Methods for controlling immune cell activity

Abstract

The present invention relates to a method for inhibiting the activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen, the method comprising the steps of: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to the subject a molecule for binding to the cellular immunotherapeutic agent, the molecule comprising or consisting of an epitope of the target antigen, wherein the epitope on the molecule competes with an epitope on the target antigen for binding to the cellular immunotherapeutic agent, thereby causing the molecule to disrupt the interaction between the cellular immunotherapeutic agent and the target antigen; thereby inhibiting the activity of the cellular immunotherapeutic agent in the subject.

Description

[Technical field]

[0001] The present invention relates to improved methods of treatment involving the administration of immune cells. Related Applications

[0002] This application claims priority to Australian Provisional Patent Application No. 2021901030, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002]

[0003] Adoptive immunotherapy, which involves the introduction of ex vivo generated antigen-specific T cells, is a promising strategy for treating cancer. The cells can be autologous, allogeneic, or derived from a third party that is HLA-matched (or partially matched). The T cells used in adoptive immunotherapy can be generated either by the expansion of antigen-specific T cells or by re-direction of T cells by genetic engineering.

[0003]

[0004] The gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) has successfully generated new specificities in T cells. CARs are synthetic receptors that consist of a targeting moiety associated with one or more signaling domains in a single fusion molecule. CARs have successfully enabled T cells to be redirected to antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors.

[0004]

[0005] In preclinical trials, various generations of CAR architectures have been developed, each with the aim of enhancing the activation signal, proliferation, cytokine production and effector function of CAR-modified T cells. For example, second generation CARs were developed to incorporate intracellular domains of one or more costimulatory molecules, such as CD28, OX40, and 4-1BB, within the endodomain, which improved the activation and expansion of antigen-specific T cells. Third generation CARs contain combinations of costimulatory endodomains. Fourth generation CARs contain activation-dependent cytokine secretion in addition to costimulatory endodomains. Fifth generation CAR-T cells contain deletions of human leukocyte antigen (HLA) and TCR genes in T cells obtained from healthy donors to avoid host immune rejection or graft-versus-host disease (GvHD) against the transplanted CAR T cells.

[0005]

[0006] Despite their promise in treating cancer, various adverse events have been reported with CAR-modified T cells. In one example, a patient died 5 days after cyclophosphamide chemotherapy followed by infusion of CAR-modified T cells recognizing the antigen ERBB2 (HER-2 / neu). Toxicity results in clinically significant release of proinflammatory cytokines, pulmonary toxicity, multiple organ failure, and ultimately death of the patient. This and other adverse events highlight the need for caution when employing CAR-modified T cells because, unlike antibodies against tumor-associated antigens, these cells are not cleared from the body in a short time period.

[0006]

[0007] Several studies have reported diverse systems aimed at improving the efficacy and safety of cellular immunotherapeutics. In one example, inhibitory chimeric antigen receptors (iCARs) were designed to stop / inhibit T cell function upon encountering off-target cells. iCARs consist of an antigen-specific single-chain variable fragment (scFv) fused to a T cell inhibitory signaling domain. Cells expressing tumor-associated antigens, but not normal tissue antigens, induce T cell activation, cytotoxicity, and cytokine signaling to kill cells on target. To function, iCAR technology relies on the preliminary selection of two antigens: one tumor-associated antigen and one normal tissue antigen.

[0007]

[0008] Another system involves the use of an anti-CD20 CAR combined with an inducible caspase 9 (iC9) suicide switch. The latter gene becomes functional in the presence of the prodrug AP1903 (tacrolimus) by binding to a mutated FK506 binding protein (FKBP1). Viral transduction introduces DNA from a vector into target cells, and the vector-derived DNA directs chemically induced dimerization (CID) and the expression of accessory proteins. In the presence of the AP1903 drug, dimerization of the CID protein occurs, thus turning on the signal cascade. In the event of severe or life-threatening toxicity caused by the administered T cells, AP1903 is injected to induce rapid destruction and elimination of CaspaCID™-enabled cells. A similar apoptosis induction system based on a multimerizing agent is described in WO 2014 / 152177.

[0008]

[0009] So-called "adapter CAR" platforms have also been developed, which involve a modular design in which the T cell is typically in an "off" state until an adapter molecule is provided that directs the T cell to a tumor-associated antigen, thereby facilitating activation of the CAR-T cell.

[0009]

[0010] Prior art approaches typically involve complex inhibitory or "off-switch" mechanisms to control immune cell activity, or alternatively require the use of complex adaptor systems to switch on CAR T cells. Furthermore, many of the approaches described in the prior art for "switching off" CAR-T cell activity result in cell death and therefore do not provide sufficient flexibility in the use of CAR-T cell-based immunotherapeutics.

[0010]

[0011] Improved approaches to control the activity of immune cell-based therapeutics, particularly CAR-T cell-based therapeutics, are needed.

[0012] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood to be relevant, considered to be relevant, and / or could reasonably be expected to be taken in combination with other prior art by a person of ordinary skill in the art. Summary of the Invention

[0011]

[0013] The present invention is based on the inventors' realization that it is possible to fine-tune the activity of immune cells that recognize and bind to specific target antigens on cancer cells.

[0014] In a first aspect, the present invention provides various methods for attenuating or temporarily "switching off" or inhibiting the activity of cellular immunotherapeutic agents, such as, but not limited to, CAR T therapy.

[0012]

[0015] In one embodiment of the first aspect of the invention, there is provided a method of inhibiting activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to a subject a molecule for binding to a cellular immunotherapeutic agent, the molecule comprising or consisting of an epitope of a target antigen. wherein an epitope on the molecule competes with an epitope on the target antigen for binding to the cellular immunotherapeutic agent, thereby causing the molecule to disrupt an interaction between the cellular immunotherapeutic agent and the target antigen; Thereby, a method is provided for inhibiting the activity of a cellular immunotherapeutic agent in a subject.

[0013]

[0016] Preferably, the inhibition is reversible, such that activity of the cellular immunotherapeutic agent is restored once the molecule is removed from the circulation of the subject. Preferably, the method does not reduce the viability of the cells of the cellular immunotherapeutic agent.

[0014]

[0017] Those skilled in the art are familiar with various cytotoxic events that can be induced or caused by cellular immunotherapeutic agents. Such cytotoxic events can include abnormal release of any cytokine or inflammatory response after administration of cellular immunotherapeutic agents. Abnormal release of cytokines can be referred to by various terms, including hypercytokinemia or "cytokine storm". "Cytokine release syndrome" (CRS) is a form of systemic inflammatory response syndrome (SIRS) that occurs when a large number of white blood cells are activated and release inflammatory cytokines, which in turn activate even more white blood cells. The term "cytokine storm" is often loosely used interchangeably with cytokine release syndrome (CRS), but is more accurately a distinguishable syndrome that may represent a severe episode of cytokine release syndrome or a component of another disease entity, such as macrophage activation syndrome. When occurring as a result of treatment, CRS symptoms may be delayed until several days or weeks after treatment. Immediate (fulminant) CRS is like a cytokine storm.

[0015]

[0018] Thus, in a further embodiment of the first aspect of the invention there is provided a method of minimising or reducing the risk of an aberrant inflammatory response in a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen on a cell, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to a subject a molecule for binding to a cellular immunotherapeutic agent, the molecule comprising or consisting of an epitope of a target antigen. wherein an epitope on the molecule competes with an epitope on the target antigen for binding to the cellular immunotherapeutic agent, thereby causing the molecule to disrupt an interaction between the cellular immunotherapeutic agent and the target antigen; Thereby, a method is provided for minimizing or reducing the risk of an aberrant inflammatory response in a subject. Preferably, the method does not reduce the viability of the cells of the cellular immunotherapeutic agent.

[0016]

[0019] Further provided is a method for treating an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to a subject a molecule for binding to a cellular immunotherapeutic agent, the molecule comprising or consisting of an epitope of a target antigen. wherein an epitope on the molecule competes with an epitope on the target antigen for binding to the cellular immunotherapeutic agent, thereby causing the molecule to disrupt an interaction between the cellular immunotherapeutic agent and the target antigen; Thereby, a method of treating an abnormal inflammatory response in a subject is provided. Preferably, the method does not reduce the viability of cells of the cellular immunotherapeutic agent.

