Reversible control of chimeric antigen receptor t cells

Abstract

Receptor traps that modulate immune cells are disclosed. The receptor traps can have an extracellular domain from a tumor cell, which is capable of binding to a chimeric antigen receptor (CAR) on a CAR-T cell, thereby reversibly inhibiting activation of the CAR-T cell.

Description

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

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as of the date of the present 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 by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights whatsoever. Technical Field

[0004] Aspects of the invention relate to compositions and methods for modulating molecules on the surface of cells, including chimeric antigen receptors (CARs) on the surface of CAR-T cells, to reversibly control CAR receptors and receptors on cancer cells, thereby inhibiting cancer cell signaling and growth. Sequence Listing

[0005] This application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [], is named [] and is [] bytes in size. Background Art

[0006] Chimeric antigen receptor (CAR) T cells have become a promising therapy for patients with hematological malignancies. However, the widespread use of CAR-based immunotherapy has been limited by potentially life-threatening toxicities, and the therapeutic effect is limited by a decrease in persistence. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are two of the most common toxicities observed after CAR-T cell therapy. Further, a decrease in persistence is typically due to immune cell exhaustion, wherein the continuous signaling performed via CAR ultimately results in low or no efficacy and treatment failure or recurrence. Therefore, desirable CAR-based immunotherapy will include limiting the toxicity caused by unwanted downstream signaling effects and controlling or limiting the activity of CAR to avoid the ability to exhaust. Summary of the Invention

[0007] Disclosed herein are novel reagents and methods for regulating molecules on the cell surface. In some embodiments, reagents and methods are used to control the level of CAR molecules on CAR-T cells. In some embodiments, 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, reagents and methods are used to control or inhibit cancer cell growth or regulate an immune response.

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

[0009] In some embodiments, the extracellular domain element can be fused with a dimerization element. The dimerization element can form a dimer of two receptor traps. Dimerization can increase the binding valence of the receptor trap. In some embodiments, the dimerization element can be the Fc portion of an antibody. In some embodiments, the binding of CAR to the trap molecule can block CAR-T cells (e.g., inhibiting the binding of CAR to molecules that can activate CAR-T cells; inhibiting CAR-T cell activation). In some embodiments, the blocking or inhibition can be reversible. In some embodiments, the binding of CAR to the trap molecule does not activate or substantially does not activate the CAR-T cell. 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.

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

[0011] In some embodiments, methods of administering an antigen / receptor trap to a subject are disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Certain illustrations, diagrams, or flow charts are provided to allow a better understanding of the present invention. However, it should be noted that the drawings only illustrate selected embodiments of the present invention and therefore should not be considered to limit the scope. There are other and equally effective embodiments and applications of the present invention.

[0013] Figure 1A schematic diagram showing CAR-T cell therapy for a patient.

[0014] Figure 2 Demonstrated are exemplary methods for using immunosuppressive agents to treat toxicities observed following CAR-T cell therapy.

[0015] Figure 3 Demonstrated are exemplary methods for treating toxicities observed following CAR-T cell therapy using suicide genes or elimination markers.

[0016] Figure 4 Demonstrated are exemplary methods for using reversible genetic switches to treat toxicities observed following CAR-T cell therapy.

[0017] Figure 5 We demonstrate a reversible CAR-T regulatory mechanism as a strategy to enhance CAR-T cell efficacy.

[0018] Figure 6 Demonstrated are exemplary methods for controlling CAR-T cell activation using receptor traps as disclosed herein.

[0019] Figure 7 Examples of expression of CD19 receptor trap monomers and dimers are shown.

[0020] Figure 8 An example of CD19 extracellular domain grafting onto human IgG1 Fc and molecular expression is shown.

[0021] Figure 9 An example of an anti-CD19 Jurkat NFAT GFP / CD19-K562 co-culture assay is shown.

[0022] Figure 10 Results show that CD19 receptor trap monomers blocked activation of anti-CD19 Jurkat CAR in a dose-dependent manner.

[0023] Figure 11 demonstrated that the CD19-Fc dimer receptor trap molecule was more effective than the monomer in blocking activation of the anti-CD19 Jurkat CAR.

[0024] Figure 12 Results show improved expression yields and reduced aggregation of engineered CD19 receptor trap variants.

[0025] Figure 13 An example of grafting of the improved CD19 extracellular domain into human IgG1 Fc and molecular expression is shown.

[0026] Figure 14 Demonstrated engineered CD19 receptor trap variants with improved IC50 Results of blocking anti-CD19 Jurkat CAR activation.

[0027] Figure 15 We demonstrate that receptor trap inhibition of Jurkat cell activation is reversible.

[0028] Figure 16 Additional results are presented showing that receptor trap inhibition of Jurkat cell activation is reversible.

[0029] Figure 17 Additional results are presented showing that receptor trap inhibition of Jurkat cell activation is reversible.

[0030] Figure 18 Results demonstrated that antigen / receptor traps inhibited Jurkat cell activation in the presence of K562 and minimally affected Jurkat CAR-T cells in the absence of activated tumor cells.

[0031] Figure 19 Additional results demonstrated that antigen / receptor traps minimally affected Jurkat CAR-T cells in the absence of activated tumor cells.

[0032] Figure 20 Additional results demonstrated that antigen / receptor traps minimally affected Jurkat CAR-T cells in the absence of activated tumor cells.

[0033] Figure 21 Additional results demonstrated that antigen / receptor traps minimally affected Jurkat CAR-T cells in the absence of activated tumor cells.

[0034] Figure 22 Additional results demonstrated that antigen / receptor traps minimally affected Jurkat CAR-T cells in the absence of activated tumor cells.

[0035] Figure 23 Expression of antigen / receptor traps containing the extracellular domain of HER2, an epitope from the HER2 protein bound by the Herceptin antibody (Herceptin BD-FC), or the extracellular domain of BCMA was demonstrated.

[0036] Figure 24 Results are shown for the expression of CARs specific for CD19, HER2, or BCMA in primary T cells.

[0037] Figure 25 Schematic diagrams of retroviral vectors used to express various CARs are shown.

[0038] Figure 26A 、 Figure 26B and Figure 26C The activities of the antigen / receptor traps against BMCA, HER2, and HER2 shown in Figure 32 are shown separately. Each figure shows the results without target cells (left) and with target cells (right).

[0039] Figure 27A 、 Figure 27B and Figure 27C Schematic diagram of a study examining the effects of CD19-specific CARTrap 9 (A) and the effects of CARTrap 9 on cytokine production in primary cells (B) and tumor cell killing (C). The data show that CARTrap inhibits / ablates interferon-γ production (B). The data show that CARTrap abrogates tumor killing, and that tumor killing is restored by removal of CARTrap.

[0040] Figure 28A-28E. Characterization of CD19 extracellular domain 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 K562 cell activation of CAR-Jurkat cells by various CD19 CAR-Trap molecules and corresponding IC50 values are shown. NFAT-GFP is used to measure CAR-Jurkat cell activation levels via flow cytometry. (C) SDS-PAGE gel characterization of five CAR-Trap proteins is shown. (D) Comparison of protein expression levels of CD19 extracellular domain variant CAR-Trap and CD19wt CAR-Trap is shown. (E) Structural representation of the CD19 / CAR scFv complex is shown, indicating that mutations in CD19 NT.1 are primarily located away from the interaction interface. Models were constructed using PyMOL based on PDB 7URV.

[0041] Figures 29A-29E. Characterization of multivalent CD19 extracellular domain fusions for inhibiting CAR-T cell activity. (A) Schematic representation of CD19 NT.1 monomers, dimers, and tetramers is shown. (B) SDS-PAGE gel confirmation of monomer, dimer, and tetramer fusion protein formation is shown. (C) Dose-dependent inhibition of K562 cell activation of CAR-Jurkat cells by different CAR-Trap molecules and corresponding IC50 values are shown. NFAT-GFP was used to quantify CAR-Jurkat cell activation levels by flow cytometry. (D) A diagram depicting a comparison of monomeric and dimeric CAR-Trap competing with tumor surface antigens for binding to CAR on the T cell membrane. Given that multiple CARs and tumor antigens are simultaneously engaged at the cancer cell-T cell interface, dimeric CAR-Trap will demonstrate a superior ability to disrupt this interaction. (E) The reversibility of CAR-Trap activation of CAR-Jurkat is shown.

[0042] Figure 30A-30D. (A) depicts a schematic representation of the effect of CAR density on baseline, ligand-independent tonic signaling, and responses to both CAR-Trap and tumor cells. (B) depicts the intensity of various CAR-T cell signals. (C) Different expression groups of Jurkat cells expressing EF1a-CAR constructs, which were untreated or exposed to different concentrations of CAR-Trap, or exposed to tumor cells at a 1:1 effector cell: T cell ratio. (D) CD69 levels for each group included in (C).

