments, the trap molecule is not associated with a cell.

[0043] In some embodiments, the antigen or epitope to which the CAR binds and used in the antigen / receptor trap is an antigen or epitope that is specific to or substantially restricted to tumor or cancer cells (e.g., a tumor-specific antigen, a tumor-associated antigen, etc.), i.e., in at least some embodiments, the antigen or epitope used in the antigen / receptor trap is not found or is minimally present on non-tumor or non-cancer cells.

[0044] In some embodiments, the trap molecule can be derived from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0045] In embodiments, the trap molecule may be derived from a blood (hematological) cancer cell or a cell derived from a solid tumor. In embodiments, the target antigen or epitope may be derived from B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL), multiple myeloma, etc. In embodiments, the target antigen or epitope may be derived from a brain tumor, breast tumor, kidney tumor, etc.

[0046] In embodiments, the target antigen or epitope can be derived from hepatocellular carcinoma, GPC3-positive hepatocellular carcinoma, liver cancer, lung cancer, advanced lung cancer, advanced solid tumor, colon cancer, colorectal cancer, EGFR-positive colorectal cancer, esophageal cancer, pancreatic cancer, prostate cancer, gastric cancer, sarcoma, osteoid sarcoma, Ewing's sarcoma, breast cancer, ovarian cancer, glioma, cervical cancer, lung squamous cell carcinoma, liver metastasis, liver tumor, gastric tumor, advanced EGFR-positive solid tumor, etc.

[0047] In some embodiments, the antigen / receptor trap molecule is based on an ectodomain or ectodomain means derived from CD19 or B-cell maturation antigen (BCMA).

[0048] In some embodiments, the engineered ectodomain can be multivalent (eg, multimeric).

[0049] In some embodiments, the density of CAR molecules on CAR-T cells can affect the CAR-Trap and CAR-T cell response to tumor cells.

[0050] In embodiments, target antigens or epitopes used in antigen / receptor traps may include CD19, B-cell maturation antigen (BCMA), human epidermal growth factor 2 (HER2), and the like.

[0051] In some embodiments, the CD19 ectodomain or ectodomain means is (SEQ ID NO: 26): It can be TIFF2025531328000002.tif35170.

[0052] In some embodiments, the CD19 ectodomain can be a variant that has a lower IC50 than the wild-type molecule. In some embodiments, the CD19 ectodomain can be a 19.1, C6.2, NT.1, or CT.2 variant of CD19 (Figure 28A).

[0053] In some embodiments, the BCMA ectodomain or ectodomain means is (SEQ ID NO: 27): LQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA It can be said that:

[0054] In embodiments, target antigens or epitopes used in antigen / receptor traps can include molecules derived from hematological cancers, such as CD123, CD138, CD20, CD22, CD38, CD5, Igκ chain, LeY, NKG2D ligand, ROR1, and WT1.

[0055] In embodiments, target antigens or epitopes used in antigen / receptor traps can include molecules derived from solid tumors and / or cancers, such as C-Met, CAIX, CD133, CD171, CD70, CEA, EGFR, EGFR vIII, Ep-CAM, EphA2, FAP, GD2, GPC3, HER2, HPV16-E6, IL13Ra2, LeY, MAGEA3, MAGEA4, MART1, mesothelin, MUC1, MUC16, NY-ESO-1, PD-L1, PSCA, PSMA, ROR1, and VEGFR2.

[0056] In some embodiments, the antigen / receptor trap molecule can regulate the cell surface molecule (e.g., CAR) to which the antigen / receptor trap molecule binds. In some embodiments, the antigen / receptor trap can regulate the cell surface molecule in cells in which the cell surface molecule is overexpressed. In some embodiments, the antigen / receptor trap can upregulate the cell surface molecule in cells in which the cell surface molecule is overexpressed.

[0057] In some embodiments, the dimerization means can be part of the CAR-Trap molecule. The dimerization means can be any region that can associate with another dimerization region, typically in a separate CAR-Trap molecule, using a covalent or non-covalent bond. The dimerized CAR-Trap molecule can be a homodimer or a heterodimer. Many protein dimerization domains are known in the art (see, for example, Dang, Dung Thanh. "Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry." Frontiers in Chemistry 10(2022):829312). Examples of dimerization domains include zipper motifs, such as leucine zippers.

[0058] In some embodiments, the dimerization means can be an Fc region from an antibody and can be fused to an ectodomain. In some embodiments, the Fc region is from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc region is (SEQ ID NO: 25): It can be TIFF2025531328000003.tif28170.

[0059] In some embodiments, the Fc region can dimerize to form homodimeric or heterodimeric structures. In some embodiments, the Fc region can have or be modified to have cysteine ​​amino acids capable of forming disulfide bonds (one or more, such as two, disulfide bonds). In some embodiments, dimers of CAR-Trap with Fc regions can be formed through disulfide bonds between cysteine ​​residues in separate CAR-Trap molecules.

[0060] In certain embodiments, the Fc region may be a variant containing amino acid substitutions that alter antigen-independent effector functions, such as the circulatory half-life of the molecule to which it is linked. Molecules linked to these Fc regions (e.g., ectodomains) may exhibit either increased or decreased binding to FcRn, potentially resulting in increased or decreased serum half-life, compared to Fc regions lacking these substitutions. Fc variants with improved affinity for FcRn are predicted to have longer serum half-lives, and such molecules are useful in methods where a longer half-life of the linked molecule is desired. In contrast, Fc variants with reduced FcRn binding affinity are predicted to have shorter half-lives, and such molecules are also useful, for example, when a shorter circulation time may be advantageous. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta. Furthermore, other applications where reduced FcRn binding affinity is desired include applications where localization to the brain, kidney, and / or liver is desired. In one embodiment, the Fc variant binding molecule is capable of reducing transport from the vasculature to the epithelial cells of the glomerulus of the kidney.

[0061] In another embodiment, the Fc variant-binding CAR trap molecule may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, the Fc region with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions in the "FcRn-binding loop" of the Fc region. The FcRn-binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in PCT Publication WO 05 / 047327, incorporated herein by reference. In an exemplary embodiment, the bispecific modulator disclosed herein comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0062] In some embodiments, the ectodomains disclosed herein may be linked to Fc variants comprising amino acid substitutions that alter glycosylation. For example, the Fc variants may have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, the Fc variants comprise reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In other embodiments, the molecule may have an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In certain embodiments, the Fc variant may have an amino acid substitution at amino acid position 228 or 299 (EU numbering). One example of an amino acid substitution that results in reduced or altered glycosylation is described in PCT Publication WO 05 / 018572, incorporated herein by reference in its entirety.