[0017]

[0020] The abnormal inflammatory response may include cytokine-related toxicity. The inflammatory response may include a systemic inflammatory response and may be classified as systemic inflammatory response syndrome (SIRS). SIRS may be classified as "cytokine release syndrome" (CRS). The abnormal inflammatory response may be hypercytokinemia or "cytokine storm".

[0018]

[0021] The abnormal inflammatory response may include one or more of the conditions listed in Table 1 herein.

[0022] In further embodiments, it will be appreciated that periodic and reversible "switching off" of the cellular therapeutic agent is desirable to increase or maximize the persistence of the cellular therapeutic agent in the subject (in other words, the inactivation period for the cellular therapeutic agent), prevent immune cell depletion, and allow the cells to regain capability.

[0019]

[0023] Accordingly, the present invention provides a method of promoting or increasing the persistence of a cellular immunotherapeutic agent in a subject, the cellular immunotherapeutic agent being for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to a subject a molecule for binding to a cellular immunotherapeutic agent, the molecule comprising or consisting of an epitope of a target antigen. wherein an epitope on the molecule competes for binding to the cellular immunotherapeutic agent, and the molecule thereby disrupts an interaction between the cellular immunotherapeutic agent and the target antigen; Thereby, a method is provided for promoting or increasing the persistence of a cellular immunotherapeutic agent in a subject.

[0020]

[0024] It is understood that by administering a molecule that competes with the target antigen for binding to the cellular immunotherapeutic agent, the method provides a period during which the cellular immunotherapeutic agent is no longer active, or has reduced activity, thereby promoting the persistence of the cellular immunotherapeutic agent, as further described herein.

[0021]

[0025] moreover, a) inhibiting the activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent for binding to a target antigen; b) minimizing or reducing the risk of an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; c) treating an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; d) minimizing or reducing the risk of tumor lysis syndrome in subjects who have undergone or are undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; e) treating tumor lysis syndrome in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; or f) increasing the persistence of a cellular immunotherapeutic agent in a subject Use of a molecule comprising or consisting of an epitope of a target antigen in the manufacture of a medicament for the cellular immunotherapeutic agent is for binding to a target antigen, and the molecule competes with the target antigen for binding to the cellular immunotherapeutic agent, thereby causing the molecule to disrupt an interaction between the cellular immunotherapeutic agent and the target antigen; Preferably, the molecule does not target cellular immunotherapeutic agents to cell-mediated killing, uses are provided.

[0022]

[0026] Furthermore, a) inhibiting the activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent for binding to a target antigen; b) minimizing or reducing the risk of an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; c) treating an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; d) minimizing or reducing the risk of tumor lysis syndrome in subjects who have undergone or are undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; e) treating tumor lysis syndrome in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent to bind to a target antigen on a cell; or f) increasing the persistence of a cellular immunotherapeutic agent in a subject A composition comprising a molecule comprising or consisting of an epitope of a target antigen, for use in particular in the cellular immunotherapeutic agent is for binding to a target antigen, and the molecule competes with the target antigen for binding to the cellular immunotherapeutic agent, thereby causing the molecule to disrupt an interaction between the cellular immunotherapeutic agent and the target antigen; Preferably, compositions are provided in which the molecules do not target cellular immunotherapeutics to cell-mediated killing.

[0023]

[0027] In a further embodiment of the first aspect of the invention there is provided a kit for use in the methods described herein, the kit comprising: - a cellular immunotherapeutic agent for binding to a target antigen; and - a molecule for binding to a cellular immunotherapeutic agent, the molecule comprising or consisting of an epitope of a target antigen; wherein an epitope on the molecule competes with a target antigen for binding to a cellular immunotherapeutic agent, and the molecule thereby disrupts the interaction between the cellular immunotherapeutic agent and the target antigen.

[0024]

[0028] Optionally, the kit comprises instructions for use in the method of the first aspect of the invention.

[0029] In any embodiment of the first aspect, the molecule reduces the ability of the cellular immunotherapeutic agent to bind to the target antigen on the cell, particularly the target antigen is expressed or present on the surface of the cancer cell.Preferably, the molecule blocks the interaction between the cellular immunotherapeutic agent and the target antigen present on the cell.In other words, the cellular immunotherapeutic agent comprises a portion for binding to the target antigen on the cancer cell, and the molecule comprises an epitope similar or identical to the target antigen, so that the molecule competes with the target antigen on the cell for binding to the cellular immunotherapeutic agent.Therefore, the method of the present invention comprises a molecule for replacing or preventing or reducing the binding of the cellular immunotherapeutic agent to its target antigen on the cancer cell.

[0025]

[0030] Preferably, the cell that comprises the target antigen is a cancer cell, and the cellular immunotherapeutic agent is for use in treating cancer.Examples of such cellular immunotherapeutic agents for binding to the target antigen on cancer cells include CAR T cells, indirect or ligand-based CAR-T cells, cells with modified TCR, and other cellular therapeutic agents as further defined herein.

[0026]

[0031] It will be understood that a variety of forms of the molecule can be utilized in accordance with the present invention, provided that the molecule contains an epitope of the target antigen that competes for binding by the cellular immunotherapeutic agent.

[0027]

[0032] In either embodiment, the molecule for binding to a cellular immunotherapeutic agent comprises or consists of a polypeptide, which may be in the form of a fusion or chimeric protein or any other polypeptide molecule.

[0028]

[0033] In certain embodiments, where the molecule is a polypeptide, the method may include providing to the subject a nucleic acid encoding the polypeptide.

[0034] It will be appreciated that a wide range of other architectures can be adopted for the epitope-containing molecule, for example, the molecule can be in the form of a polypeptide that contains the epitope and can be conjugated or fused to any suitable carrier moiety, in certain embodiments, the carrier moiety can be selected from carbohydrates, lipids, liposomes, peptides, and aptamers, as further defined herein.

[0029]

[0035] In some embodiments, the epitope may be provided in the context of a liposome that contains the epitope on its surface by PEG coupling (e.g., a PEGylated liposome). This provides the liposome with an epitope coated within it that is recognized and bound by the cellular immunotherapeutic agent.

[0030]

[0036] In an embodiment of either of the first aspects, the molecule is in the form of a fusion protein that includes an epitope that is specific for the target antigen. The fusion protein may include the epitope and an additional sequence to promote improved solubility of the polypeptide.

[0031]

[0037] The further sequence may include the Fc region of the antibody. However, according to the first aspect of the present invention, it is understood that the Fc region of the antibody is preferably modified so that it does not exhibit effector function or any detectable effector function. For example, the Fc region of the antibody may retain FcRn binding, but may be modified so that the region does not bind to Fcγ receptors (FcγR). The Fc region of the antibody may be modified so that the region does not bind to various cells involved in mediating antibody-dependent cell killing (ADCC), such as NK cells (expressing only FcγRIII) or monocytes (expressing FcγRI, FcγRII and FcγRIII) or hematopoietic cells. Preferably, the Fc region is modified so that it does not induce complement-dependent cytotoxicity (CDC).

[0032]

[0038] Additional sequences may include serum albumin, transferrin, the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta chain, imprecise repeat peptide sequences, polypeptide sequences composed of proline-alanine-serine polymers, elastin-like peptide (ELP) repeat sequences, homopolymers of glycine residues, or gelatin-like proteins.

[0033]

[0039] In a second aspect, the present invention provides various methods for irreversible or permanent "switching off" of the activity of a cellular immunotherapeutic agent (e.g., but not limited to, CAR T therapy) to bind to a target antigen, preferably a target antigen on a cancer cell.