[0043] Figure 31A-Figure 31D.CAR-Trap reversibly regulates primary CAR-T cells. (A) Schematic diagram of the setting of the primary CAR-T cell co-culture assay. The secreted IFN-γ level was measured to determine the CAR-T cell activation level in the presence of CD19+A375 cells and CAR-Trap; Live cell fluorescence microscopy was used to determine the anti-tumor effect. (B) Measurement of human primary CAR-T cell IFN-γ release in the co-culture assay described in (a) using IFNγ split-luciferase assay (Promega). The data represent 2 independent experiments. (C) Fluorescence microscopy of mCherry-labeled A375 cells, which shows reversible control of CAR-T cell-mediated A375 killing with CAR-Trap. (D) Overlay of bright field and mCherry channel images, which shows that CAR-T cell-mediated A375 killing activity is restored over time after CAR-Trap washout.

[0044] 32A-32C. Engineering of BCMA CAR-Trap. (A) Structure of the BCMA extracellular domain derived from PDB 4ZFO. (B) Schematic representation of the BCMA CAR-Trap and its corresponding SDS-PAGE validation. (C) Dose-dependent inhibition of BCMA CAR-Trap on activation of anti-BCMA CAR-Jurkat cells by H929 cells and corresponding IC50 values. DETAILED DESCRIPTION

[0045] Chimeric antigen receptor (CAR) T cells have emerged as a promising therapy for patients with hematological malignancies ( Figure 1 However, in some cases, CAR-T therapies can cause side effects in patients receiving these cells, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0046] Several regulatory mechanisms have been developed to control CAR-T cells in vivo to address these adverse events. However, these strategies have not satisfactorily addressed current needs, as toxicity and deaths continue to be reported in CAR-T clinical trials.

[0047] Disclosed herein are novel, modular, and reversible strategies for modulating CAR-T cell activity. Typically, these methods do not require additional genetic engineering of CAR-T cells. In some embodiments, these strategies can modulate CAR-T toxicity. In some embodiments, these strategies can increase the efficacy of CAR-T cell therapy. In some embodiments, these strategies are based on reversible blocking or internalization of CAR receptors.

[0048] Disclosed herein are recombinant protein switches based on the extracellular domains of cell surface molecules that chimeric antigen receptors (CARs) can bind to. These protein switches can be referred to as "CAR-Traps." In some embodiments, the extracellular domain can be a tumor cell surface antigen. CAR-Trap can reversibly and quantitatively "adjust" CAR-T cell activity.

[0049] In some embodiments, antigen traps (including receptor traps) may include antigens or ligands that a CAR receptor can bind to. Receptor traps can act as a CAR OFF switch by binding to the CAR receptor and blocking its interaction with antigens or ligands that would normally be found on tumor cells to which the CAR-T cells would bind (triggering CAR-T cell activation).

[0050] For example, the antigen / receptor trap molecule can be a molecule that the CAR of a CAR-T cell specifically binds to. In some embodiments, the binding of the CAR to the antigen / receptor trap molecule inhibits the binding of the CAR to the same or similar molecules on the surface of the tumor cell. In some embodiments, the binding of the CAR to the antigen / receptor trap prevents or reduces the activation of the CAR-T cell. In some embodiments, the binding of the CAR to the antigen / receptor trap can activate the CAR-T cell.

[0051] In some embodiments, receptor traps do not require engineering of CAR-T receptors or CAR-T cells and can be applied to CAR-T therapies that are already approved or in clinical development.

[0052] In other embodiments, the receptor trap can be reversible. Reversibility can provide fine-tuning of CAR-T cell activity, for example, for toxicity management and / or for rejuvenating cells for continued therapy.

[0053] In some embodiments, receptor traps can be customized for CAR-T cells targeting different tumor antigens, for example, by replacing the components used in the design (i.e., the traps can be modular). These antigen traps can be designed by engineering the extracellular domains of tumor surface antigens. Antigen traps can interrupt the interaction between CAR-T cells and tumor cells.

[0054] Also disclosed are methods for enhancing CAR-T efficacy. Temporal "resting" of CAR-T cells can reverse CAR-T exhaustion. In some embodiments, the disclosed reversible CAR modulators can increase the efficacy of CAR-T cells by alternating CAR-T cells between "active" and "resting" states.

[0055] A detailed description of one or more embodiments is provided herein. However, it should be understood that the invention can be embodied in many forms. Therefore, the specific details disclosed herein should not be construed as limiting, but rather as a basis for the claims and a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.

[0056] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In the claims and / or the specification, use of the word "a" or "an," when used in conjunction with the term "comprising," may mean "one," but is also consistent with "one or more," "at least one," and "one or more than one."

[0057] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they should be understood to be followed by the phrase "without limitation." Similarly, "example," "exemplary," etc. should be understood as non-limiting.

[0058] The term "substantially" allows deviations from the descriptive terms that do not adversely affect the intended purpose. Descriptive terms should be understood as being modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0059] The terms "comprising" and "including," as well as "having" and "involving" (and similarly "comprises," "includes," "has," and "involves") and the like are used interchangeably and have the same meaning. In particular, the definition of each of these terms is consistent with the common definition of "comprising" under U.S. patent law and is therefore to be interpreted as open-ended terms meaning "at least the following," and is also to be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it should be understood to mean "one or more" unless such an interpretation is meaningless in the context.

[0060] As used herein, the term "about" can mean approximately, roughly, roughly, or within the range of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the numerical values set forth above. The term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent upward or downward (higher or lower). CAR-T cells, toxicity, and controlling toxicity

[0061] Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers. Figure 1). However, due to the lack of control over the infused CAR-T cells, the widespread use of the therapy may be limited by potentially life-threatening toxicities. Toxicity is an obstacle to the development of CAR-T therapies for blood cancers and solid tumors. Reported deaths caused by CAR-T therapies have recently been discussed in the literature (Neelapu, Sattva S. et al. "Toxicity management after chimeric antigen receptor T cell therapy: one size does not fit'ALL' [Toxicity management after chimeric antigen receptor T cell therapy: one size does not fit'ALL']." Nature Reviews Clinical Oncology [Nature Reviews Clinical Oncology] 15.4 (2018): 218-218).

[0062] Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the two most common toxicities observed after CAR-T cell therapy.

[0063] CRS can be characterized by high fever, hypoxia, hypotension, or multi-organ toxicity; it develops in 37%-93% of patients with lymphoma and 77%-93% of patients with leukemia. ICANS is characterized by confusion, delirium, seizures, or cerebral edema; it develops in 23%-67% of patients with lymphoma and 40%-62% of patients with leukemia. Severe CRS and ICANS require monitoring and treatment in an intensive care setting, and multiple deaths have been reported due to unmanageable CRS or ICANS toxicity.

[0064] Three types of treatments for these toxicities are currently in use.

[0065] In some cases, immunosuppressants ( Figure 2) treat patients with immunosuppressants, including systemic corticosteroids, IL-6 receptor antibodies (e.g., tocilizumab), lymphocytotoxic anti-CD52 antibodies (e.g., alentuzumab), tyrosine kinase inhibitors (e.g., dasatinib) (LCK inhibitors do not inhibit activated T cells), etc. However, these treatments have limitations. For example, treatment with high-dose steroids can limit the time span for CAR-T cells to work and can induce hematological hypoplasia and toxicity. Anti-IL6 receptor antibodies have a variety of biological activities and can non-specifically suppress the immune system (Bonifant, Challice L. et al. "Toxicity and management in CAR T-cell therapy [toxicity and management in CAR T cell therapy]." Molecular Therapy-Oncolytics [molecular therapy-oncology] 3 (2016): 16011).

[0066] In some cases, suicide genes or marker elimination ( Figure 3 ) to treat patients, including iCasp9, anti-CD20 (e.g., rituximab), anti-EGFR (e.g., cetuximab), etc. However, these treatments have limitations. For example, these treatments can irreversibly and / or permanently eliminate CAR-T cells from the body (Brandt, JB et al. “Emerging approaches for regulation and control of CAR T cells: a minireview.” Frontiers in Immunology 11 (2020): 326.

[0067] In some cases, CAR-T cells with switchable CAR receptors can be used in patients ( Figure 4 ), including split CAR, SMaSh-CAR, CAR PROTAC, etc. However, these treatments have limitations. For example, these treatments may impair CAR-T activity, the switch may be leaky, and the switch may be immunogenic (Labanieh, Louai et al. "Enhanced safety and efficacy of protease-regulated CAR-T cell receptors." Cell [Cell] 185.10 (2022): 1745-1763).