[0063] In some embodiments, the molecules disclosed herein may be modified to remove glycosylation and may be referred to as "agly" molecules. An exemplary aglycosylation is the aglycosylation of the Fc region of an IgG4 antibody, which lacks Fc effector functions and eliminates the potential for Fc-mediated toxicity to normal tissues and cells. In yet other embodiments, the molecules disclosed herein may have an altered carbohydrate chain. For example, the number of fucose residues on the N-glycan at Asn297 in the Fc region may be reduced, i.e., the molecules may be afucosylated. In some embodiments, the number of sialic acid residues on the N-glycan at Asn297 in the Fc region may be altered.

[0064] In some embodiments, the CH2 or CH3 region of an Fc antibody domain can be truncated or modified to adjust the half-life of the molecule. In some embodiments, the Fc truncation comprises CH3 or CH2 (e.g., Gehlsen, Kurt R., et al. "Pharmacokinetics of engineered human monomeric and dimeric CH2 domains." MAbs. Vol. 4. No. 4. Taylor & Francis, 2012; Ying, Tianlei, et al. "Engineered soluble monomeric IgG1 CH3 domain: generation, mechanisms of function, and implications for design of biological therapeutics." Journal of Biological Chemistry 288.35(2013):25154-25164).

[0065] In some embodiments, the Fc region can have or be modified to have cysteine ​​amino acids capable of forming disulfide bonds. In some embodiments, dimers or tetramers of CAR-Trap molecules can be formed through disulfide bonds between cysteine ​​residues in the Fc regions of separate CAR-Trap molecules (e.g., Figures 13, 28A, or 29A). In some embodiments, other types of chemical bonds can be formed to obtain multimeric molecules. In some embodiments, bonds can be formed between regions of CAR-Trap molecules that are not Fc regions. These dimers can be homodimers. In some embodiments, heterodimers can be formed. These multimers can have multiple ectodomains (e.g., can be multivalent with respect to the ectodomain). In some embodiments, these CAR-Trap molecules can have 2, 3, 4, 5, 6, 7, 8, or more ectodomains.

[0066] In some embodiments, a linkage (e.g., a linker) can be placed between various sections of the CAR-Trap molecule. In some embodiments, this linkage can be placed between the ectodomain or ectodomain means and the dimerization means. In some embodiments, the linkage can be a glycine-rich linker ("GS linker"). In some embodiments, the "GS" linker can be a combination of glycine and serine amino acids. In some embodiments, the GS linker can be GSSGGSGGSGGS (SEQ ID NO: 28). Other sequences are possible. In some embodiments, the GS linker can be SGGGG (SEQ ID NO: 29), SGGGSGGG (SEQ ID NO: 30), GSSGGSGGSGGS (SEQ ID NO: 31), GSGS (SEQ ID NO: 32), GSGGS (SEQ ID NO: 33), GSSGSS (SEQ ID NO: 34), GSSSSSS (SEQ ID NO: 35), etc. In some embodiments, the GS linker can have at least four amino acids that are glycine and / or serine. In some embodiments, other amino acids can be part of the GS linker, as long as glycine and serine are in the majority.

[0067] In some embodiments, the antigen / receptor trap molecules disclosed herein can include the following nucleotide and amino acid sequences and molecules that are at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to the following nucleotide and amino acid sequences: Specifically, the amino acid sequences of the antigen / receptor trap molecules can be labeled as follows: The underlined Times New Roman font is the signal peptide, The bold Times New Roman font is from an external domain and The italic Times New Roman font is, in some embodiments, a linker encoded by a restriction enzyme site generator; The underlined and bold Times New Roman font is the GS linker. The underlined, italic, bold Times New Roman font is a severable linker, The underlined Courier New font is in the Fc region, The bold Courier New font is the TEV section. The underlined, bold Courier New font is His-Tag, The underlined, italic, bold Courier New font is Avi-Tag.

[0068] pDP10 CD19-WT FC (SEQ ID NO: 1) TIFF2025531328000004.tif115170

[0069] (SEQ ID NO: 2) TIFF2025531328000005.tif49170

[0070] pDP11 CD19-WT (SEQ ID NO: 3) TIFF2025531328000006.tif60170

[0071] (SEQ ID NO: 4) TIFF2025531328000007.tif33170

[0072] pDP32-CD19-FC-CD19 WT (SEQ ID NO: 5) TIFF2025531328000008.tif157170

[0073] (SEQ ID NO: 6) TIFF2025531328000009.tif71170

[0074] pDP33_CD19.1_Fc (SEQ ID NO: 7) TIFF2025531328000010.tif136170

[0075] (SEQ ID NO: 8) TIFF2025531328000011.tif49170

[0076] pDP34_CD19 C6.2-Fc (SEQ ID NO: 9) TIFF2025531328000012.tif117170

[0077] (SEQ ID NO: 10) TIFF2025531328000013.tif47170

[0078] pDP35_CD19 NT.1-Fc (SEQ ID NO: 11) TIFF2025531328000014.tif114170

[0079] (SEQ ID NO: 12) TIFF2025531328000015.tif47170

[0080] pDP36_CD19 CT.2-Fc (SEQ ID NO: 13) TIFF2025531328000016.tif114170

[0081] (SEQ ID NO: 14) TIFF2025531328000017.tif47170

[0082] pDP45-CD19 NT.1-FC-CD19 NT.1 tetramer (SEQ ID NO: 15) TIFF2025531328000018.tif152170