[0034]

[0040] In one embodiment of the second aspect of the invention, there is provided a method of inhibiting activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to the subject a molecule for binding to a cellular immunotherapeutic agent. and the molecule comprises i) an epitope of the target antigen that competes for binding to the cellular immunotherapeutic agent, whereby the molecule disrupts the interaction between the cellular immunotherapeutic agent and the target antigen; ii) Compounds for inducing cell death wherein upon binding of the molecule to the cellular therapeutic agent, the cellular immunotherapeutic agent undergoes cell death or is targeted for cell death; Thereby, a method is provided for inhibiting the activity of a cellular immunotherapeutic agent in a subject.

[0035]

[0041] Preferably, the inhibition is irreversible, so that the method optionally reduces the viability of the cells of the cellular immunotherapeutic agent by introducing a toxin into the cellular immunotherapeutic agent or by targeting the cells for killing, for example, by NK cells and other cytotoxic cells.

[0036]

[0042] Those skilled in the art are familiar with various cytotoxic events that can be induced or caused by cellular immunotherapeutic agents. Such cytotoxic events can include any abnormal release of cytokines or inflammatory responses after administration of cellular immunotherapeutic agents. Abnormal release of cytokines can be referred to by various terms, including hypercytokinemia or "cytokine storm". "Cytokine release syndrome" (CRS) is a form of systemic inflammatory response syndrome (SIRS) that occurs when a large number of white blood cells are activated and release inflammatory cytokines, which in turn activate even more white blood cells. The term "cytokine storm" is often loosely used interchangeably with cytokine release syndrome (CRS), but is more accurately a distinguishable syndrome that may represent a severe episode of cytokine release syndrome or a component of another disease entity, such as macrophage activation syndrome. When occurring as a result of treatment, CRS symptoms may be delayed until several days or weeks after treatment. Immediate (fulminant) CRS is like a cytokine storm.

[0037]

[0043] Thus, in a further embodiment of the second aspect of the invention there is provided a method of minimising or reducing the risk of an aberrant inflammatory response in a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to the subject a molecule for binding to a cellular immunotherapeutic agent. and the molecule comprises i) an epitope of the target antigen that competes for binding to the cellular immunotherapeutic agent, whereby the molecule disrupts the interaction between the cellular immunotherapeutic agent and the target antigen; ii) Compounds for inducing cell death wherein upon binding of the molecule to the cellular therapeutic agent, the cellular immunotherapeutic agent undergoes cell death or is targeted to cell-mediated killing; Thereby, methods are provided that minimize or reduce the risk of an aberrant inflammatory response in a subject.

[0038]

[0044] Preferably, the method reduces the viability of the cells of the cellular immunotherapeutic agent, preferably targeting the cells for degradation / death, thereby avoiding or reducing the risk of further aberrant immune responses that may otherwise be elicited by the cellular immunotherapeutic agent.

[0039]

[0045] Further provided is a method of treating an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; - administering to a subject a molecule for binding to a cellular immunotherapeutic agent, the molecule comprising: i) an epitope of the target antigen that competes for binding to the cellular immunotherapeutic agent, whereby the molecule disrupts the interaction between the cellular immunotherapeutic agent and the target antigen; ii) Compounds for inducing cell death wherein upon binding of the molecule to the cellular therapeutic agent, the cellular immunotherapeutic agent undergoes cell death or is targeted to cell-mediated killing; Thereby, a method is provided for treating an abnormal inflammatory response in a subject.

[0040]

[0046] Preferably, the method reduces the viability of cells of a cellular immunotherapeutic agent, preferably targeting the cells for destruction / death.

[0047] The abnormal inflammatory response may include cytokine-related toxicity. The inflammatory response may include a systemic inflammatory response and may be classified as systemic inflammatory response syndrome (SIRS). SIRS may be classified as "cytokine release syndrome" (CRS). The abnormal inflammatory response may be hypercytokinemia or "cytokine storm".

[0041]

[0048] The abnormal inflammatory response may include one or more of the conditions listed in Table 2 herein.

[0049] moreover, a) inhibiting the activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent for binding to a target antigen; b) minimizing or reducing the risk of an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; or c) treating an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; d) minimizing or reducing the risk of tumor lysis syndrome in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; or e) Treating tumor lysis syndrome in subjects who have undergone or are undergoing treatment with cellular immunotherapeutic agents for binding to target antigens. Use of a molecule comprising an epitope of a target antigen in the manufacture of a medicament for The molecule is i) an epitope of the target antigen that competes with the target antigen for binding to the cellular immunotherapeutic agent, whereby the molecule disrupts the interaction between the cellular immunotherapeutic agent and the target antigen; ii) Compounds that induce cell death wherein upon binding of the molecule to the cellular therapeutic agent, the cellular immunotherapeutic agent undergoes cell death or is targeted to cell-mediated killing.

[0042]

[0050] Furthermore, a) inhibiting the activity of a cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent for binding to a target antigen; b) minimizing or reducing the risk of an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; c) treating an abnormal inflammatory response in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; or d) minimizing or reducing the risk of tumor lysis syndrome in subjects who have undergone or are undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen; e) Treating tumor lysis syndrome in subjects who have undergone or are undergoing treatment with cellular immunotherapeutic agents for binding to target antigens. A composition comprising a molecule comprising an epitope of a target antigen for use in The molecule is i) an epitope of a target antigen that competes with the target antigen on a cell for binding to a cellular immunotherapeutic agent, whereby the molecule disrupts the interaction between the cellular immunotherapeutic agent and the target antigen; ii) Compounds that induce cell death wherein upon binding of the molecule to the cellular therapeutic agent, the cellular immunotherapeutic agent undergoes cell death or is targeted to cell-mediated killing.

[0043]

[0051] In a further embodiment of the second aspect of the invention there is provided a kit for use in the methods described herein, the kit comprising: - a cellular immunotherapeutic agent for binding to a target antigen; and - Molecules containing epitopes of the target antigen and the molecule comprises i) an epitope of a target antigen that competes with the target antigen on a cell for binding to a cellular immunotherapeutic agent, whereby the molecule disrupts the interaction between the cellular immunotherapeutic agent and the target antigen; ii) Compounds that induce cell death wherein upon binding of the molecule to the cellular therapeutic agent, the cellular immunotherapeutic agent undergoes cell death or is targeted for cell-mediated killing.

[0044]

[0052] Optionally, the kit comprises instructions for use in the method of the second aspect of the invention.

[0053] Preferably, the target antigen is a cancer cell, and the cellular immunotherapeutic agent is for use in the treatment of cancer.Preferably, the molecule blocks the interaction between the cellular immunotherapeutic agent and the target antigen present on the cell.In other words, the cellular immunotherapeutic agent comprises a portion for binding to the target antigen on the cancer cell, and the molecule comprises an epitope similar or identical to the target antigen, so that the molecule competes with the target antigen on the cell for binding to the cellular immunotherapeutic agent.Therefore, the method of the present invention comprises a molecule for replacing, preventing or reducing the binding of the cellular immunotherapeutic agent to the target antigen on the cancer cell, and targeting the cellular immunotherapeutic agent to cell death or cell-mediated killing.

[0045]

[0054] According to the second aspect of the invention, the compound that induces cell death can be any toxin or chemotherapeutic agent that induces cytotoxicity. Thus, the molecule can be in the form of a protein conjugate that includes a first portion that includes an epitope of the target antigen and a second portion in the form of a cytotoxic compound (e.g., a toxin or chemotherapeutic agent that induces apoptosis upon binding of the molecule). In a further embodiment, the molecule can be in the form of an anti-idiotypic antibody for binding to a cellular immunotherapeutic agent (i.e., preferably for binding to a region of the cellular immunotherapeutic agent that is involved in binding to the target antigen). In such an embodiment, the anti-idiotypic antibody can include a toxin or chemotherapeutic agent conjugated thereto. Suitable toxins or chemotherapeutic agents are known to those skilled in the art (and may have found use in other contexts, such as antibody-drug conjugates).

[0046]

[0055] In a preferred embodiment of the second aspect, the molecule is in the form of a fusion protein that comprises an epitope specific for the target antigen and an amino acid sequence for inducing cell-mediated killing. The amino acid sequence for inducing cytotoxic killing can be any sequence that facilitates the targeting of the cellular immunotherapeutic agent to cell-mediated killing by cytotoxic immune cells, such as cytotoxic T lymphocytes or NK cells, when the cellular immunotherapeutic agent is bound to the polypeptide.