[0068] However, it is known that reversible CAR-T regulatory mechanisms can be used to enhance CAR-T efficacy ( Figure 5 ). Constitutive CAR-T cells can show an increase in the level of exhaustion-related proteins. However, in some embodiments, transient "resting" can reverse the exhaustion phenotype. In some embodiments, the regulated CAR can be reversibly turned off and on to switch CAR-T cells between "off" and "on" states (Weber, Evan W. et al. "Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling." Science [Science] 372.6537 (2021): eaba1786; Labanieh, Louai et al. "Enhanced safety and efficacy of protease-regulated CAR-T cell receptors [Enhanced safety and efficacy of protease-regulated CAR-T cell receptors]." Cell [Cell] 185.10 (2022): 1745-1763). This can be a method for treating CAR-T cell exhaustion.

[0069] 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. Antigen or receptor trap

[0070] In some embodiments, disclosed herein are strategies for regulating molecules (e.g., the amount of molecules) on the cell surface and / or for regulating the activity of molecules on the cell surface using antigen traps or receptor trap methods. For example, this strategy may involve binding to cell surface molecules (e.g., proteins) of "trap" molecules. In some embodiments, the trap molecule may be an extracellular domain or extracellular domain element to which a chimeric antigen receptor (CAR) on the surface of a CAR-T cell can bind. In some embodiments, the extracellular domain element may be bound by a CAR-T cell in substantially the same manner as the extracellular domain can be bound by a CAR, but the extracellular domain element may be derived from a variant of the extracellular domain, etc.

[0071] In an embodiment, the binding of a trap molecule can affect the ability of a common ligand of a cell surface molecule to bind to a cell surface molecule (e.g., CAR) and / or regulate the molecule. In an embodiment, a trap molecule can bind to a cell surface molecule so that the binding of a regulatory protein to a cell surface molecule (e.g., an activator or repressor of a cell surface molecule; e.g., an epitope to which CAR can bind) is inhibited. In an embodiment, the binding of the trap molecule itself to a cell surface molecule minimally affects the regulation of the cell surface protein (e.g., the trap binding does not affect or minimally affects the upregulation and / or downregulation of the cell surface or membrane molecule / protein). In some embodiments, the trap molecule does not associate with the cell (e.g., free in solution). In some embodiments, the trap molecule can also have an Fc region of an antibody fused to a trap molecule or its extracellular domain.

[0072] In some embodiments, the cell surface molecule or protein can be a CAR molecule. In some embodiments, the CAR molecule can be on a CAR-T cell. In some embodiments, the strategy for regulating CAR-T activity includes blocking the CAR receptor with one or more antigen / receptor trap molecules. In some embodiments, the blocking of the CAR receptor by the trap is reversible. In some embodiments, the receptor trap can act as a CAR OFF switch (by binding to the CAR receptor on the T cell and blocking its interaction with the molecule to which the CAR receptor normally binds (e.g., a molecule on the cell) Figure 6 In some embodiments, the receptor trap can inhibit the activation of the CAR-T cell that binds to it. In some embodiments, the antigen-receptor trap molecule specific for CAR can be referred to as a CAR-Trap molecule.

[0073] In some embodiments, the receptor trap can be any antigen or epitope that a chimeric antigen receptor (CAR) specifically binds, provided that: i) the receptor trap molecule blocks binding of at least some "normal" antigens / epitopes that the CAR is intended to bind; and / or ii) it does not activate or repress or minimally activates or represses the CAR-T cell by 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 intended to bind). In some embodiments, the receptor trap can reversibly regulate the CAR-T cell.

[0074] In an embodiment, the receptor trap can be a modification of the antigen / epitope to which the CAR is intended to bind (e.g., an extracellular domain element). In some embodiments, the trap molecule can be similar to the antigen on the cell surface to which the CAR is intended to bind. In some embodiments, the IC50 of the extracellular domain element binding to the CAR can be at least 1 / 10, 1 / 20, 1 / 30, 1 / 40, or 1 / 50 of the wild-type extracellular domain that the same CAR can bind. In some embodiments, the trap molecule does not associate with the cell.

[0075] In some embodiments, the antigens or epitopes that the CAR binds to and uses in the antigen / receptor trap are those that are unique or substantially exclusive to tumors or cancer cells (e.g., tumor-specific antigens, tumor-associated antigens, etc.) That is, at least in some embodiments, the antigens or epitopes used in the antigen / receptor trap are not found or are minimally present on non-tumor or non-cancer cells.

[0076] In some embodiments, the trap molecule can be derived from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0077] In an embodiment, the trap molecule can be derived from a blood (hematology) cancer cell or a cell from a solid tumor. In an embodiment, the target antigen or epitope can 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 an embodiment, the target antigen or epitope can be derived from a brain tumor, a breast tumor, a kidney tumor, etc.

[0078] In an embodiment, the target antigen or epitope can be from hepatocellular carcinoma, GPC3-positive hepatocellular carcinoma, liver cancer, lung cancer, advanced lung cancer, advanced solid tumors, 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, squamous cell lung cancer, liver metastasis, liver tumor, gastric tumor, advanced EGFR-positive solid tumor, etc.

[0079] In some embodiments, the antigen / receptor trap molecule is based on the extracellular domain or extracellular domain element from CD19 or B cell maturation antigen (BCMA).

[0080] In some embodiments, the engineered extracellular domain can be multivalent (eg, a multimer).

[0081] In some embodiments, the density of CAR molecules on CAR-T cells can affect the response of CAR-T cells to CAR-Trap and tumor cells.

[0082] In an embodiment, the target antigen or epitope used in the antigen / receptor trap may include CD19, B cell maturation antigen (BCMA), human epidermal growth factor 2 (HER2), etc.

[0083] In some embodiments, the CD19 extracellular domain or extracellular domain element can be (SEQ ID NO: 26):

[0084] PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSL GLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRD SLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK

[0085] In some embodiments, the CD19 extracellular domain can be a variant with a lower IC50 than the wild-type molecule. In some embodiments, the CD19 extracellular domain can be a 19.1, C6.2, NT.1, CT.2 variant of CD19 ( Figure 28A ).

[0086] In some embodiments, the BCMA extracellular domain or extracellular domain element can be (SEQ ID NO: 27):

[0087] LQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNS VKGTNA

[0088] In an embodiment, the target antigen or epitope used in the antigen / receptor trap may include a molecule from a hematological cancer. In an embodiment, the target antigen or epitope used in the antigen / receptor trap may include CD123, CD138, CD20, CD22, CD38, CD5, Ig kappa chain, LeY, NKG2D ligand, ROR1, WT1, etc.

[0089] In an embodiment, the target antigen or epitope used in the antigen / receptor trap may include molecules from solid tumors and / or cancers. In an embodiment, the target antigen or epitope used in the antigen / receptor trap may include 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, VEGFR2, etc.

[0090] In some embodiments, the antigen / receptor trap molecule can modulate the cell surface molecule (e.g., CAR) to which it binds. In some embodiments, the antigen / receptor trap can modulate 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.

[0091] In some embodiments, the dimerization element can be part of a CAR-Trap molecule. A dimerization element can be any region that can be associated with another dimerization region (typically in a separate CAR-Trap molecule) using a covalent bond or a non-covalent bond. The dimerized CAR-Trap molecule can be a homodimer or a heterodimer. There are many protein dimerization domains known in the art (e.g., see Dang, Dung Thanh. "Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry [Molecular methods for protein dimerization: supramolecular chemistry opportunities]." Frontiers in Chemistry [Chemistry Frontiers] 10 (2022): 829312). Exemplary dimerization domains can include a zipper motif, like a leucine zipper.

[0092] In some embodiments, the dimerization element can be an Fc region from an antibody that can be fused to an extracellular domain. In some embodiments, the Fc region is from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc region can be (SEQ ID NO: 25):

[0093] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0094] In some embodiments, the Fc region can dimerize to form a homodimer or heterodimer structure. In some embodiments, the Fc region can have or can be modified to have a cysteine amino acid capable of forming a disulfide bond (1 or more, such as 2 disulfide bonds). In some embodiments, a dimer of a CAR-Trap with an Fc region can be formed by disulfide bonds between cysteine residues in separate CAR-Trap molecules.

[0095] In certain embodiments, the Fc region can be a variant comprising an amino acid substitution that changes the effector function independent of the antigen, such as the circulating half-life of the molecule connected thereto. Compared to the Fc region lacking these substitutions, the molecules connected to these Fc regions (e.g., extracellular domains) can exhibit increased or decreased binding to FcRn, and can have an increased or decreased half-life in serum, respectively. Fc variants with improved FcRn affinity are expected to have a longer serum half-life, and such molecules have useful applications in methods requiring the connected molecule to have a long half-life. By contrast, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules are also useful in cases where a shortened circulation time may be advantageous. Fc variants with reduced FcRn binding affinity are also unlikely to pass through the placenta. In addition, other applications of the FcRn binding affinity that may need to be reduced include those applications that need to be located in the brain, kidney, and / or liver. In one embodiment, the molecule linked to the Fc variant may exhibit reduced transport from the vasculature across the glomerular epithelium.