[0083] (SEQ ID NO: 16) TIFF2025531328000019.tif70170

[0084] pDP47-CD19 NT.1_monomer (SEQ ID NO: 17) TIFF2025531328000020.tif61170

[0085] (SEQ ID NO: 18) TIFF2025531328000021.tif37170

[0086] pDP37-HER2 ETD_Fc (SEQ ID NO: 19) TIFF2025531328000022.tif165170

[0087] (SEQ ID NO: 20) TIFF2025531328000023.tif80170

[0088] pDP38-HerceptinBD-FC (SEQ ID NO: 21) TIFF2025531328000024.tif87170

[0089] (SEQ ID NO: 22) TIFF2025531328000025.tif33170

[0090] pDP39-BCMA ECD-FC (SEQ ID NO: 23) TIFF2025531328000026.tif79170

[0091] (SEQ ID NO: 24) TIFF2025531328000027.tif30170

[0092] antibody "Recombinant," with respect to a polypeptide (e.g., an antibody) or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, including, but not limited to, those that can be created by combining polynucleotides or polypeptides that do not normally occur together. As used herein, "polypeptide" encompasses the singular form "polypeptide" as well as the plural form "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to a chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or other terms used to refer to chains or chains of two or more amino acids can refer to "polypeptides" herein, and the term "polypeptide" can be used instead of or interchangeably with any of these terms. "Polypeptide" can also refer to the product of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. They may be generated by any method, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.Such conservatively modified variants of the antibodies disclosed herein may exhibit increased cross-reactivity compared to the unmodified antibody.

[0093] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain: a basic side chain (e.g., lysine, arginine, histidine), an acidic side chain (e.g., aspartic acid, glutamic acid), an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), a beta-branched side chain (e.g., threonine, valine, isoleucine), or an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0094] Some embodiments also feature antibodies that share a specified percentage of amino acid or nucleotide sequence identity with the antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity when compared to a specific region or the entire length of any one of the antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleic acid sequence identity when compared to a specific region or the entire length of any one of the antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment using methods known in the art, for example, software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity of the nucleic acids and proteins of the invention.

[0095] An embodiment of the present invention provides an isolated antibody. As used herein, the term "isolated," with respect to a cell, a nucleic acid, such as DNA or RNA, refers to a molecule separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. For example, an "isolated nucleic acid" can include a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in its natural state. "Isolated" can also refer to a cell or polypeptide that is separated from other cellular proteins or tissues. Isolated polypeptides include both purified and recombinant polypeptides.

[0096] As used herein, "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody, an antigen-binding fragment, or a single chain thereof. For example, "antibody" can include molecules containing proteins or peptides that constitute at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" refers to an antibody that reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides.

[0097] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to F (ab’)2 , F (ab)2 , F ab ', Fab Antibody fragments are portions of antibodies, such as Fvs, scFvs, and the like. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" also includes aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab'), Fd, Fvs, single-chain Fvs (scFvs), single-chain antibodies, dAbs (domain antibodies), minibodies, disulfide-linked Fvs (sdFvs), fragments consisting of either the VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0098] "Single-chain variable fragment" or "scFv" refers to a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L (scFv) refers to a fusion protein of the variable regions of a single-chain Fv ("scFv") polypeptide molecule. A single-chain Fv ("scFv") polypeptide molecule, a covalently linked VH:VL heterodimer, can be expressed from a gene fusion containing VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883.) In some embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility or contain serine or threonine for solubility, and the V H N-terminus and V LIt is also possible to link the C-terminus of the antibody V region to the C-terminus of the antibody V-region, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Various methods have been described for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules and identifying chemical structures that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, for example, U.S. Pat. Nos. 5,091,513; 5,892,019; 5,132,405; and 4,946,778, which are incorporated herein by reference in their entireties.

[0099] Antibody molecules obtained from humans are classified into five immunoglobulin classes, including one or more of IgG, IgM, IgA, IgE, and IgD, which differ from one another in the nature of the heavy chains present in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). Particular classes also contain subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, and IgG5, are well characterized and are known to confer functional specificity. With regard to IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 daltons. The four chains are held together by disulfide bonds in a Y-shape, with the light chains continuing from the mouth of the "Y" through the variable regions and sandwiching the heavy chains. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) or subclass of immunoglobulin molecule.

[0100] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either kappa or lambda light chains. For example, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. The amino acid sequence of the heavy chains runs from the N-terminus at the forked end of the Y-shape to the C-terminus at the end of each chain.

[0101] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. The variable domains of both the light (VL) and heavy (VH) chains determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement fixation. The term "antigen-binding site" or "binding portion" may refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Within the V regions of the heavy and light chains are three highly divergent regions called "hypervariable regions," which are sandwiched between highly conserved side chains called "framework regions" or "FR." Thus, the term "FR" may refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to one another in three dimensional space to form an antigen-binding surface that is complementary to the three dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs."

[0102] The six CDRs present in each antigen-binding domain are short, noncontiguous amino acid sequences that are specifically arranged to form the antigen-binding domain when the antibody assumes a three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show little inter-molecular variability. The framework regions largely adopt a beta-sheet structure, and the CDRs form loops that connect to, and in some cases form part of, the beta-sheet structure. The framework regions form a scaffold that orients the CDRs through interchain noncovalent interactions. The antigen-binding domain formed by the arranged CDRs provides a surface complementary to the epitope on the immunoreactive antigen, facilitating noncovalent binding of the antibody to its cognate epitope. The amino acids constituting the CDRs and framework regions, respectively, have already been defined and can be readily identified for heavy or light chain variable regions by those skilled in the art (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDapartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0103] Where there is more than one definition of a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless expressly stated to the contrary. As a specific example, the term "complementarity-determining region" ("CDR") is used to describe the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This region is described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), both of which are incorporated herein by reference in their entireties. The Kabat and Chothia definitions of CDRs include overlapping amino acid residues and subsets when comparing amino acid residues. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs as defined by each of the above references are set forth in the table below for comparison. The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR from the amino acid sequence of the variable region of an antibody. TIFF2025531328000028.tif67170

[0104] Kabat et al. defined a numbering system for variable domain sequences that is applicable to all antibodies. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence without reliance on experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system defined by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0105] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue from the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue from the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins at approximately residue 24 (i.e., following the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue from the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at about the 33rd residue from the end of CDR-L2 (ie, following the cysteine ​​residue), contains about 7 to 11 residues, and ends with the sequence F or WGXG (X is any amino acid).

[0106] As used herein, the term "epitope" may include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable region, defining a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. Epitope determinants are composed of chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains.