[0047]

[0056] The further sequence preferably comprises the Fc region of the antibody, and in such an embodiment, the polypeptide may be referred to as "Fc fusion protein comprising epitopes specific for dysfunctional P2X7 receptor". According to the second aspect of the present invention, it is understood that the Fc region of the antibody preferably exhibits effector function and binds to Fcγ receptor (FCγR). The Fc region of the antibody preferably comprises sequences that are bound by various cells involved in mediating antibody-dependent cell killing (ADCC), such as NK cells (expressing only FcγRIII) or monocytes (expressing FcγRI, FcγRII and FcγRIII) or hematopoietic cells. The Fc region of the antibody may also comprise sequences that induce complement-dependent cytotoxicity (CDC).

[0048]

[0057] According to the first or second aspect of the invention, the target antigen may be any antigen associated with cancer cells and may be used as a target for binding by a cellular immunotherapeutic agent.

[0049]

[0058] Examples of target antigens for binding by cellular immunotherapeutic agents include, but are not limited to, dysfunctional (nf) P2X7 receptor, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSA, CD19, CD20, Clec9a, CD276, PD-L1 and PD-L2. Other examples of target antigens are further described herein.

[0050]

[0059] In embodiments of either of the first and second aspects, the cellular immunotherapeutic agent comprises an immune cell or a precursor thereof, the immune cell expressing a receptor comprising an antigen recognition domain and a signaling domain, the antigen recognition domain binding to a target antigen expressed on the surface of a cancer cell.

[0051]

[0060] In an alternative embodiment of the first and second aspects, the cellular immunotherapeutic agent comprises an immune cell or a precursor thereof, the immune cell expressing a receptor comprising an antigen recognition domain and a signaling domain, the antigen recognition domain binding to a ligand that promotes binding of the immune cell to a target antigen expressed on the surface of a cancer cell.

[0052]

[0061] The immune cell or its precursor can be a T cell, a NK cell, or any other immune cell that expresses a receptor or binding domain for binding to a target antigen on a cancer cell.

[0053]

[0062] Typically, the antigen recognition domain of the receptor expressed by the cellular immunotherapeutic agent recognizes only tumor-associated antigens as defined herein.

[0063] In some embodiments, the signaling domain comprises a portion derived from an activating receptor. In some embodiments, the activating receptor is a member of the CD3 coreceptor complex or is an Fc receptor. In some embodiments, the portion derived from the CD3 coreceptor complex is CD3-ζ. In some embodiments, the portion derived from an Fc receptor is FcεRI or FcγRI.

[0054]

[0064] In some embodiments, the signaling domain comprises a portion derived from a costimulatory receptor. In some embodiments, the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. In some embodiments, the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137) and ICOS.

[0055]

[0065] In either embodiment, the receptor expressed by the immune cell may be a chimeric antigen receptor (CAR) that binds to a target antigen expressed on the cell surface, and thus the cellular immunotherapeutic agent includes an immune cell expressing a CAR. Other receptors (such as modified TCRs or ligand-based CARs) are also contemplated within the scope of the present invention.

[0056]

[0066] In any embodiment, the immune cell expressing the antigen receptor can be any immune cell described herein, or a cell that can be differentiated into an immune cell (e.g., a precursor of an immune cell). The cell that can be differentiated into an immune cell (e.g., a T cell expressing a CAR) can be a stem cell, a multilineage progenitor cell, or an induced pluripotent stem cell.

[0057]

[0067] In any embodiment, the immune cell expressing CAR can be any immune cell described herein, or a cell that can be differentiated into an immune cell (e.g., a precursor of an immune cell). The cell that can be differentiated into an immune cell (e.g., a T cell expressing CAR) can be a stem cell, a multilineage progenitor cell, or an induced pluripotent stem cell. Thus, in a particularly preferred embodiment, the cellular immunotherapeutic agent comprises a CAR-T cell that binds to a tumor-associated antigen.

[0058]

[0068] In certain embodiments, the cellular immunotherapeutic agent is intended to bind to a dysfunctional P2X7 receptor on a cancer cell, and thus the antigen recognition domain of the receptor expressed by the cellular immunotherapeutic agent binds to the dysfunctional P2X7 receptor.

[0059]

[0069] In any embodiment, antigen recognition domain binds to epitope associated with adenosine triphosphate (ATP) binding site of dysfunctional P2X7 receptor.In some embodiments, dysfunctional P2X7 receptor has reduced ability to bind ATP at ATP binding site compared with the ATP binding ability of functional P2X7 receptor (e.g., receptor with wild-type sequence and ATP-binding receptor conformation or fold).In some embodiments, dysfunctional P2X7 receptor cannot bind ATP at ATP binding site.

[0060]

[0070] In any embodiment, dysfunctional P2X7 receptor has conformational change that causes receptor to malfunction.In some embodiments, conformational change is the amino acid change from trans conformation to cis conformation.In some embodiments, the amino acid that changes from trans conformation to cis conformation is proline at amino acid position 210 of dysfunctional P2X7 receptor.

[0061]

[0071] In any embodiment, the antigen recognition domain binds to an epitope that includes a proline at amino acid position 210 of a dysfunctional P2X7 receptor. In some embodiments, the antigen recognition domain binds to an epitope that includes one or more amino acid residues ranging from a glycine at amino acid position 200 to a cysteine ​​at amino acid position 216 (inclusive) of a dysfunctional P2X7 receptor.

[0062]

[0072] The antigen recognition domain of receptor can be any suitable molecule that can interact with and specifically bind to dysfunctional P2X7 receptor.However, in some embodiments, the antigen recognition domain comprises amino acid sequence homology with the amino acid sequence of the antibody or its fragment that binds to dysfunctional P2X7 receptor.In some embodiments, the antigen recognition domain comprises amino acid sequence homology with the amino acid sequence of the fragment-antigen binding (Fab) part of the antibody that binds to dysfunctional P2X7 receptor.In some embodiments, the antibody is a humanized antibody.

[0063]

[0073] In any embodiment, the antigen recognition domain comprises amino acid sequence homology with the amino acid sequence of the single chain variable fragment (scFv) or multivalent scFv that binds to the dysfunctional P2X7 receptor. In some embodiments, the multivalent scFv is a bivalent or trivalent scFv.

[0064]

[0074] In either embodiment, the antigen recognition domain comprises amino acid sequence homology to a single antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor.

[0075] In any embodiment, the antigen recognition domain comprises a binding polypeptide that comprises amino acid sequence homology with one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide comprises one or more VDRs of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L In a preferred embodiment, the binding polypeptide comprises amino acid sequence homology to the CDR1, 2 and 3 domains of the V chain of an antibody. H and / or V L The amino acid sequence of the CDR of the chain, or the V of the antibody H and / or V LThe antibody or fragment thereof may comprise any of the amino acid sequences of the polypeptide chains, or the amino acid sequence of an antibody or fragment thereof, which antibody or fragment thereof may be any of the amino acid sequences described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 8,710, 8,820, 8,930, 8,940, 8,950, 8,960, 8,970, 8,980, 9,020, 9,040, 9,060, 9,0 ... Nos. 9,663,584, or 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010-0036101), PCT / AU2008 / 001364 (or U.S. Patent Nos. 8,440,186, 9,181,320, 9,944, No. 10,597,451), PCT / AU2008 / 001365 (or corresponding U.S. Pat. No. 8,293,491 or U.S. Pat. No. 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Pat. No. 8,597,643, U.S. Pat. No. 9,328,155 or U.S. Pat. No. 10,238,716), PCT No. 9,127,059, U.S. Pat. No. 9,688,771, or U.S. Pat. No. 10,053,508), and PCT / AU2010 / 001741 (or any one of the corresponding WO 2011 / 075789 or U.S. Pat. No. 8,835,609).Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 as described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or the CDR amino acid sequence of BPM09 as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Patent Application Publication No. 2010-0036101) and produced by hybridoma AB253 deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101.