[0096] In another embodiment, the CAR-Trap molecules connected to Fc variants can show a reduction in transport from the brain through the blood-brain barrier (BBB) into the vascular space. In one embodiment, the Fc region with the FcRn combination of changes is included in the Fc domain with one or more amino acid substitutions in the "FcRn binding loop" of the Fc domain. The FcRn binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in PCT Publication No. WO 05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the bispecific modulator disclosed herein includes one or more Fc domains with the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0097] In some embodiments, the extracellular domain disclosed herein can be connected to an Fc variant comprising an amino acid substitution that changes glycosylation. For example, an Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation). In some embodiments, the Fc variant comprises reduced glycosylation of N-linked glycans typically found at amino acid position 297 (EU numbering). In another embodiment, these molecules can have an amino acid substitution near or within a glycosylation motif, such as an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In a specific embodiment, the Fc variant can have an amino acid substitution at amino acid position 228 or 299 (EU numbering). Exemplary amino acid substitutions that confer reduced or altered glycosylation are described in PCT Publication No. WO 05 / 018572, which is incorporated herein by reference in its entirety.

[0098] In some embodiments, the molecules disclosed herein can be modified to eliminate glycosylation and can be referred to as "aglycosylated" molecules. Exemplary aglycosylated molecules can have an aglycosylated Fc region of an IgG4 antibody, which lacks Fc effector functions, thereby eliminating the possibility of Fc-mediated toxicity to normal important tissues and cells. In yet other embodiments, the molecules disclosed herein can have altered glycans. For example, a reduced number of fucose residues can be present on the N-glycan at Asn297 of the Fc region, i.e., afucosylated. In some embodiments, a modified number of sialic acid residues can be present on the N-glycan at Asn297 of the Fc region.

[0099] In some embodiments, the CH2 or CH3 region of the Fc antibody domain can be truncated or modified to modulate the half-life of the molecule. In some embodiments, the Fc truncation includes 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).

[0100] In some embodiments, the Fc region may have or may be modified to have cysteine amino acids capable of forming disulfide bonds. In some embodiments, dimers or tetramers of CAR-Trap molecules may be formed by disulfide bonds between cysteine residues in the Fc region of individual CAR-Trap molecules (e.g., Figure 13 , Figure 28A or Figure 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 the CAR-Trap molecule that are not Fc regions. These dimers can be homodimers. In some embodiments, heterodimers can be formed. These multimers can have multiple extracellular domains (e.g., can be multivalent for the extracellular domains). In some embodiments, these CAR-Trap molecules can have 2, 3, 4, 5, 6, 7, 8 or more extracellular domains.

[0101] In some embodiments, a connection (e.g., a joint) can be located between different segments of the CAR-Trap molecule. In some embodiments, this connection can be located between the extracellular domain or an extracellular domain element and a dimerization element. In some embodiments, the connection can be a glycine-rich joint ("GS" joint). In some embodiments, a "GS" joint can be a combination of glycine and serine amino acids. In some embodiments, the GS joint can be GSSGGSGGSGGS (SEQ ID NO: 28). Other sequences are possible. In some embodiments, the GS joint 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 joint can have at least 4 amino acids as glycine and / or serine. In some embodiments, other amino acids can be part of a GS joint, as long as glycine and serine account for the majority.

[0102] 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% identical to the following nucleotide and amino acid sequences. Specifically, the amino acid sequence of the antigen / receptor trap molecule can be labeled as follows:

[0103] The underlined Times New Roman font is the signal peptide ;

[0104] The bold Times New Roman font is the extracellular domain;

[0105] Times New Roman font in italics is a linker, which in some embodiments is encoded by the creation of a restriction enzyme site;

[0106] The underlined and bold Times New Roman font is the GS connector ;

[0107] Times New Roman fonts that are underlined, italicized, and bold are cuttable joints. ;

[0108] The underlined Courier New font is the Fc area ;

[0109] The bold Courier New font is the TEV site;

[0110] The underlined and bold Courier New font is the His tag ;and

[0111] The underlined, italicized, and bold Courier New font is an Avi tag. .

[0112] pDP10 CD19-WT FC (SEQ ID NO: 1)

[0113]

[0114] (SEQ ID NO:2)

[0115]

[0116] pDP11 CD19-WT(SEQ ID NO:3)

[0117]

[0118] (SEQ ID NO:4)

[0119]

[0120] pDP32-CD19-FC-CD19 WT(SEQ ID NO:5)

[0121]

[0122] (SEQ ID NO:6)

[0123]

[0124] pDP33_CD19.1_Fc(SEQ ID NO:7)

[0125]

[0126] (SEQ ID NO:8)

[0127]

[0128] pDP34_CD19 C6.2-Fc(SEQ ID NO:9)

[0129]

[0130] (SEQ ID NO:10)

[0131]

[0132] pDP35_CD19 NT.1-Fc(SEQ ID NO:11)

[0133]

[0134] (SEQ ID NO:12)

[0135]

[0136] pDP36_CD19 CT.2-Fc(SEQ ID NO:13)

[0137]

[0138] (SEQ ID NO: 14)

[0139]

[0140] pDP45-CD19 NT.1-FC-CD19 NT.1 tetramer (SEQ ID NO: 15)

[0141]

[0142] (SEQ ID NO: 16)

[0143]

[0144] pDP47-CD19 NT.1_monomer (SEQ ID NO: 17)

[0145] ATGCGAATGCAGCTGCTGCTGCTGATTGCGCTGAGCCTGGCGCTGGTGACCAACAGCACTAGTcccgaggaacctctagtggtgaaggtggaagagggagataccgctgctctgtggtgcctcaaggggacctcagatggccccactcagcagctgacctggtctcgggagtccccgcttaaacccttcttaaaatacagcctgggggtgccaggcctgggagtccacgtcaggcccgatgccatctctgtcgtcatcaggaacgtctctcaacagatggggggcttctacctgtgccagccggggcccccctctgagaaggcctggcagcctggctggacagtcaatgtggagggcagcggggagctgttccggtggaatgtttcggacctaggtggcctgggctgtggcctgaagaacaggtcctcagagggccccagctccccttccgggaagctcatgagccccaagctgtatgtgtgggccaaagaccgccctgagatctgggagggagagcctccgtgtctcccaccgagggacagcctgaaccagagcctcagcagggacctcaccgtagcccctggctccacactctggctgtcctgtggggtaccccctgactctgtgtccaggggccccctctcctggacccatgtgcaccccaaggggcctaagtcattgctgagcctagagctgaaggacgatcgcccggccagagatatgtgggtaatgggtacgtcactgatgttgccccgggccacagctcaagacgctggaaagtggtattgtcaccgtggcaacgtaaccacctcattccacctggaggtaatcgctcggccagtaaaggctcactcagacctgaggactggtggctggaagACTAGTTCTGGTGGTGGTGGTGGTGAGAATCTGTACTTTCAGAGCTCGGGCGGAGGATCGGGTGGAGGCCACCACCATCATCACCACCATCACGGATCCGGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAAGGCTAA

[0146] (SEQ ID NO:18)

[0147]

[0148] pDP37-HER2 ETD_Fc(SEQ ID NO:19)

[0149]

[0150] (SEQ ID NO:20)

[0151]

[0152] pDP38-Herceptin BD-FC(SEQ ID NO:21)

[0153]

[0154] (SEQ ID NO:22)

[0155]

[0156] pDP39-BCMA ECD-FC(SEQ ID NO:23)

[0157]

[0158] (SEQ ID NO:24)

[0159] Antibody

[0160] When relevant to polypeptide (such as antibody) or polynucleotide, " recombinant " refers to the form of polypeptide or polynucleotide that does not exist in nature, and its non-limiting example can be created by combining polynucleotide or polypeptide that do not normally occur together. As used herein, " polypeptide (polypeptide) " can encompass the singular " polypeptide (polypeptide) " as well as the plural " polypeptide (polypeptides) ", and refers to the molecule consisting of monomers (amino acids) connected linearly by amide bonds (also referred to as peptide bonds). The term " polypeptide " refers to any one chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, " protein ", " amino acid chain " or any other term for referring to one chain or multiple chains of two or more amino acids may be referred to as " polypeptide " in this article, and the term " polypeptide " can be used to replace any one of these terms or be used interchangeably with any one of these terms. "Polypeptide" can also refer to the product of post-expression modification of the polypeptide, and these modifications include but are not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis. For amino acid sequences, those skilled in the art will readily recognize that single substitutions, deletions or additions (which change, add, delete or replace a single amino acid or a small percentage of amino acids in the encoded sequence) carried out on nucleic acids, peptides, polypeptides or protein sequences are collectively referred to herein as "conservatively modified variants". In some embodiments, the change results in amino acid substitution by chemically similar amino acids. Providing a conservative substitution table of functionally similar amino acids is well known in the art. Compared to unmodified antibodies, such conservatively modified variants of the antibodies disclosed herein can exhibit increased cross-reactivity.