[0107] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ) and K d A smaller K represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method is to measure the rates of formation and dissociation of the antigen-binding site / antigen complex; these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and "off rate constant" (K off Both the K and K can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). off / K onThe ratio of α to β allows all parameters unrelated to affinity to be cancelled out, resulting in the equilibrium binding constant K D (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay such as a radioligand binding assay or similar assay known to those skilled in the art, such as BIAcore or Octet (BLI). D ) is 1 μM or less, 10 μM or less, 10 nM or less, 10 pM or less, or 100 pM or less to about 1 pM. For example, in some embodiments, D Approximately 1E-12M~K D In some embodiments, K D Approximately 1E-11M~K D In some embodiments, K D Approximately 1E-10M~K D In some embodiments, K D Approximately 1E-9M~K D In some embodiments, K D Approximately 1E-8M~K D In some embodiments, K D Approximately 1E-7M~K D For example, in some embodiments, K D is about 1E-12M, but in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, but in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, but in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, but in other embodiments, K D is about 1E-5M. In some embodiments, for example, K Dis about 3E-11M, but in other embodiments, K D is about 3E-12M. In some embodiments, K D is approximately 6E-11M. "Specifically binds" or "having specificity" refers to an antibody that binds to an epitope via its antigen-binding domain, and means that the binding is complementary between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" if it binds to an epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.

[0108] For example, antibodies can be monovalent or bivalent, and single-chain or double-chain. Functionally, antibodies have a binding affinity of 10 -5 M to 10 -12 For example, the binding affinity of an antibody is in the range of 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 - M to 10 -10 M to 10 -7 M to 10 -10 M to 10 -8 M to 10 -10 M to 10 -9 M to 10 -10 M to 10 -5 M to 10 -9 M to 10 -6 M to 10-9 M to 10 -7 M to 10 -9 M to 10 -8 M to 10 -9 M to 10 -5 M to 10 -8 M to 10 -6 M to 10 -8 M to 10 -7 M to 10 -8 M to 10 -5 M to 10 -7 M to 10 -6 M to 10 -7 Up to M or 10 -5 M to 10 -6 Up to M.

[0109] Those skilled in the art will recognize that whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention can be determined without undue experimentation by determining whether the former interferes with the specific binding of the latter. For example, if it is shown that the human monoclonal antibody under test competes with a human monoclonal antibody of the invention, resulting in reduced binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or closely related epitopes.

[0110] Another method for determining whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the epitope to which it normally reacts, followed by the addition of the human monoclonal antibody to be tested and determining whether the ability of the human monoclonal antibody to be tested to bind to the epitope is inhibited. If the ability of the human monoclonal antibody to be tested is inhibited, then that antibody has the same, or a functionally equivalent, epitope specificity as the monoclonal antibody of the present invention. Screening for human monoclonal antibodies of the present invention can also be performed by using an epitope to determine whether the test monoclonal antibody can neutralize a polypeptide containing the epitope.

[0111] Various procedures known in the art can be used to produce polyclonal or monoclonal antibodies to a protein of the invention, or to a derivative, fragment, analog, homolog, or ortholog thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0112] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen, or epitope thereof, against which the immunoglobulin is sought, can be immobilized on a column, and immune-specific antibodies purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0113] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules that contain only one molecular species of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. For example, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. mAbs have an antigen-binding site and are capable of immunoreacting with a specific epitope of an antigen.

[0114] Monoclonal antibodies can be prepared using hybridoma methods such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0115] Nucleic acids, vectors and cells expressing receptor traps Nucleic acids encoding all or part of the receptor traps described herein are also disclosed. Various vectors (e.g., plasmids, viruses, etc.) containing the nucleic acids are also disclosed. Various cells (e.g., prokaryotes, eukaryotes) that contain the nucleic acids or vectors and can express the fusion proteins are also disclosed.

[0116] method Disclosed herein are methods for administering the antigen / receptor traps described herein to a subject. In some embodiments, the antigen / receptor traps can target a CAR receptor on a CAR-T cell. In some embodiments, the methods are used to treat toxicity (e.g., toxicity due to cytokine release) in a subject who has received a CAR-T cell infusion for the treatment of cancer, or to treat CAR-T cell exhaustion in similar subjects. In some embodiments, the methods are used to improve the efficacy of CAR-T cells administered to a subject to treat cancer.

[0117] In some embodiments, the subject being treated for cancer may be undergoing CAR-T cell therapy for a cancer selected from the group consisting of B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL), multiple myeloma, and the like.

[0118] In some embodiments, the reagents and methods disclosed herein can be used with cells that are not cancer cells.

[0119] therapeutic preparations Aspects of the present disclosure relate to therapeutic preparations. As used herein, the term "therapeutic preparation" may refer to any compound or composition that can be used or administered for a therapeutic effect (e.g., receptor trapping). As used herein, the term "therapeutic effect" may refer to an effect sufficient to improve symptoms, such as treating, curing, preventing, or improving an associated pathology, or increasing the rate of treating, curing, preventing, or improving such a pathology.

[0120]

[0013] The embodiments described herein may be administered to a subject in the form of a pharmaceutical composition or therapeutic formulation prepared for the intended route of administration. Such compositions and preparations may include, for example, an active ingredient and a pharmaceutically acceptable carrier. Such compositions and preparations may be in a form adapted for oral, subcutaneous, parenteral (e.g., intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, nasal, transdermal, vaginal, buccal, ocular, or pulmonary administration, e.g., a form adapted for administration by a peripheral route, suitable for oral administration, or suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal, and intravenous, and such compositions can be prepared by methods well known to those skilled in the art, for example, as described in "Remington's Pharmaceutical Sciences," 17th Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 and latest editions, and in Marcel Dekker's "Drugs and the Pharmaceutical Sciences" series of monographs. The compositions and formulations can be provided in conventional forms, such as solutions and suspensions for injection, capsules and tablets, enteric-coated formulations such as those disclosed in U.S. Pat. No. 5,350,741, and for oral administration.