[0065]

[0076] According to the first and second aspects of the invention, the methods involve the use of a molecule or polypeptide that competes for binding to a cellular immunotherapeutic agent and displaces or blocks binding of the cellular immunotherapeutic agent to a target antigen, preferably the target antigen is expressed or present on the surface of a cancer cell.

[0066]

[0077] The molecule or polypeptide preferably contains an epitope of the target antigen such that it is preferentially bound by the cellular immunotherapeutic agent, thereby relieving the cellular immunotherapeutic agent from binding to the cancer cell.

[0067]

[0078] It is well within the skill of the art to generate a molecule or polypeptide (or optionally a nucleic acid encoding a polypeptide) that contains an epitope of a target antigen for binding by a cellular immunotherapeutic agent. For example, in the context of a cellular immunotherapeutic agent for binding to CD19, the molecule or polypeptide contains the analogous epitope of the CD19 molecule that is recognized by the cellular immunotherapeutic agent, such that the cellular immunotherapeutic agent preferentially binds to the molecule or polypeptide rather than to CD19 present on the surface of the target cell.

[0068]

[0079] Although the following description relates to specific examples in which the target antigen is a dysfunctional P2X7 receptor, it will be understood that the scope of the invention is not limited to these examples.

[0080] In any embodiment of the first or second aspect of the present invention, the cellular immunotherapeutic agent is for binding to the dysfunctional P2X7 receptor on cancer cell.Therefore, in such an embodiment, the molecule or polypeptide comprises the epitope of the dysfunctional P2X7 receptor that competes for binding to the cellular therapeutic agent.

[0069]

[0081] In any embodiment of the first or second aspect, the epitope of the target antigen on the molecule comprises the amino acid sequence that is substantially the same or homologous to the epitope on the dysfunctional P2X7 receptor that is bound by cellular immunotherapeutic agent.In other words, even if the amino acid sequence of the two epitopes may be different, it is sufficiently homologous for the cellular immunotherapeutic agent to bind to both the polypeptide and the dysfunctional P2X7 receptor on the cell.In any embodiment, the epitope on the polypeptide comprises or consists of the amino acid sequence of the epitope on the dysfunctional P2X7 receptor that the cellular immunotherapeutic agent binds to.

[0070]

[0082] In any embodiment of the first or second aspect, the epitope of dysfunctional P2X7 receptor comprises or consists of the epitope that is only found on dysfunctional P2X7 receptor, but not found on functional form of P2X7 receptor.In other words, preferably, the polypeptide comprises or consists of the epitope that is specific to dysfunctional P2X7 receptor.

[0071]

[0083] In a further embodiment of the first or second aspect, the polypeptide comprises an epitope corresponding to E200, E300 or combined E200 / E300 epitope as defined herein. It is within the skill of the art to obtain various polypeptides for use according to the present invention, particularly in the context of "blocking" or reducing the binding potency of anti-nfP2X7CAR. For example, the skilled artisan will understand that it is possible to include additional amino acids at the N-terminus or C-terminus of the polypeptide region that contains the epitope bound by CAR. In a non-limiting example, and in the context of E200, which is typically defined as having an amino acid sequence substantially as defined in SEQ ID NO:2 (and having a minimal sequence as defined in SEQ ID NO:11), additional amino acids derived from the native sequence of P2X7 receptor can be included in the polypeptide, for example, at the residues "DFP" at the N-terminus of the epitope in the P2X7 receptor sequence, and / or at the residues "TFHKT" at the C-terminus of the epitope in the P2X7 receptor sequence. In any embodiment, the polypeptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 amino acids from the P2X7 receptor sequence in addition to the sequence of E200 or E300 or a composite epitope.

[0072]

[0084] In a preferred embodiment, the sequence of the E200 epitope is further modified to replace the cysteine ​​residue (residue 17 of SEQ ID NO:2) with a serine residue. One of skill in the art will appreciate that this can be done to reduce the possibility of any disulfide bonds between the polypeptide and another molecule.

[0073]

[0085] It is also within the skill of the art to include additional amino acid residues in the E200, E300 or composite epitopes (or extended epitopes discussed in the paragraph above), such as by adding at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 additional amino acid residues to the N- and C-terminal regions of a peptide consisting of the amino acid sequence of the relevant epitope. Typically, such additional amino acids can be derived from linker sequences (such as peptides containing glycine and serine residues); or from the hinge region of an immunoglobulin. Typically, no more than 30, no more than 25, or no more than 20 amino acid residues are added to the N- and / or C-terminal residues of the E200, E300 or composite epitopes defined herein.

[0074]

[0086] It is understood that according to the first aspect of the invention, the molecule may be in the form of a polypeptide consisting of an epitope of the dysfunctional P2X7 receptor.

[0087] Examples of peptides and polypeptides that comprise an epitope of a dysfunctional P2X7 receptor and are suitable for use in accordance with the present invention are provided in Table 1 (defined according to any of SEQ ID NOs: 2-70).

[0075]

[0088] As used herein, unless the context otherwise requires, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, components, integers or steps.

[0076]

[0089] Further aspects of the invention and further embodiments of the aspects set out in the preceding paragraphs will become apparent from the following description, given by way of example with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0077] [Figure 1]

[0090] A. Percentage change in viability of MOLM-13 cells after co-culture in the direct CAR system with anti-nfP2X7 CAR T cells + / - a peptide containing an epitope recognized by the anti-nfP2X7 CAR. The presence of different peptide variants reduced the killing of MOLM-13 cells by CAR T cells. B. Percentage change in viability of MOLM-13 cells after co-culture in the indirect CAR T cell system (anti-nfP2X7 CAR T cells + a polypeptide containing an epitope of the anti-nfP2X7 CAR and an anti-CD33 binding domain) + / - a peptide containing an epitope recognized by the anti-nfP2X7 CAR. The presence of different peptide variants reduced the killing of MOLM-13 cells by CAR T cells. [Diagram 2]

[0091] Changes in viability of MOLM-13 cells after co-culture with anti-nfP2X7 CART cells + / - peptides containing epitopes recognized by anti-nfP2X7 CAR. [Diagram 3]

[0092] Killing of JeKo-1 cells by T cells and T cells expressing anti-nfP2X7 CAR in the presence or absence of a peptide containing the epitope recognized by the CAR. Only T cells expressing nfP2X7 CAR completely prevented the proliferation of JeKo-1 cells over a 5-day time course. Addition of a peptide (containing the sequence of the epitope recognized by the nfP2X7 CAR) reduced the killing of JeKo-1 cells by CAR T cells. [Figure 4]

[0093] The reduction in killing of MOLM-13 cells by nfP2X7 CART cells was dose-dependent and could be controlled by varying the concentration of the peptide containing the epitope recognized by nfP2X7 CAR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] Sequence information

[0094]

[0079] [Table 1-1]

[0080] [Table 1-2]

[0081] [Table 1-3]

[0082] [Table 1-4]

[0083] [Table 1-5]

[0084] [Table 1-6]

[0085] Detailed Description of the Preferred Embodiments

[0095] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0086]

[0096] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and the present invention is in no way limited to the methods and materials described.

[0087]

[0097] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features which are mentioned or evident from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0088]

[0098] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0099] One aspect of the present invention described herein is based on the identification that it is possible to reduce the ability of cellular immunotherapeutic agents (e.g., CAR T cells) that express receptors that bind to dysfunctional P2X7 receptors to bind to dysfunctional P2X7 receptors on the surface of cancer cells.The inventors have determined that this can be achieved by providing a polypeptide that competes with dysfunctional P2X7 receptors to bind to cellular immunotherapeutic agents.For example, the in vivo activity of T cells expressing CARs that contain an antigen binding domain that binds to dysfunctional P2X7 receptors can be reduced by administering a polypeptide that contains the same epitope of dysfunctional P2X7 receptors that the antigen binding domain of CARs binds to.This reduction or inhibition is reversible, and the viability of cellular immunotherapeutic agents can be maintained.