[0161] For example, a "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced by another amino acid residue from the same side chain family. In another embodiment, an amino acid chain can be replaced by a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0162] Some embodiments also feature antibodies with a specified percentage of identity or similarity to the amino acid or nucleotide sequences of the antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When 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, when compared to a specified region or the full length of any of the antibodies described herein, the antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity. In some embodiments, the present invention provides the method for the present invention to determine the sequence identity or similarity of the nucleic acid of the present invention and protein.For example, when compared with any one of the designated region or the total length of the antibody as described herein, the antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher nucleic acid identity. The sequence identity or similarity with the nucleic acid of the present invention and protein can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described below: Ausubel et al., edits (2007) Current Protocols in Molecular Biology. [contemporary molecular biology program]. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment or visual inspection can be used to determine the sequence identity or similarity percentage of the nucleic acid of the present invention and protein.

[0163] Aspects of the present invention provide isolated antibodies. As used herein, the term "isolated" refers to molecules isolated from other DNA or RNA present in the natural origin of macromolecules. The term "isolated" can also refer to nucleic acids or peptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or that are substantially free of chemical precursors or other chemicals when chemically synthesized. For example, "isolated nucleic acid" can include nucleic acid fragments that are not naturally occurring as fragments and that are not found in the natural state. "Isolated" can also refer to cells or polypeptides separated from other cellular proteins or tissues. Isolation polypeptides can include both purified and recombinant polypeptides.

[0164] As used herein, "antibody" or "antigen-binding polypeptide" may refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a complete antibody and any antigen-binding fragment or single chain thereof. For example, an "antibody" may include any protein- or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule with biological activity that binds to an antigen. Non-limiting examples are the complementary determining regions (CDRs) of a heavy or light chain or its ligand-binding portion, 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" may refer to immunoglobulin molecules and immunoactive portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds to (immunoreacts with) an antigen. "Specific binding" or "immunoreacts with..." may refer to an antibody that reacts with one or more antigenic determinants of a desired antigen and does not react with other polypeptides.

[0165] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as an F (ab′)2 、F (ab)2 、F ab ′、F ab, Fv, scFv, etc. Regardless of the structure, antibody fragments will bind to the same antigen recognized by the intact antibody. The term "antibody fragment" may include aptamers (e.g., mirror image aptamers), miniantibodies, 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')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAbs (domain antibodies), miniantibodies, disulfide-linked Fv (sdFv), fragments comprising VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies.

[0166] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V L ) fusion protein of the variable region of the single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer that can be expressed by a gene fusion comprising a VH-encoding gene and a VL-encoding gene connected by a linker encoding a peptide. (See Huston et al. (1988) Proc Nat Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, rich in serine or threonine for solubility, and the V H The N-terminus of V L The C-terminus of the constant region is linked to the C-terminus of the antibody, and vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin. Many methods have been described for chemical structure analysis to convert naturally aggregated, but chemically separated, light and heavy polypeptide chains from antibody V regions into scFv molecules, which will fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site. See, for example, U.S. Patent Nos. 5,091,513; 5,892,019; 5,132,405; and U.S. Patent No. 4,946,778, each of which is incorporated by reference in its entirety.

[0167] Antibody molecules obtained from humans are divided into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ from each other by the nature of the heavy chains present in the molecule. It will be understood by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), as well as some subclasses thereof (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specialization. For IgG, a standard immunoglobulin molecule comprises 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. These four chains are connected by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains like brackets, starting at the mouth of the "Y" and continuing to the end of the variable region. The immunoglobulin or antibody molecules described herein can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0168] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be combined with a κ or λ light chain. For example, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0169] Both light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. The variable domains of the light (VL) and heavy (VH) chains determine antigen recognition and specificity. In contrast, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental transport, Fc receptor binding, complement fixation, etc. The term "antigen binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly distinct segments within the V regions of the heavy and light chains, called hypervariable regions, are interspersed between more conserved flanking segments called "framework regions" or "FRs." Therefore, the term "FR" can refer to the amino acid sequences naturally present between and adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs."

[0170] The six CDRs present in each antigen-binding domain are short discontinuous amino acid sequences that are specifically positioned to form antigen-binding domains when the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids (FR regions) in the antigen-binding domain show less intermolecular variability. The framework region mainly adopts a β-sheet conformation, and CDR forms a loop connecting the β-sheet structure, and in some cases forms a part of the β-sheet structure. The framework region plays the role of forming a scaffold that positions the CDR in the correct orientation by interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDR provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. One of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, for either the heavy or light chain variable region, as they have been previously defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0171] When there are two or more definitions for the terms used and / or accepted in the art, the definition of the term as used herein is intended to include all of these meanings, unless clearly stated otherwise. A particular example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen combining sites found in the variable region of heavy and light chain polypeptides. This region has been described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. [Journal of Molecular Biology] 196:901-917 (1987) (incorporated herein in its entirety by reference). The CDR definition according to Kabat and Chothia includes overlapping or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of an antibody or its variants is intended to fall within the scope of the terms defined and used herein. The appropriate amino acid residues of the CDRs defined by the references cited above are listed in the table below as a comparison. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR. CDR Kabat numbering Chothia Number VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0172] Kabat et al. have defined a numbering system for variable domain sequences that is applicable to any antibody. Technicians can clearly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0173] 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 fifteenth residue after 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 thirty-third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after the cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue after the end of CDR-L2 (ie, after the cysteine residue); comprises approximately 7-11 residues and ends at the sequence F or WGXG, where X is any amino acid.

[0174] As used herein, the term "epitope" may include any protein determinant that can specifically bind to an immunoglobulin, scFv or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, a subset of the VL domain and VH domain, or complementary determining regions (CDRs) of an antibody forms a variable region that defines a three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site present at each arm end of the Y. The epitope determinant can be composed of a chemically active surface molecule group (such as an amino acid or sugar side chain) and can have specific three-dimensional structural characteristics and specific charge characteristics. For example, antibodies can be produced against the N-terminal peptide or C-terminal peptide of a polypeptide. More particularly, 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 chain and the VL chain.

[0175] As used herein, the terms "immunobinding" and "immunobinding properties" may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of the immunobinding interaction can be measured by the dissociation constant (K) of the interaction. d ) to represent the smaller K d=A greater affinity indicates greater affinity. Methods well known in the art can be used to quantify the immunological binding properties of a selected polypeptide. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K on ) and the “dissociation rate constant” (K off ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). off / K on The ratio allows the elimination of all parameters not related to affinity and is equal to the equilibrium binding constant K D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) When the equilibrium binding constant (K) is quantified, the binding constant (K) is expressed as a function of the binding activity of the binding site. D )≤1 μM, ≤10 μM, ≤10 nM, ≤10 pM, or ≤100 pM to about 1 pM, the antibodies of the present invention can specifically bind to the epitope. For example, in some embodiments, K D Between approximately 1E-12M and approximately 1E-11M K D In some embodiments, K D Between approximately 1E-11M and approximately 1E-10MK D In some embodiments, K D Between approximately 1E-10M and approximately 1E-9M K D In some embodiments, K D Between approximately 1E-9M and approximately 1E-8M K D In some embodiments, K D Between approximately 1E-8M and approximately 1E-7M K D In some embodiments, K D Between approximately 1E-7M and approximately 1E-6M K D For example, in some embodiments, K D is about 1E-12M, while in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, while in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, while in other embodiments, KD is about 1E-7M. In some embodiments, K D is about 1E-6M, while in other embodiments, K D is about 1E-5M. For example, in some embodiments, K D is about 3E-11M, while in other embodiments, K D is about 3E-12M. In some embodiments, K D is about 6E-11 M. "Specifically binds" or "is specific for..." can refer to an antibody that binds to an epitope via its antigen-binding domain, and that such binding requires some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope.

[0176] For example, antibodies can be monovalent or bivalent and can comprise a single or double chain. Functionally, the binding affinity of an antibody is in the range of 10 -5 M to 10 -12 M range. For example, the binding affinity of an antibody is 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, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 - 9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M, or 10 -5 M to 10 -6 M.

[0177] Those skilled in the art will recognize that, without undue experimentation, one can determine whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the human monoclonal antibody blocks the specific binding of the human monoclonal antibody of the invention. For example, if the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as indicated by reduced binding of the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or a closely related epitope.

[0178] Another way to determine whether a human monoclonal antibody has the specificity of the human monoclonal antibodies of the present invention is to pre-incubate the human monoclonal antibodies of the present invention with an epitope with which they normally react, and then add the human monoclonal antibody to be tested to determine whether the ability of the human monoclonal antibody to be tested to bind to the epitope is inhibited. If the human monoclonal antibody to be tested is inhibited, it has the same or 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 utilizing the epitope and determining whether the test monoclonal antibody can neutralize a polypeptide containing the epitope.