[0121] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, and phosphates; and tonics such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0122] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ), phosphate-buffered saline (PBS), and the like. In all cases, the composition will be sterile and fluid to the extent that easy syringability exists. In embodiments, it will be stable under the conditions of manufacture and storage and will be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof, or a pharmaceutically acceptable polyol. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0123] Sterile injectable solutions can be prepared by incorporating the compound in the required amount in a suitable solvent with one or a combination of the ingredients listed herein, followed by filtration sterilization, if necessary. Dispersions are prepared by incorporating the active compound in a sterile solvent containing a basic dispersion medium and the required other ingredients from the ingredients listed herein. In the case of sterile powders for preparing sterile injectable solutions, useful methods include vacuum drying and freeze-drying, which can yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0124] Oral compositions may contain an inert diluent or an edible carrier. They may also be placed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is orally administered, gargled, and expectorated or swallowed. Oral formulations of drugs may be administered, for example, once a day, twice a day, three times a day, or four times a day, depending on the half-life of the drug.

[0125] Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition administered to a subject. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch, lactose, or disintegrating agents such as alginic acid, Primogel® (sodium starch glycolate), or corn starch; lubricants such as magnesium stearate or stearotate; lubricants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; and flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0126] Systemic administration can also be via transmucosal or transdermal routes. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are known in the art, and include, for example, detergents, bile salts, fusidic acid derivatives, etc. for transmucosal administration. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams known in the art.

[0127] In embodiments, administration may include introducing a pharmaceutical composition into a subject by a method or route that results in at least partial localization of the composition at the desired site so that the desired effect occurs.

[0128] For example, the pharmaceutical composition may be administered by bolus injection or infusion. Bolus injection refers to a route of administration in which a syringe is connected to a venous access device and the agent is infused directly into the subject. The term "infusion" may refer to intravascular injection.

[0129] The embodiments described herein can be administered to a subject once (e.g., as a single injection, bolus, or deposition). Alternatively, they can be administered to a subject once or twice daily for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to one year. In embodiments, administration can continue for the life of the subject. They can also be administered to a subject once or twice daily, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or any combination thereof.

[0130] In embodiments, the compositions described herein can be administered chronically to a subject. "Chronic administration" can refer to administration in a continuous manner, such as maintaining a therapeutic effect (activity) over an extended period of time.

[0131] The specific dosage and treatment regimen for a particular patient will depend on a variety of factors, including the particular antibody, variant, or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the particular disease being treated. Assessment of such factors by a medical professional is within the ordinary skill of those in the art. The amount will also vary depending on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0132] A therapeutically effective amount of a reagent or therapeutic composition of the present invention can be the amount necessary to achieve a therapeutic goal. As described herein, this is the binding interaction between the reagent or therapeutic composition and its target, which, in some cases, inhibits the function of the target. The amount required for administration further depends on the binding affinity of the reagent or therapeutic composition for its specific target and the rate at which the administered reagent or therapeutic composition depletes the free volume of the target to which it is administered. The binding polypeptides described herein can be administered to a subject (e.g., a patient) in the range of about 0.1 mg / kg to 100 mg / kg of the patient's body weight, 0.1 mg / kg to 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to immune responses to foreign polypeptides. Therefore, it is often possible to administer lower and less frequent doses of human antibodies. Furthermore, the dosage and frequency of administration of a reagent or therapeutic composition of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) through modifications such as lipidation. A typical therapeutically effective dose range for an antibody or antibody fragment of the invention can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies can range, for example, from twice daily to once weekly.

[0133] When a fragment (e.g., an antibody fragment) is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of an antibody. Such peptides can be synthesized chemically and / or by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds as needed for the particular indication being treated, e.g., those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine (e.g., IL-15), chemotherapeutic agent, growth inhibitory agent, etc. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0134] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules) or macroemulsions. Sustained-release formulations can also be prepared.

[0135] The pharmaceutical or therapeutic carrier or diluent used can be a conventional solid or liquid carrier.Non-limiting examples of solid carriers include lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ether of cellulose.Non-limiting examples of liquid carriers include syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water.Similarly, carriers or diluents include glyceryl monostearate or glyceryl distearate, and sustained-release materials known in the art can be used alone or mixed with wax.

[0136] If a solid carrier is used for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier varies widely but can be from about 25 mg to about 1 g.

[0137] If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

[0138] The composition and / or preparation may also be in a form suitable for local or systemic injection or infusion, and may therefore be formulated with sterile water or isotonic saline or glucose solution. The composition may be in a form adapted for peripheral administration only, excluding forms that can be administered centrally. The composition and / or preparation may be in a form adapted for central administration.

[0139] The composition and / or preparation can be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution can be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation can be combined with a sterile aqueous solution before administration. The composition and / or preparation can contain pharmaceutically and / or therapeutically acceptable auxiliary substances necessary to approximate physiological conditions, such as buffers, tonicity adjusters, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0140] Embodiment Examples of the embodiments disclosed herein are disclosed below in numbered paragraphs.

[0141] 1. An antigen trap or receptor trap as disclosed herein.

[0142] 2. The antigen or receptor trap of embodiment 1, comprising an antigen or epitope to which a chimeric antigen receptor (CAR) binds.

[0143] 3. The antigen or receptor trap of embodiment 2, wherein the CAR is present on a T cell (CAR-T cell).

[0144] 4. Binding of the CAR to the antigen or trap a. substantially blocks binding of other antigens or epitopes to which the CAR can bind; and / or b. Does not substantially activate or suppress CAR-T cells 4. The antigen or receptor trap of embodiment 3.

[0145] 5. An antigen or epitope to which the CAR on a CAR-T cell can bind, CAR binding to the antigen a. inhibits the binding of the CAR to its cognate antigen displayed on the surface of a target cell; and / or b. Does not substantially activate or suppress CAR-T cells Antigen or epitope.

[0146] 6. The antigen or epitope of embodiment 5, which is not associated with a cell.

[0147] 7. The antigen or epitope of embodiment 5, further comprising an antibody Fc region fused to the antigen or epitope.

[0148] 8. The antigen or epitope of embodiment 5, which is derived from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0149] 9. The antigen or epitope of embodiment 5, which is derived from a blood (hematological) cancer cell or derived from a cell from a solid tumor.

[0150] 10. The antigen or epitope of embodiment 5, which is derived from a cell from B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL), or multiple myeloma.

[0151] 11. The antigen or epitope of embodiment 5, which is derived from cells derived from a brain tumor, a breast tumor, or a kidney tumor.