[0089]

[0100] Another aspect of the present invention described herein is to irreversibly inhibit or reduce the in vivo activity of cellular immunotherapeutic agents by targeting the cellular immunotherapeutic agents to kill cells.Targeting is achieved by administering a polypeptide that comprises (a) the epitope of dysfunctional P2X7 receptor that cellular immunotherapeutic agents can bind to, and (b) the sequence that promotes the targeting of cellular immunotherapeutic agents to cell-mediated killing.For example, the in vivo activity of T cells expressing CAR that comprises an antigen binding domain that binds to dysfunctional P2X7 receptor can be irreversibly reduced by administering a polypeptide that comprises (a) the same epitope of dysfunctional P2X7 receptor that CAR's antigen binding domain binds to, and (b) the sequence (e.g., Fc region) that targets CAR T cells for cell-mediated killing (e.g., cytotoxic T cell or NK cell-mediated killing).

[0090] definition

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091]

[0102] For purposes of interpreting this specification, the following definitions shall generally apply and, where appropriate, terms used in the singular shall also include the plural and vice versa.

[0103] An "antibody" or "immunoglobulin" or "Ig" is a gamma globulin protein found in the blood or other body fluids of vertebrates that functions in the immune system to bind to antigens, thus identifying and / or neutralizing foreign substances.

[0092]

[0104] Antibodies are generally heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H). Each L chain is linked to an H chain by one covalent disulfide bond. Depending on the H chain isotype, the two H chains are linked to each other by one or more disulfide bonds. Each H and L chain also has regularly spaced intrachain disulfide bridges.

[0093]

[0105] The H and L chains define specific Ig domains. More specifically, each H chain contains a variable domain (V H ), followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) followed by a constant domain (CL) at the other end. L is V H Aligned with C L is aligned with the first constant domain of the heavy chain (CHL).

[0094]

[0106] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in 3 / 4 sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The L chains of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of the constant domain.

[0095]

[0107] The constant domain comprises the Fc portion, which comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody, such as ADCC, are determined by the sequence of the Fc region, which is also the portion recognized by the Fc receptors (FcR) found on certain cells.

[0096]

[0108] V who joined together H and V L The pairing of these forms the "variable region" or "variable domain" which comprises the amino terminal domain of either the heavy or light chain of an antibody. The variable domain of the heavy chain is called the "V H The variable domain of the light chain can be called "VL The V domain contains the "antigen-binding site" that influences antigen binding and defines the specificity of a particular antibody for a particular antigen. The V region spans about 110 amino acid residues and consists of relatively invariant stretches of 15-30 amino acids (generally about 4), called framework regions (FR), separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3), each generally 9-12 amino acids long. The FRs adopt a predominantly β-sheet conformation, and the hypervariable regions form loops that connect the β-sheet structures and in some cases form part of the β-sheet structures.

[0097]

[0109] "Hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions; H Three of them (H1, H2, H3) and V L Includes three of them (L1, L2, L3).

[0098]

[0110] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0111] "Antigen binding site" generally refers to a molecule that contains at least the hypervariable and framework regions necessary to confer antigen binding function to a V domain. The antigen binding site may be in the form of an antibody or antibody fragment (e.g., mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD-Fv, Fv, F(ab')2 or scFv) in the manner described herein.

[0099]

[0112] An "intact" or "whole" antibody comprises an antigen-binding site, as well as a C L and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0100]

[0113] "Whole antibody fragments containing the variable domain" include SD-mAbs, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0101]

[0114] A "Fab fragment" is a fragment that contains the entire light chain and the variable region domain of the heavy chain (V H ) and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0102]

[0115] "Fab' fragments" differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0103]

[0116] The "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments which have divalent antigen-binding activity and are still capable of cross-linking antigen.

[0117] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.

[0104]

[0118] In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. Folding of these two domains gives rise to six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0105]

[0119] A "single-chain Fv" is also abbreviated as "sFv" or "scFv" and refers to V chains connected to form a single polypeptide chain. H and V L An scFv polypeptide is an antibody fragment that contains an antibody domain. H Domain and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0106]

[0120] A "single variable domain" is typically one half of an Fv (containing only the three CDRs specific for an antigen) that has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0107]

[0121] "Diabody" refers to an antibody fragment that has two antigen-binding sites, the fragments being bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to a heavy chain variable domain (V H Small antibody fragments include V domains such that interchain, but not intrachain, pairing of the V domains is achieved, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. H Domain and V L They are prepared by constructing an sFv fragment (see previous paragraph) with a short linker (about 5-10 residues) between the domains.

[0108]

[0122] Diabodies can be bivalent or bispecific. Bispecific diabodies are diabodies that combine the V H Domain and V L They are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Triabodies and tetrabodies are also commonly known in the art.

[0109]

[0123] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its existing environment. Contaminating components are substances that would interfere with therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0110]

[0124] "Human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering antigen to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled.

[0111]

[0125] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0112]

[0126] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the population containing the individual antibodies is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies can be prepared by the hybridoma method. "Monoclonal antibodies" can also be isolated from phage antibody libraries using molecular engineering techniques.

[0113]

[0127] As used herein, "tumor-associated antigen" refers to an antigen expressed by a cancer cell (the term "tumor-antigen" can also be used to refer to the same). Tumor antigens are proteins produced by tumor cells that induce an immune response, particularly a T-cell mediated immune response. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, hK4 prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, P501S prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0114]

[0128] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a large number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to each individual tumor. B-cell differentiation antigens, such as CD19, CD20 and CD37, are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy with monoclonal antibodies, although with limited success. The type of tumor antigen referred to in this invention can also be a tumor-specific antigen (TSA). TSAs are unique to tumor cells and are not present on other cells in the body. Tumor-associated antigens (TAA) are not unique to tumor cells, but are instead expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or antigens that are normally present at very low levels on normal cells, but expressed at much higher levels on tumor cells. Tumor-associated antigens of most clinical interest are differentially expressed compared to corresponding non-tumor tissues, allowing preferential recognition of tumor cells by specific T cells or immunoglobulins.

[0115]

[0129] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3\CA 27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.Particularly preferred examples of target cell antigens according to the invention include CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LIA), CD184 (CXCR4 ... CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and in particular EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSACD276 and dysfunctional (nf) P2X7 receptor.

[0116]

[0130] "Purinergic receptor" generally refers to a receptor that uses purines (eg, ATP) as a ligand.

[0131] "P2K7 receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, at least one of which has the sequence set forth in SEQ ID NO:1:

[0132] SEQ ID NO:1 MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWMDPQSKGIQ TGRCVVYEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNFRYAK YYKENNVEKRTLIKVFGIRFDILVFGTGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLAL HDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY The amino acid sequence of the present invention is substantially as shown in

[0117]

[0133] Insofar as P2X7 receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X7 receptor" encompasses naturally occurring variants of P2X7 receptor, for example, P2X7 monomer isoforms (e.g., forms consisting of extracellular domain sequence or truncated forms thereof), including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of monomers forming P2X7 receptor, naturally occurring variant forms (e.g., alternative splice forms) and naturally occurring allelic variants. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO:1. In certain embodiments, P2X7 receptor can have modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO:1 can be replaced, deleted, or residues can be inserted.

[0118]

[0134] "Functional P2X7 receptor" generally refers to a form of P2X7 receptor that has three intact binding sites or grooves for binding ATP. When bound to ATP, functional receptors form non-selective sodium / calcium channels that transform into pore-like structures, allowing calcium ions and molecules up to 900 Da to enter the cytosol, which may result in the induction of programmed cell death. In normal homeostasis, the expression of functional P2X7 receptors is generally restricted to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages and monocytes. Some expression of functional P2X7 receptors may also be found on erythrocytes and other cell types.