[0179] Various procedures known in the art can be used to generate polyclonal or monoclonal antibodies to the proteins of the invention or their derivatives, fragments, analogs, homologs or orthologs (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).

[0180] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provides the IgG fraction of immune serum. Subsequently or alternatively, the specific antigen or its epitope as the target of the immunoglobulin sought can be fixed on a column to purify the immunospecific antibody by immunoaffinity chromatography. The purification of immunoglobulins is discussed, for example, by D.Wilkinson (The Scientist, Inc., Philadelphia, Pennsylvania, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0181] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" may refer to a population of antibody molecules comprising 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. A MAb contains an antigen binding site that can immunoreact with a specific epitope of an antigen, the epitope being characterized by having a unique binding affinity for the antigen binding site.

[0182] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. Nucleic acids, vectors, and cells expressing receptor traps

[0183] Also disclosed are nucleic acids encoding all or part of the receptor traps described herein. Also disclosed are various vectors (e.g., plasmids, viruses, etc.) comprising the nucleic acids. Also disclosed are various cells (e.g., prokaryotic cells, eukaryotic cells) that contain the nucleic acids or vectors and can express the fusion proteins. method

[0184] Disclosed herein are methods for administering the antigen / receptor traps described herein to a subject. In some embodiments, the antigen / receptor traps can target CAR receptors on CAR-T cells. In some embodiments, these methods are used to treat toxicity (e.g., toxicity due to cytokine release) in subjects who have received CAR-T cell infusions for cancer treatment or to treat CAR-T cell exhaustion in similar subjects. In some embodiments, these methods are used to improve the efficacy of CAR-T cells that have been administered to a subject to treat cancer.

[0185] In some embodiments, the subject being treated for cancer may have 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), multiple myeloma, etc.

[0186] In some embodiments, the reagents and methods disclosed herein can be used with cells that are not cancer cells. therapeutic preparations

[0187] Aspects of the present disclosure relate to therapeutic agents. As used herein, the term "therapeutic agent" may refer to any compound or composition (e.g., a receptor trap) that can be used or administered to obtain a therapeutic effect. As used herein, the term "therapeutic effect" may refer to an effect sufficient to result in an improvement in symptoms, such as the treatment, healing, prevention, or improvement of a related medical condition, or an increase in the rate of treatment, healing, prevention, or improvement of such a condition.

[0188] As described herein, the embodiments can be used to administer to a subject in the form of a pharmaceutical composition or therapeutic preparation prepared for an intended route of administration. Such compositions and preparations can include, for example, one or more active ingredients and a pharmaceutically acceptable carrier. Such compositions and preparations can be suitable for oral, subcutaneous, parenteral (such as intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, skin, vagina, cheek, eye or lung administration, such as for a form suitable for administration by a peripheral route, or for oral administration or for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions can be prepared in a manner well known to those skilled in the art, for example, as described in "Remington's Pharmaceutical Sciences," 17th edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, Pennsylvania, USA, 1985 and recent editions, and "Drugs and the Pharmaceutical Sciences," series of monographs, Marcel Dekker. These compositions and preparations may appear in conventional forms, such as solutions and suspensions for injection, capsules and tablets, in the form of enteric formulations (eg, as disclosed in US Pat. No. 5,350,741) and for oral administration.

[0189] Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0190] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM TM (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be fluid to the extent that it is easy to inject. In embodiments, it can be stable under manufacturing and storage conditions and can prevent the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium comprising the following substances: for example, water, ethanol, pharmaceutically acceptable polyols (such as, glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size under dispersion conditions, and by using a surfactant. Protection against the effects of microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal). In many cases, it can be useful to include isotonic agents such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption (for example, aluminum monostearate and gelatin) in the composition.

[0191] Sterile injectable solutions can be prepared by incorporating the compound in the desired amount into an appropriate solvent optionally with one or a combination of the ingredients enumerated herein, followed by sterilization by filtration. Dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0192] Oral compositions can include inert diluents or edible carriers. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral treatment, the active compound can be incorporated into and used in the form of tablets, lozenges, or capsules along with excipients. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is orally applied and gargled (swished) and spit out or swallowed. For example, depending on the half-life of the drug, the oral formulation of the drug can be used once a day, twice a day, three times a day, or four times a day.

[0193] Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition for administration to a subject. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, (sodium starch glycolate) or corn starch; a lubricant, such as magnesium stearate or sterotes; a glidant, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring, such as peppermint, methyl salicylate, or orange flavoring.

[0194] Systemic administration can also be carried out through mucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are known in the art and, for transmucosal administration, include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, these active compounds are formulated into ointments, salves, gels, or creams (as known in the art).

[0195] In embodiments, administration can include placing the pharmaceutical composition into a subject by a method or route that results in at least partial localization of the composition at a desired site such that a desired effect is produced.

[0196] For example, the pharmaceutical composition can be administered by bolus injection or by infusion. Bolus injection can refer to an administration route in which a syringe is connected to an IV access device and the drug is injected directly into the subject. The term "infusion" can refer to intravascular injection.

[0197] The embodiments described herein can be administered to a subject once (e.g., as a single injection, bolus, or deposition). Alternatively, the subject can be administered 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 lifetime of the subject. It can also be administered once or twice daily for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times / year, or a combination thereof.

[0198] In embodiments, the compositions described herein can be administered to a subject chronically."Chronic administration" can refer to administration in a continuous manner, such as to maintain a therapeutic effect (activity) over an extended period of time.

[0199] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof, the patient's age, weight, overall health, sex and diet, as well as the time of administration, rate of excretion, drug combination and the severity of the specific disease being treated. The judgment of the medical caregiver on these factors is within the scope of those of ordinary skill in the art. The amount will also depend on the individual patient to be 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.

[0200] The therapeutically effective amount of the reagent of the present invention or therapeutic composition can be the amount required for achieving therapeutic purposes. As described herein, this can be the binding interaction between the reagent or therapeutic composition and its target, and in some cases, the binding interaction interference target plays a function. In addition, the amount to be used will depend on the binding affinity of the reagent or therapeutic composition to its specific target, and will also depend on the rate of exhaustion of the free volume of the experimenter to which the reagent or therapeutic composition is applied. The dosage of the binding polypeptide as described herein applied to the experimenter (for example, patient) is about 0.1mg / kg to 100mg / kg patient weight, between 0.1mg / kg and 20mg / kg patient weight or 1mg / kg to 10mg / kg patient weight. Due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, it is generally possible to use lower doses of human antibodies and lower frequency. Further, the dosage and frequency of the reagent or therapeutic composition used in this disclosure can be reduced by enhancing the uptake and tissue permeability (for example, entering the brain) of these antibodies (for example, by modification such as, for example, lipidation). By way of non-limiting example, a typical range of therapeutically effective doses of the antibodies or antibody fragments of the invention may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may range, for example, from twice daily to once weekly.

[0201] When using a fragment (e.g., an antibody fragment), it is preferred that the minimum inhibitory fragment specifically binds to the target protein binding domain. For example, based on the variable region sequence of an antibody, a peptide molecule can be designed so that it retains the ability to bind to the target protein sequence. Such a peptide can be produced chemically and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 90: 7889-7893 (1993)). The formulation can also contain more than one active compound according to the needs of the specific indication being treated, such as those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent or a growth inhibitor. Such a molecule is suitably present in an amount effective for the intended purpose.

[0202] The active ingredient can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively. Sustained-release formulations can be prepared.

[0203] The pharmaceutical or therapeutic carrier or diluent adopted can be conventional solid or liquid carrier.The limiting examples of solid carrier are the lower alkyl ethers of lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid or cellulose.The limiting examples of liquid carrier are syrup, peanut oil, olive oil, phospholipids, fatty acid, fatty acid amine, polyethylene oxide and water.Similarly, carrier or diluent can comprise any sustained release material as known in the art separately or mixed with wax, for example glyceryl monostearate or glyceryl distearate.

[0204] When a solid carrier is used for oral administration, the preparation may be tableted, placed in a hard gelatin capsule in powder or pellet form or it may be in the form of a troche or lozenge.The amount of solid carrier will vary widely but may be from about 25 mg to about 1 g.

[0205] When 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.

[0206] The compositions and / or formulations 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. These compositions may be in a form suitable only for peripheral administration in addition to a form suitable for central administration. The compositions and / or formulations may be in a form suitable for central administration.

[0207] The compositions and / or formulations can be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution can be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized formulation can be combined with a sterile aqueous solution before administration. The compositions and / or formulations can contain pharmaceutically and / or therapeutically acceptable auxiliary substances as needed to approximate physiological conditions, such as buffers, tonicity regulators, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.