[0152] 12. The antigen or epitope according to epitope 5, which is derived from a cell derived from hepatocellular carcinoma, GPC3-positive hepatocellular carcinoma, liver cancer, lung cancer, advanced lung cancer, advanced solid tumor, colon cancer, colorectal cancer, EGFR-positive colorectal cancer, esophageal cancer, pancreatic cancer, prostate cancer, gastric cancer, sarcoma, osteoid sarcoma, Ewing's sarcoma, breast cancer, ovarian cancer, glioma, cervical cancer, lung squamous cell carcinoma, liver metastasis, liver tumor, gastric tumor, or advanced EGFR-positive solid tumor.

[0153] 13. The antigen or epitope of embodiment 5, wherein the CAR comprises a CD19-specific, human epidermal growth factor receptor 2 (HER2)-specific, or B-cell maturation antigen (BMCA)-specific CAR.

[0154] 14. The antigen or epitope of embodiment 5, which binds to a CAR specific for CD123, CD138, CD20, CD22, CD38, CD5, Igκ chain, LeY, NKG2D ligand, ROR1, or WT1.

[0155] 15. The antigen or epitope of embodiment 5, which is bound to a CAR specific for C-Met, CAIX, CD133, CD171, CD70, CEA, EGFR, EGFR vIII, Ep-CAM, EphA2, FAP, GD2, GPC3, HER2, HPV16-E6, IL13Ra2, LeY, MAGEA3, MAGEA4, MART1, mesothelin, MUC1, MUC16, NY-ESO-1, PD-L1, PSCA, PSMA, ROR1, or VEGFR2.

[0156] 16. The antigen or epitope of embodiment 5, comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or an amino acid sequence which is at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical thereto.

[0157] 17. The antigen or epitope of embodiment 5, wherein the nucleotide sequence encoding the antigen or epitope comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or a nucleotide sequence at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical thereto.

[0158] 18. A nucleic acid encoding an antigen or epitope according to embodiment 5.

[0159] 19. A vector comprising the nucleic acid of embodiment 18.

[0160] 20. A cell comprising the vector of embodiment 19.

[0161] 21. A method for treating a disease associated with CAR-T therapy or for increasing the efficacy of CAR-T therapy, comprising administering an antigen or epitope according to any one of embodiments 5 to 17. [Example]

[0162] In order that the present invention may be more fully understood, the following examples are set forth. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be utilized to achieve similar results.

[0163] Example 1 – Receptor trapping and expression The soluble ectodomain of the CAR antigen can prevent CAR-T cell interaction with antigens on tumor cells. CD19 is expressed on normal and malignant B cells. In one embodiment, the ectodomain of CD19 was tested as a receptor trap.

[0164] We first used the full-length extracellular domain of CD 19. In some embodiments, the wild-type CD19 ectodomain can be difficult to express.

[0165] The CD19 ectodomain described above was expressed both as a monomer (CD19ecto) and as an Fc-fused dimer (CD19ecto-Fc). Upon expression of these polypeptides, we observed higher-order protein aggregation and / or clustering (Figure 7). We also observed higher-order aggregation / clustering when the Fc-fused dimers were arranged as tetramers (Figure 8).

[0166] Example 2 - Function of receptor traps To test the ability of various receptor traps to affect CAR-T cell activity, we used Jurkat cells expressing a chimeric antigen receptor (CAR) specific for the extracellular domain of CD19. When activated by binding to the extracellular domain of CD19 on tumor cells, the Jurkat cell line expressed GFP, which could be detected using flow cytometry. We used a second cell line, K562, which expresses the extracellular domain of CD19 on its surface. In this assay, when Jurkat CAR binds to CD19 on the surface of K562 cells, the activation of Jurkat cells is measured by the expression of GFP in Jurkat cells.

[0167] In the assay, inactivated Jurkat cells alone (without K562) produced a peak of background fluorescence (red peak in Figure 9). Upon addition of K562 cells and binding of Jurkat CAR to the extracellular CD19 domain on the surface of the K562 cells, the Jurkat cells became activated and could be detected as a fluorescent cell population (blue peak in Figure 9) that exceeded the background fluorescence of inactivated Jurkat cells (red peak).

[0168] To test for receptor traps, we added them to a combination of Jurkat cells and K562 cells. In the absence of CD19ecto protein, co-incubation of CD19+ K562 leukemia cells with anti-CD19-CAR-Jurkat NFAT-GFP cells induced Jurkat activation (as indicated by high GFP expression) (Figure 9). Upon addition of CD19ecto (Figure 10) or CD19ecto-Fc protein (Figure 11), a dose-dependent decrease in GFP fluorescence was observed, indicating that these proteins can block CAR-T / tumor interactions. Furthermore, removal of the proteins restored the inhibitory effect. Furthermore, both CD19ecto and CD19ecto-Fc showed negligible activation of CAR-Jurkat cells in the absence of tumor cells.

[0169] In other studies, engineered CD19 variants were used to improve expression yields and reduce aggregation. Several variants of this type are described in Klesmith, Justin R., et al., "Retargeting CD19 chimeric antigen receptor T cells via engineered CD19-fusion proteins." Molecular pharmaceutics 16.8 (2019): 3544-3558. An example of the expression of this type of variant is shown in Figure 12. Data showed that these variants improved CD19 expression and reduced aggregation (Figure 13).

[0170] To test the activity of the variants, the Jurkat-K562 cell assay described above was used. Figure 14 shows that the variants have improved IC compared to the previous receptor trap. 50 The data shown in Figures 15-17 indicate that the inhibition caused by receptor traps is reversible. The data in Figures 18-22 indicate that receptor traps minimally affect Jurkat CAR-T cells in the absence of activating K562 tumor cells.

[0171] Example 3 – Receptor traps for HER2-specific and BCMA-specific CARs Figure 23 shows gel analysis of receptor traps specific for Her2-specific and BCMA-specific CARs.

[0172] Figure 24 shows the expression of CARs, including Her2- and BCMA-specific CARs, in primary T cells using retroviral vectors.

[0173] Figure 25 shows an overview of the retroviral vector used to express the CAR in the previous figure.

[0174] Figure 26A shows the effect of a BCMA-specific receptor trap.

[0175] FIG. 26B shows the effect of a HER2-specific receptor trap.

[0176] Figure 26C shows the effect of another HER-2 specific receptor trap.