[0119]

[0135] "Dysfunctional P2X7 receptor" generally refers to a form of P2X7 receptor that has a different conformation from functional P2X7, whereby the receptor cannot form an apoptotic pore, but can still act as a non-selective channel by maintaining a single functional ATP binding site located between adjacent monomers. One example occurs when one or more monomers have cis isomerization at Pro210 (according to SEQ ID NO: 1). Isomerization can result from any molecular event that results in misfolding of the monomer, including, for example, mutation of the monomer primary sequence or abnormal post-translational processing. One consequence of this isomerization is that the receptor cannot bind ATP at one, more specifically two, of the ATP binding sites on the trimer, and as a result, cannot extend the opening of the channel. In such a situation, the receptor cannot form a pore, which limits the extent to which calcium ions can enter the cytosol. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial, mesenchymal, embryonic, neurogenic and hematopoietic cancers. As used herein, the term "dysfunctional P2X7 receptor" can be used interchangeably with the terms "non-functional P2X7 receptor" or "nfP2X7 receptor."

[0120]

[0136] A "cancer-associated P2X7 receptor" is a P2X7 receptor that is generally found on cancer cells (including precancerous, neoplastic, malignant, benign or metastatic cells) but not on non-cancerous or normal cells.

[0121]

[0137] "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NOs: 2-11; 15-70).

[0138] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NOs: 12 and 13).

[0122]

[0139] "Composite epitope" generally refers to an epitope formed from the adjoining E200 and E300 epitopes or portions of these epitopes. An example of a composite epitope comprising the E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14).

[0123]

[0140] The term "anti-P2X7 receptor antibody" or "antibody that binds to P2X7 receptor" refers to an antibody that can bind to P2X7 receptor with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting P2X7 receptor, typically non-functional P2X7 receptor or cancer-related P2X7 receptor.Preferably, the degree of binding of P2X7 receptor antibody to unrelated protein is less than about 10% of the binding of antibody to P2X7 receptor, for example, as measured by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Biacore or flow cytometry.In certain embodiments, the antibody that binds to P2X7 receptor has a dissociation constant (Kd) of <1μM, <100nM, <10nM, <1nM, or <0.1nM. Anti-nfP2X7 receptor antibodies are generally antibodies that have some or all of these serological characteristics and bind to dysfunctional P2X7 receptors but not to functional P2X7 receptors.

[0124]

[0141] An "affinity matured" antibody is an antibody with one or more modifications in one or more hypervariable regions thereof that result in improved affinity of the antibody for the antigen compared to a parent antibody that does not have those modification(s). Preferred affinity matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are generated by means known in the art.

[0125]

[0142] A "blocking antibody" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0126]

[0143] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0144] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0127]

[0145] As used herein, the term "antigen" is intended to include substances that bind to or induce the production of one or more antibodies, and can include, but are not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and recognized by the adaptive immune system, including, but not limited to, antibodies or TCRs, or engineered molecules, including, but not limited to, transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single-chain antibodies or multimers thereof, or any other molecule that can achieve binding to a structure with high affinity.

[0128]

[0146] "Epitope" generally refers to that part of an antigen that is bound by the antigen-binding site of an antibody. An epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to a sequence of amino acids as in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope", i.e., binds to residues when the hypervariable loops of the CDRs are present in a tertiary or quaternary protein structure.

[0129]

[0147] The term "disorder" or "condition" refers to a functional abnormality or impairment in a subject, such as cancer, an autoimmune disorder, or a viral, bacterial, parasitic, or other infection.

[0130]

[0148] For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can also exist in a non-native environment, such as, for example, a host cell.

[0131]

[0149] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey or human. Preferably, the subject is a human. The subject may be a subject (patient) suffering from a disorder such as cancer, but the subject may also be a healthy subject. As used herein, the terms "subject", "individual" and "patient" can be used interchangeably.

[0132]

[0150] As used herein, the term "autologous" refers to any material derived from the same subject into which it is subsequently reintroduced.

[0151] The term "allogeneic" as used herein refers to any material derived from a different subject of the same species as the subject into which the material is reintroduced.

[0133]

[0152] The term "therapeutically effective amount" or "therapeutically effective population" refers, for example, to an amount of a cell population that provides a therapeutic benefit in a subject.

[0153] The terms "bind", "specifically bind" or "specific" in relation to a targeting moiety, for example when used in a CAR, refer to an antigen binding domain that recognizes and binds to a particular antigen, but does not substantially recognize or bind to other molecules in a sample. An antigen binding domain or targeting moiety that specifically binds to an antigen from one species may also bind to an antigen from another species. This cross-species reactivity is typical of many antibodies and therefore does not violate the definition that an antigen binding domain is specific. An antigen binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (such as allelic variants, splice variants, isoforms, etc.) or homologous variants of this antigen from the same gene family. This cross-reactivity is typical of many antibodies and therefore does not violate the definition that an antigen binding domain is specific.

[0134]

[0154] The terms "engineered cells" and "genetically modified cells" as used herein can be used interchangeably. These terms refer to the fact that cells contain and / or express foreign genes or nucleic acid sequences that in turn modify the genotype or phenotype of the cells or their progeny. In particular, these terms refer to the fact that cells, preferentially immune cells, can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins that are not naturally expressed in these cells. For example, immune cells are engineered to express artificial constructs such as chimeric antigen receptors on their cell surface. For example, CAR sequences can be delivered into cells using adenovirus, adeno-associated virus (AAV)-based, retrovirus or lentivirus vectors, or any other pseudotyped variations thereof, or any other gene delivery mechanism such as electroporation or lipofection with CRISPR / Cas9, transposons (e.g., Sleeping-Beauty) or variations thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0135]

[0155] The term "immune cell" or "immune effector cell" refers to a cell that is part of the immune system and can perform a specific effector function, for example, alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic progenitor cell, such as pluripotent stem cells and early progenitor cell subsets that can mature or differentiate into somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificially modified cells / genetically modified cells (such as iPSCs and other artificial cell types). Preferred immune cells are cells with cytotoxic effector functions, for example, alpha-beta T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells or gamma-delta T cells. By "effector function" is meant a special function of a cell; for example, in T cells, the effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0136]

[0156] As used herein, the term "treating" a disorder means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0137]

[0157] The term "prevention" as used herein is intended to refer to at least a reduction in the likelihood of risk (or susceptibility) of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease that do not develop in an individual who may be exposed to or predisposed to the disease but has not yet experienced or presented symptoms of the disease). The biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians. For example, prevention of abnormal immune response can be characterized by the lack of increased release of cytokines after treatment with cellular immunotherapeutic agents.

[0138]

[0158] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell. Cellular immunotherapy

[0159] As described herein, the invention includes the use of molecules to bind to cellular immunotherapeutic agents, the cellular immunotherapeutic agents comprising immune cells expressing a receptor comprising an antigen recognition domain, preferably a signaling domain, which recognizes a target antigen. Preferably, the target antigen is expressed on the cell surface and is a tumor-associated or tumor-specific antigen, as further defined herein.

[0139]

[0160] Those skilled in the art will appreciate that the methods of the present invention can be applied to a wide variety of different cellular immunotherapeutic agents. In certain embodiments, the cellular immunotherapeutic agent comprises an immune cell expressing a chimeric antigen receptor (CAR) (or a variant thereof, including a ligand-based CAR) or a modified T cell receptor (modified TCR), whereby the immune cell is a CAR, a TCR, or a variant thereof. Furthermore, in preferred embodiments, it is understood that the antigen recognition domain typically binds directly to a target antigen on a target cell (e.g., a so-called "direct CAR"). However, the methods of the present invention can be applied in the context of a cellular immunotherapeutic agent that indirectly interacts with a target cell (e.g., via binding to a ligand that facilitates interaction with a target antigen on the target cell). Examples of such indirect cellular immunotherapeutic agents are reviewed in Arndt et al., (2020) Cancers (Basel), vol. 12:1302, which is incorporated herein by reference.

[0140]

[0161] The cell may be an "engineered cell," "genetically modified cell," "immune cell," or "immune effector cell," as described herein. Furthermore, the cell may be capable of differentiating into an immune cell. The cell capable of differentiating into an immune cell (e.g., a T cell expressing a CAR) may be a stem cell, a multilineage progenitor cell, or an induced pluripotent stem cell.

[0141]

[0162] The immune cells of the present invention can be any suitable immune cell, or its precursor, or can be a homogenous or heterogenous cell population. In some embodiments, the cells are leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells, natural killer T cells, or γδ T cells.