[0208] Example

[0209] Exemplary embodiments disclosed herein are disclosed in the following numbered paragraphs.

[0210] 1. An antigen trap or receptor trap as disclosed herein.

[0211] 2. The antigen or receptor trap of Example 1, comprising an antigen or epitope bound by a chimeric antigen receptor (CAR).

[0212] 3. The antigen or receptor trap of embodiment 2, wherein the CAR is on a T cell (CAR-T cell).

[0213] 4. The antigen or receptor trap of embodiment 3, wherein the binding of the CAR to the antigen or receptor trap:

[0214] a. substantially blocking the binding of other antigens or epitopes to which the CAR can bind; and / or

[0215] b. substantially does not activate or suppress the CAR-T cells.

[0216] 5. An antigen or epitope to which a CAR on a CAR-T cell can bind, wherein the binding of the CAR to the antigen:

[0217] a. inhibiting the binding of the CAR to its cognate antigen displayed on the surface of the target cell; and / or

[0218] b. substantially does not activate or suppress the CAR-T cells.

[0219] 6. The antigen or epitope of embodiment 5, wherein the antigen or epitope is not associated with cells.

[0220] 7. The antigen or epitope of embodiment 5, further comprising an Fc region of an antibody fused to the antigen or epitope.

[0221] 8. The antigen or epitope of embodiment 5, wherein the antigen or epitope is from a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).

[0222] 9. The antigen or epitope of embodiment 5, wherein the antigen or epitope is from a blood (hematological) cancer cell or a cell from a solid tumor.

[0223] 10. The antigen or epitope of embodiment 5, wherein the antigen or epitope is from a cell from B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL), or multiple myeloma.

[0224] 11. The antigen or epitope of embodiment 5, wherein the antigen or epitope is from a cell derived from a brain tumor, a breast tumor, or a kidney tumor.

[0225] 12. An antigen or epitope as described in epitope 5, wherein the antigen or epitope is from a cell from hepatocellular carcinoma, GPC3-positive hepatocellular carcinoma, liver cancer, lung cancer, advanced lung cancer, advanced solid tumors, 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, squamous cell lung cancer, liver metastasis, liver tumor, gastric tumor or advanced EGFR-positive solid tumor.

[0226] 13. The antigen or epitope of embodiment 5, wherein the CAR comprises a CD19-specific CAR, a human epidermal growth factor receptor 2 (HER2)-specific CAR, or a B cell maturation antigen (BMCA)-specific CAR.

[0227] 14. The antigen or epitope of embodiment 5, wherein the antigen or epitope is bound to a CAR specific for CD123, CD138, CD20, CD22, CD38, CD5, Igκ chain, LeY, NKG2D ligand, ROR1, or WT1.

[0228] 15. The antigen or epitope of embodiment 5, wherein the antigen or epitope 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.

[0229] 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 at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0230] 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% identical thereto.

[0231] 18. A nucleic acid encoding the antigen or epitope of embodiment 5.

[0232] 19. A vector comprising the nucleic acid of embodiment 18.

[0233] 20. A cell comprising the vector of embodiment 19.

[0234] 21. A method for treating a disease associated with CAR-T therapy or for increasing the efficacy of CAR-T therapy, the method comprising administering the antigen or epitope of any one of Examples 5-17. Examples

[0235] Provide example below to help more fully understand the present invention.The following example illustrates the exemplary mode of making and practicing the present invention.Yet the scope of the present invention is not limited to the specific embodiment disclosed in these examples, and these examples are only for illustrative purposes, because can utilize alternative method to obtain similar result. Example 1 - Receptor trap and expression

[0236] The soluble extracellular domain of the CAR antigen can prevent the interaction of CAR-T cells with antigens on tumor cells. CD19 is expressed on normal and malignant B cells. In one embodiment, the use of the extracellular domain of CD19 as a receptor trap was tested.

[0237] First, the full-length extracellular domain of CD 19 was used. In some embodiments, the wild-type CD 19 extracellular domain may be difficult to express.

[0238] The extracellular domain of CD19 is expressed as both a monomer (CD19ecto) and an Fc-fused dimer (CD19ecto-Fc). In the expression of these polypeptides, higher order protein aggregation and / or clustering was observed ( Figure 7 Higher order aggregation / clustering was also observed when Fc-fusion dimers were arranged as tetramers ( Figure 8 ). Example 2 - Functionality of receptor traps

[0239] To test the ability of various receptor traps to affect the activity of CAR-T cells, Jurkat cells expressing a chimeric antigen receptor (CAR) specific for the extracellular domain of CD19 were used. The Jurkat cell line expresses GFP when activated by binding to the extracellular domain of CD19 on tumor cells, which can be detected using flow cytometry. A second cell line, K562, expressing the extracellular domain of CD19 on its surface was used. In the assay, when the Jurkat CAR binds to CD19 on the surface of K562 cells, the activation of the Jurkat cells is measured by the expression of GFP in the Jurkat cells.

[0240] In the assay, inactivated Jurkat cells alone (without K562) produced a background fluorescence peak ( Figure 9 When K562 cells are added and Jurkat CAR binds to the extracellular CD19 domain on the surface of K562 cells, Jurkat cells are activated and can be seen as a fluorescent cell population (above the background fluorescence (red peak) of inactivated Jurkat cells). Figure 9 The blue peak in the figure is detected.

[0241] To test the receptor trap, the receptor trap was added to a combination of Jurkat and K562 cells. In the absence of CD19ecto protein, co-incubation of CD19+ K562 leukemia cells and anti-CD19-CAR-Jurkat NFAT-GFP cells induced Jurkat activation (indicated by high GFP expression) ( Figure 9 ). When adding CD19ecto( Figure 10 ) or CD19ecto-Fc protein ( Figure 11 A dose-dependent decrease in GFP fluorescence was observed in the absence of CAR-T cells (CD19ecto), indicating that these proteins can block CAR-T / tumor interactions. Furthermore, protein removal reversed the inhibitory effect. Furthermore, both CD19ecto and CD19ecto-Fc showed negligible activation of CAR-Jurkat cells in the absence of tumor cells.

[0242] In other studies, engineered CD19 variants were used to increase expression yield and reduce aggregation. Some 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 The data showed that these variants increased CD19 expression and reduced aggregation ( Figure 13 ).

[0243] To test the activity of the variants, the Jurkat-K562 cell assay described above was used. Figure 14 Data are presented showing that the variant has an improved IC compared to previous receptor traps 50 . Figure 15-17 The data presented show that the inhibition caused by receptor traps is reversible. Figures 18-22 The data in [ 14 ] show that receptor traps minimally affect Jurkat CAR-T cells in the absence of activated K562 tumor cells. Example 3 - Receptor traps for HER2-specific CAR and BCMA-specific CAR

[0244] Figure 23 Gel analysis of receptor traps specific for Her2-specific CAR and BCMA-specific CAR is shown.

[0245] Figure 24 Shown is the expression of CARs (including Her2-specific CAR and BCMA-specific CAR) in primary T cells using retroviral vectors.

[0246] Figure 25 Schematic diagram of the retroviral vector used to express the CAR in the previous figure is shown.

[0247] Figure 26A The effect of a BCMA-specific receptor trap is shown.

[0248] Figure 26B The effect of a HER2-specific receptor trap is shown.

[0249] Figure 26C The effect of another HER-2 specific receptor trap is shown. Example 4 - Effects of CAR-specific receptor traps (CAR-Trap) on CAR-T cells

[0250] Figure 27A A schematic diagram of the study is shown.

[0251] Figure 27B Results are shown, demonstrating that CAR-specific antigen traps interfere with interferon gamma production by primary CAR-T cells.

[0252] Figure 27C Results are shown, indicating that CAR-specific antigen traps interfere with tumor cell killing by CAR-T cells. The data also show that tumor cell killing is restored when the antigen traps are removed. Example 5 - Characterization of CD19 extracellular domain variants for inhibiting CAR-T cell activity CD19 CAR-Trap (wtCD19CAR-Trap, Figure 28 A) (Figure 28 A) was engineered by cloning and expressing the extracellular domain (CD19-wt) of wild-type CD19 fused to the Fc portion of IgG1 (composed of amino acids 20-291) (CD19wt-Fc). In order to evaluate the inhibitory effect of the protein, Jurkat / tumor co-culture assay was used. The CAR-Jurkat cell line expressing nuclear factor of activated T cells (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 overexpressing CD19 represented cancer cells. Overnight co-incubation of these cell lines resulted in a 59-fold increase in the NFAT-GFP signal in CAR-Jurkat, indicating that tumor antigen-induced CAR-Jurkat activation was robust. The increasing dose of CD19wt-Fc added to the co-culture resulted in a dose-dependent reduction in Jurkat activation (Figure 28 B). However, the IC50 values were suboptimal (>200 nM), and only about 30% of NFAT-GFP was downregulated at the maximum concentration tested.