[0177] Example 4 – Effect of CAR-specific receptor trap (CAR-Trap) on CAR-T cells Figure 27A shows a schematic of this study.

[0178] Figure 27B shows results demonstrating that CAR-specific antigen traps blocked interferon gamma production by primary CAR-T cells.

[0179] Figure 27C shows results demonstrating that the CAR-specific antigen trap prevented tumor cell killing by CAR-T cells. The data also show that tumor cell killing resumed when the antigen trap was removed.

[0180] Example 5 –Characterization of CD19 ectodomain variants for inhibiting CAR-T cell activity CD19 CAR-Trap (wtCD19 CAR-Trap, Figure 28A) was engineered by cloning and expressing the ectodomain of wild-type CD19 (CD19-wt), consisting of amino acids 20–291, fused to the Fc portion of IgG1 (CD19wt-Fc) (Figure 28A). To evaluate the inhibitory effect of this protein, we used a Jurkat / tumor coculture assay. A CAR-Jurkat cell line expressing the nuclear factor of activated T cell (NFAT)-GFP activation marker was engineered and used as a CAR-T cell model to evaluate the inhibitory effect of CD19 CAR-Trap, while the human immortalized myeloid leukemia cell line K562, which overexpresses CD19, served as the tumor cell. Overnight co-incubation of these cell lines resulted in a 59-fold increase in the NFAT-GFP signal in CAR-Jurkat, indicating robust tumor-antigen-induced CAR-Jurkat activation. Adding increasing doses of CD19wt-Fc to the cocultures resulted in a dose-dependent decrease in Jurkat activation (Fig. 28B), however, the IC50 value was suboptimal (>200 nM) and only ∼30% of NFAT-GFP was downregulated at the highest concentration tested.

[0181] Protein SDS-PAGE electrophoresis revealed higher-order oligomers of the CD19wt-Fc protein, indicating misfolding or the formation of nonspecific intermolecular interactions (Figure 28C). Consistently, the expression yield of this protein was very low (Figure 28D). This suggests that the CD19-wt ectodomain may not be inherently stable.

[0182] Highly stable CD19 mutants (19.1, C6.2, NT.1, and CT.2; Figure 28A) were expressed as fusions to IgG1 Fc (Figure 28A). These mutants appeared as homogenous bands on SDS-PAGE gels (Figure 28C) and demonstrated significantly improved inhibitory potency, with IC50 values ​​in the 5-7 nM range, approximately 50-fold improvement compared to CD19wt-Fc (Figure 28B). Protein expression yields were increased 79- to 208-fold compared to CD19wt-Fc (Figure 28D).

[0183] CD19NT.1 was selected for further analysis based on the observed IC50 and protein expression yield (Figure 28B, C). Structural analysis showed that the mutations in CD19NT.1 were located away from the interaction interface, suggesting that these mutations primarily contribute to the increased stability of the CD19 ectodomain (Figure 28E).

[0184] Other experiments investigated the role of multivalency in inhibiting CAR-Jurkat / tumor cell interactions. Multivalency of a molecule allows it to simultaneously bind to multiple targets, thereby increasing its overall binding strength, or "avidity." Because CAR-T / cancer cell interactions involve the binding of numerous CD19 molecules to CARs at the cell-cell interface, multivalent CAR-Trap molecules may be more effective than monovalent molecules at blocking this interaction. The inhibitory activity of monomeric, dimeric, and tetrameric CAR-Trap molecules expressing the CD19NT.1 domain alone, at the N-terminus of IgG1 Fc, or at the N- and C-termini of IgG1 was evaluated using the inhibition assay described above (Figures 29A and 29B). While the dimer and tetramer exhibited comparable IC50 values, the monomer exhibited >80x lower potency, highlighting the significance of multivalency (Figures 29C and 29D). These results indicate that improving the stability and avidity of the native CD19 ectodomain leads to a potent protein-based OFF switch for regulating anti-CD19 CAR-Jurkat cells.

[0185] Example 6 - CAR density influences T cell responses to CAR-Trap and tumor cells Recent findings indicate that the density of CAR molecules within the T cell membrane can affect CAR signaling and the overall efficacy of CAR-T therapy. To assess whether CAR density can affect baseline, ligand-independent, sustained signaling and responses to both CAR-Trap and tumor cells (Figure 30A, B), Jurkat cells were infected with an EF1a-CAR construct incorporating a CD28 costimulatory domain and sorted into 10 distinct expression groups (Figure 30C). These groups were either untreated or exposed to various concentrations of CAR-Trap or tumor cells at a 1:1 effector:T cell ratio. The level of CD69, an early T cell activation marker, was measured to assess T cell activation status under various treatment conditions.

[0186] As shown in Figure 30D, baseline CAR activation gradually increased with increasing density of CAR molecules. The correlation between CD69 levels and CAR expression followed an exponential curve, indicating that more signaling-competent complexes were formed as CAR expression increased.

[0187] Treatment with CAR-Trap can amplify CAR-T cell activity by promoting CAR dimerization (Figure 30D). The relationship here appeared nearly linear, demonstrating a direct correlation between CAR density and CAR-Trap-induced activation. Conversely, responses to tumor cells did not increase uniformly across all CAR expression levels. As CAR expression increased, tumor-induced activation appeared to reach a limit, with no further activation observed in the highest expression group.

[0188] To extrapolate these findings to primary CAR-T cells, we compared the expression levels of primary CAR-T cells with 10 CAR-Jurkat groups. We isolated primary human CD8+ T cells using an established Ficoll isolation protocol, generated CAR-T cells using lentivirus, and compared the cell surface CAR expression of CAR-T cells with the CAR-Jurkat group. The surface expression of CAR in primary T cells was similar to that in Group 1, suggesting that CAR-Trap likely leads to minimal baseline activation and may serve as an effective inhibitor in this setting.

[0189] To investigate the behavior of clinically generated CAR-T cells and their response to CAR-Trap, we quantified the cell surface CAR density in our cell line system and compared it to the number reported for clinical CAR-T cells. The clinical CAR-T cell product had a CAR density similar to the lowest expressing population in our experimental setup, 1 × 10 per T cell. 3 ~1×10 5 Therefore, CAR-Trap can function as an inhibitor in these settings, inducing minimal baseline activation.