[0253] Protein SDS-PAGE electrophoresis revealed high-order oligomers of CD19wt-Fc protein, indicating the formation of misfolding or nonspecific molecular interactions (Figure 28C). Consistently, the expression yield of this protein was extremely low (Figure 28D). This suggests that the CD19-wt extracellular domain may not be inherently stable.

[0254] CD19 mutants exhibiting high stability (19.1, C6.2, NT.1, and CT.2; FIG. 28A ) were expressed as fusions with IgG1 Fc ( FIG. 28A ). These mutants were displayed as homogenous bands on SDS-PAGE gels ( FIG. 28C ) and exhibited significantly improved inhibitory potency with IC50s ranging from 5-7 nM—approximately 50-fold higher than CD19wt-Fc ( FIG. 28B ). Protein expression yields were increased 79-208-fold compared to CD19wt-Fc ( FIG. 28D ).

[0255] Based on IC50 and observed protein expression yield, CD19NT.1 was selected for further analysis (Figure 28B, Figure 28C). Structural analysis showed that the mutations in CD19NT.1 were positioned away from the interaction interface, indicating that these mutations mainly contribute to enhancing the stability of the CD19 extracellular domain (Figure 28E).

[0256] Other experiments have studied the multivalence that inhibits CAR-Jurkat / tumor cell interaction.The multivalence of the molecule can make it possible to combine multiple targets simultaneously, thereby increasing its overall binding strength or "avidity".Because CAR-T / cancer cell interaction requires multiple copies of CD19 and CAR to be combined at the cell-cell interface, multivalent CAR-Trap molecules can block this interaction more effectively than monovalent molecules.The inhibitory activity (Figure 29 A, Figure 29 B) of monomers, dimers and tetramers CAR-Trap expressing separate, N-terminal or CD19NT.1 domains at the N-terminal and C-terminal ends of IgG1Fc was evaluated using the above-mentioned inhibition assay.Dimers and tetramers show a suitable IC50, while monomers show a low> 80x effect, which highlights the importance of multivalence (Figure 29 C, Figure 29 D).These results show that enhancing the stability and avidity of the natural CD19 extracellular domain can produce a protein-based OFF switch for regulating the effective power of anti-CD19 CAR-Jurkat cells. Example 6 - CAR density affects T cell responses to CAR-Trap and tumor cells

[0257] Recent findings show that the density of CAR molecules in T cell membranes can affect the overall efficacy of CAR signal transduction and CAR-T therapy. In order to assess whether CAR density can affect baseline, ligand-independent tonic signal transduction and the response to both CAR-Trap and tumor cells (Figure 30 A, Figure 30 B). Jurkat cells are infected with the EF1a-CAR constructs incorporating CD28 costimulatory domains and sorted into ten different expression groups (Figure 30 C). These groups are untreated or exposed to different concentrations of CAR-Trap, or with 1: 1 effector cells: T cell ratios are exposed to tumor cells. The level of CD69 (a kind of early T cell activation marker) is measured to assess the T cell activation state under different treatment conditions.

[0258] As shown in Figure 30D, baseline CAR activation gradually increased as the density of CAR molecules increased. The correlation between CD69 levels and CAR expression followed an exponential curve, indicating that as CAR expression increased, more signaling-competent complexes were formed.

[0259] CAR-Trap treatment can enhance CAR-T cell activity by promoting CAR dimerization (Figure 30D). The relationship here appears to be almost linear, indicating that there is a direct correlation between CAR density and CAR-Trap-induced activation. On the contrary, at all CAR expression levels, the response to tumor cells did not increase uniformly. Tumor-induced activation appears to reach a saturation point as CAR expression increases, and no further activation is observed in the highest expression group.

[0260] To extrapolate these findings to primary CAR-T cells, their expression levels were compared with those of ten CAR-Jurkat groups. Primary human CD8+ T cells were isolated using an established Ficoll separation protocol, CAR-T cells were generated using lentivirus, and their cell surface CAR expression was compared with that of the CAR-Jurkat group. CAR surface expression in primary T cells was similar to that of group 1, suggesting that CAR-Trap may result in minimal baseline activation and act as an effective inhibitor in these situations.

[0261] To investigate the behavior of clinically generated CAR-T cells and their response to CAR-Trap, the density of cell-surface CARs in the cell line system was quantified and compared to the number reported for clinical CAR-T cells. The clinical CAR-T cell product had a CAR density similar to that of the lowest expressing population in the experimental setting, containing 1 x 10 per T cell. 3 -1x 10 5 Therefore, in these cases, the CAR-Trap can act as an inhibitor, inducing minimal baseline activation.

[0262] These results demonstrate the importance of CAR density in determining baseline tonic signaling, responses to CAR-Trap, and responses to tumor cells. Quantification and comparison of cell-surface CAR in primary CAR-T cells and clinical CAR-T products demonstrate that CAR-Trap does not significantly trigger tonic signaling in these conditions but rather acts primarily as an inhibitor. Example 7 - CAR density affects T cell responses to CAR-Trap and tumor cells

[0263] 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 12 hours later. The cells were then incubated overnight with or without CAR-Trap, and NFAT-GFP was measured the next day. As shown in Figure 29E, for all concentrations tested, CAR-Jurkat cells recovered their activation in the absence of CAR-Trap, demonstrating the reversibility of the CAR-Trap switch.

[0264] To determine whether CD19 CAR-Trap can control the activity of primary human CAR-T cells, CD19+A375 cells were used in elution assays, as depicted 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 anti-tumor effects. Fluorescence microscopy of mCherry-labeled A375 cells revealed CAR-T cell-mediated A375 cell killing after 48 hours of co-culture (Figure 31C). When co-incubated with CAR-Trap, dose-dependent inhibition of IFN-γ release was observed, with an IC50 of 2nM (Figure 31B). Example 8-BCMA-CAR protein switch

[0265] BCMA CAR-Trap was developed by replacing the CD19NT.1 domain of the CD19NT.1-Fc dimer with the extracellular domain of BCMA (aa2 to 54) within the dimeric CAR-Trap molecular structure (Figures 32A, 32B). The BCMAwt-Fc fusion protein was well expressed and appeared as homogenous bands on SDS-PAGE gels (Figures 32A, 32B).

[0266] To evaluate the inhibitory effect of BCMAwt-Fc, CAR-Jurkat cells expressing BCMA-CAR with CD3ζ signaling motifs were incubated overnight with NCI-H929 cells (a human plasma cell line that overexpresses BCMA and is widely used in the study of multiple myeloma). As shown in Figure 32E, a 3-fold increase in the CD69 signal in CAR-Jurkat was observed after overnight incubation, indicating that cancer cells were robust in activating CAR-Jurkat cells. Adding increasing doses of BCMAwt-Fc to the co-culture resulted in 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). ***** 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. Such equivalents are considered to be within the scope of the present invention.

Claims

1. A receptor trap, comprising: an extracellular domain element derived from a tumor-specific antigen (TSA) or tumor-associated antigen (TAA) that binds to a chimeric antigen receptor (CAR) on a CAR-T cell, and dimerization element; The binding of the antigen trap to the CAR inhibits the binding of the CAR to the TSA or TAA on the tumor cell and inhibits the activation of the CAR-T cell.

2. The receptor trap of claim 1, wherein the dimerization element comprises an IgG antibody Fc domain fused to the extracellular domain element.

3. The receptor trap of claim 2, wherein the connection between the extracellular domain element and the IgG antibody Fc domain comprises a glycine-rich linker.

4. The receptor trap of any one of claims 1-3, wherein the receptor trap comprises two or more extracellular domain elements.

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 1-5, wherein the extracellular domain element comprises an extracellular domain from CD19 or B cell maturation antigen (BCMA).

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

8. The receptor trap of any one of claims 1-7, wherein the extracellular domain element comprises a variant CD19 extracellular domain having an IC50 that is at least 1 / 10, 1 / 20, 1 / 30, 1 / 40, or 1 / 50 that of a wild-type CD19 extracellular domain.

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

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 lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL), and multiple myeloma.

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 1-8, for use in treating side effects of CAR-T therapy or CAR-T cell exhaustion in cancer patients.

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

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

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

16. The receptor trap of any one of claims 13-15, wherein the extracellular domain comprises a variant of the extracellular domain of CD19 or BCMA having an IC50 that is at least 10-fold, 1-fold, 1-fold, 1-fold, 1-fold, 1-fold, or 1-fold lower than that of the wild-type CD19 or BCMA extracellular domain.

17. The receptor trap of any one of claims 13-16, comprising SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, or an amino acid sequence that is 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 according to any one of claims 1 to 8 or 13 to 18.

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 that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

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

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