[0190] These results demonstrate the importance of CAR density in determining baseline tonic signaling, response to CAR-Trap, and response to tumor cells. Quantification and comparison of cell surface CARs in primary CAR-T cells and clinical CAR-T products demonstrates that CAR-Trap does not significantly trigger tonic signaling in these settings, but rather functions primarily as an inhibitor.

[0191] Example 7 - CAR density influences T cell responses to CAR-Trap and tumor cells To assess the reversibility of the inhibitory activity described above, anti-CD19 CAR-Jurkat cells and K562 cells were co-incubated with CAR-Trap for 12 hours. CAR-Trap was removed from the co-culture assay after 12 hours. Cells were then incubated overnight with or without CAR-Trap, and NFAT-GFP was measured the following day. As shown in Figure 29E, for all concentrations tested, CAR-Jurkat cells resumed activation in the absence of CAR-Trap, demonstrating the reversibility of the CAR-Trap switch.

[0192] To determine whether CD19 CAR-Trap could regulate the activity of primary human CAR-T cells, CD19+ A375 cells were used in a washout assay, as shown in Figure 31A. In the absence of CAR-Trap, CD19+ A375 cells triggered the activation of primary human CAR-T cells and induced the release of interferon gamma (IFN-γ) (Figure 31B). These CAR-T cells mediated antitumor effects. Fluorescence microscopy of mCherry-labeled A375 cells revealed CAR-T cell-mediated killing of A375 cells after 48 hours of coculture (Figure 31C). When co-cultured with CAR-Trap, a dose-dependent inhibition of IFN-γ release was observed, with an IC50 of 2 nM (Figure 31B).

[0193] Example 8 –BCMA-CAR protein switch BCMA CAR-Trap was developed by replacing the CD19 NT.1 domain of the CD19 NT.1-Fc dimer with the BCMA ectodomain (aa 2-54) within the dimeric CAR-Trap molecule (Figure 32A, B). This BCMAwt-Fc fusion protein was well expressed and appeared as a homogenous band on an SDS-PAGE gel (Figure 32A, B).

[0194] To evaluate the inhibitory effect of BCMAwt-Fc, CAR-Jurkat cells expressing BCMA-CAR with a CD3zeta signaling motif were incubated overnight with NCI-H929 cells, a BCMA-overexpressing human plasma cell line widely used to study multiple myeloma. As shown in Figure 32E, a three-fold increase in CD69 signaling in CAR-Jurkat cells was observed after overnight incubation, indicating robust activation of CAR-Jurkat cells by cancer cells. Addition of increasing doses of BCMAwt-Fc to the coculture led to a dose-dependent decrease in Jurkat cell activation, with an IC50 of 36 nM (Figure 32C). BCMA CAR-Trap also inhibited CAR-Jurkat cell activation mediated by MM1.S cells, another B-lymphoblastoid cell line.

[0195] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein which are considered to be within the scope of this invention.

Claims

1. Chimeric Antigen Receptor (CAR) - an ectodomain derived from a tumor-specific antigen (TSA) or tumor-associated antigen (TAA) means that binds to the CAR on T cells; a dimerization means; A receptor trap comprising: Binding of the antigen trap to the CAR inhibits binding of the CAR to the TSA or TAA on tumor cells, thereby inhibiting activation of the CAR-T cells. The receptor trap.

2. The receptor trap of claim 1 , wherein said dimerization means comprises an IgG antibody Fc domain fused to said ectodomain means.

3. The receptor trap of claim 2 , wherein the link between the ectodomain means and the IgG antibody Fc domain comprises a glycine-rich linker.

4. A receptor trap according to any one of claims 1 to 3, comprising two or more ectodomain means.

5. A dimer of the receptor trap according to any one of claims 1 to 4.

6. The receptor trap of any one of claims 1 to 5, wherein the ectodomain means comprises an ectodomain derived from CD19 or B-cell maturation antigen (BCMA).

7. 7. The receptor trap of any one of claims 1 to 6, wherein the ectodomain means comprises a molecule having the amino acid sequence of SEQ ID NO: 26 or 27 or an amino acid sequence which is 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

8. 8. The receptor trap of any one of claims 1 to 7, wherein the ectodomain means comprises a variant CD19 ectodomain having an IC50 that is at least 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than the IC50 of a wild-type CD19 ectodomain.

9. 10. A method for treating side effects of CAR-T cell therapy or CAR-T cell exhaustion in a cancer patient, the method comprising administering to said cancer patient a receptor trap according to any one of claims 1 to 8.

10. 10. The method of claim 9, wherein the cancer patient has received CAR-T cell therapy for a cancer selected from the group consisting of B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin's lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL), and multiple myeloma.

11. 11. The method of claim 9 or 10, wherein the side effects include cytokine release syndrome (CRS) or neurotoxicity.

12. The receptor trap of any one of claims 1 to 8 for use in treating side effects of CAR-T therapy or CAR-T cell exhaustion in cancer patients.

13. Recombinant protein comprising a multivalent ectodomain derived from CD19 or B-cell maturation antigen (BCMA) or a variant thereof and an IgG antibody Fc domain A receptor trap comprising: The receptor trap, wherein binding of the multivalent ectodomain or a variant thereof by a CAR on a chimeric antigen receptor (CAR)-T cell reversibly inhibits activation of the CAR-T cell.

14. The receptor trap of claim 13 , wherein the link between the multivalent ectodomain or variant and the IgG antibody Fc domain comprises a glycine-rich linker.

15. 15. The receptor trap of claim 13 or 14, wherein the ectodomain comprises SEQ ID NO: 26 or 27, or a sequence which is 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

16. 16. The receptor trap of any one of claims 13 to 15, wherein the ectodomain comprises a variant of a CD19 or BCMA ectodomain that has an IC50 that is at least 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than the IC50 of wild-type CD19 or BCMA ectodomain.

17. 17. The receptor trap of any one of claims 13 to 16, comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, or 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

18. A dimer of the receptor trap according to any one of claims 13 to 17.

19. A nucleotide sequence encoding the receptor trap of any one of claims 1 to 8 or 13 to 18.

20. 20. The nucleotide sequence of claim 19, comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, or a nucleotide sequence which is 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

21. A pharmaceutical composition comprising a receptor trap or a receptor trap dimer according to any one of claims 1 to 8 or 13 to 18.

Images