[0142]

[0163] In either embodiment, the cell may be a T cell, and optionally the T cell does not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0143]

[0164] In any embodiment, the cell may be an immune cell, and optionally the cell does not express accessory molecules that may be checkpoint, exhaustion or apoptosis-related signaling receptors, as well as ligands, such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0144]

[0165] In some embodiments, the genetically modified cell comprises two or more different CARs or different TCRs. As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs that have either non-identical antigen recognition domains and / or non-identical signaling domains. In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize a dysfunctional P2X7 receptor), but have different signaling domains, such as one CAR that has a signaling domain that includes a part of an activating receptor, and the other CAR that has a signaling domain that includes a part of a costimulatory receptor. As will be understood, at least one of the two or more CARs in this embodiment has an antigen recognition domain that recognizes a dysfunctional P2X7 receptor, and the other CAR(s) can take any suitable form and be directed against any suitable antigen.

[0145]

[0166] Generally, CAR or modified TCR can include an extracellular domain (extracellular portion) that includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain. The extracellular domain can be linked to the transmembrane domain by a linker. The extracellular domain can also include a signal peptide.

[0146]

[0167] The extracellular domain of the antigen recognition domain preferably recognizes a target antigen expressed on a cancer cell. It is understood that any number of different cellular immunotherapeutic agents expressing different antigen recognition domains to bind different target antigens can be utilized in accordance with the present invention, but it is necessary to utilize molecules that contain epitopes that compete for binding to the cellular immunotherapeutic agent.

[0147]

[0168] For example, cellular immunotherapeutic agents include CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD146 (MCAM), CD155 (PVR), CD171 (LI CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and in particular a receptor having an antigen recognition domain for binding to any one of EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, CD276, and PCSA.

[0148]

[0169] In certain embodiments, the cellular immunotherapeutic agent is an immune cell that expresses CAR (or its variant) to bind to dysfunctional P2X7 receptor.The extracellular portion of CAR or its variant can comprise the nfP2X7 binding domain that recognizes E200 (or E300 or E200-300 complex) epitope as disclosed herein.

[0149]

[0170] Typically, the antigen recognition domain comprises a binding polypeptide that comprises amino acid sequence homology with one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide comprises one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L It contains amino acid sequence homology with the CDR1, 2 and 3 domains of the chain.

[0150]

[0171] In such embodiments, the binding polypeptide is a polypeptide as described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or U.S. Patent Nos. 5,111, 5,112, 5,113, 5,114, 5,115, 5,116, 5,117, 5,118, 5,119, 5,120, 5,132, 5,136, 5,138, 5,140, ​​5,142, 5,144, 5,146, 5,147, 5,148, 5,149, 5,150, 5,152, 5,156, 5,158, 5,159, 5,160, 5,162, 5,164, 5,166, 5,168, 5,169, 5,170, 5,172, 5,174, 5,176, 5,178, 5,179, 5,200, 5,201, 5,202, 5,203, 5,204, 5,205, 5,206, 5,207, 5,208, 5,210, 5,211, 5,212, 5,213, 5,214, 5,215, 5,220 No. 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701 or in any one of U.S. Patent No. 10,597,451), PCT / AU2008 / 001365 (or in any one of the corresponding U.S. Patent No. 8,293,491 or U.S. Patent No. 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding U.S. Patent No. 8,597,643, U.S. Patent No. 9,328,155 or U.S. Patent No. 10,238,716), PCT / A U2010 / 001070 (or in any one of the corresponding WO 2011 / 020155, U.S. Pat. No. 9,127,059, U.S. Pat. No. 9,688,771, or U.S. Pat. No. 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding WO 2011 / 075789 or U.S. Pat. No. 8,835,609). H and / or V L Preferably, the binding polypeptide comprises the amino acid sequence of the CDRs of the V chain of antibody 2-2-1 described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Pat. Nos. 9,127,059, 9,688,771, or 10,053,508).H and / V L The amino acid sequence of the CDRs of the BPM09 chain, or the amino acid sequence of the CDRs of BPM09 described in PCT / AU2007 / 001541 (or in corresponding U.S. Patent Application Publication No. 2010-0036101) and deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101, and produced by hybridoma AB253 described in WO 2013185010 or WO 2019056023. Alternatively, the binding polypeptide of the CAR may comprise the amino acid sequence of the CDRs of antibodies sdAb2-2-3, 2-472-2, or 2-2-12, described in WO 2017 / 041143 (also published as U.S. Patent Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Patent Application No. 17 / 057,060), which are incorporated by reference herein.

[0151]

[0172] CAR binding polypeptides can be prepared using methods described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or U.S. Patent No. 1,621,645, the entire contents of which are incorporated herein by reference). Nos. 8,067,550, ... No. 10,597,451), PCT / AU2008 / 001365 (or corresponding U.S. Pat. No. 8,293,491 or U.S. Pat. No. 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Pat. No. 8,597,643, U.S. Pat. No. 9,328,155 or U.S. Pat. No. 10,238,716 ... No. 10 / 001070 (or in any one of the corresponding WO 2011 / 020155, U.S. Pat. No. 9,127,059, U.S. Pat. No. 9,688,771, or U.S. Pat. No. 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding WO 2011 / 075789 or U.S. Pat. No. 8,835,609). H and / or V L Preferably, the binding polypeptide comprises the amino acid sequence of the V chain of antibody 2-2-1 described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Pat. Nos. 9,127,059, 9,688,771, or 10,053,508). H and / or VL The amino acid sequence of the CDR of BPM09 described in PCT / AU2007 / 001541 (or in corresponding U.S. Patent Application Publication No. 2010-0036101) and deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101, and produced by hybridoma AB253 described in WO 2013185010 or WO 2019056023. Alternatively, the binding polypeptide of the CAR may be selected from the group consisting of antibodies sdAb2-2-3, 2-472-2, or 2-2-12, which are described in WO 2017 / 041143 (also published as U.S. Patent Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Patent Application No. 17 / 057,060), which are incorporated herein by reference. H and / or V L The amino acid sequence of the chain may be included.

[0152]

[0173] CAR binding polypeptides can be prepared using methods described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,4 50,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent Application Publication No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010-0036101), PCT / AU2008 / 001364 (or U.S. Patent No. 8,440,186, U.S. Patent No. 9,181,320, U.S. Patent No. 9,944,701 or U.S. Patent No. 10,597,451 No. 8,293,491 or U.S. Pat. No. 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716), PCT / AU2010 / 001070 (or any one of the corresponding U.S. Pat. Nos. 8,293,491, 8,658,385 ... No. 9,127,059, U.S. Pat. No. 9,688,771, or U.S. Pat. No. 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding WO 2011 / 075789 or U.S. Pat. No. 8,835,609).Preferably, the binding polypeptide comprises the amino acid sequence of sdAb2-2-1 described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or the amino acid sequence of antibody BPM09 described in PCT / AU2007 / 001541 (or in the corresponding U.S. Patent Application Publication No. 2010-0036101), deposited at the European Collection of Cultures (ECACC) under accession number 06080101, and produced by hybridoma AB253 described in WO2013185010 or WO2019056023. Alternatively, the binding polypeptide may comprise the amino acid sequence of sdAb2-2-3, 2-472-2, or 2-2-12, as described in WO 2017 / 041143 (also published as U.S. Patent Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Patent Application No. 17 / 057,060), which are incorporated by reference herein.

[0153]

[0174] "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a particular organelle (eg, the endoplasmic reticulum) and / or to the cell surface.

[0154]

[0175] Generally, "antigen binding domain" refers to the region of a CAR that specifically binds to an antigen (and thereby can target cells containing the antigen). The CAR of the present invention may comprise one or more antigen binding domains. Generally, the target region on a CAR is extracellular. The antigen binding domain may comprise an antibody or an antibody-binding fragment thereof. The antigen binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a diabody. Any molecule that specifically binds to a given antigen can be used as an antigen binding domain, such as an affibody or a ligand binding domain from a naturally occurring receptor. Often the antigen binding domain is an scFv. Usually, in an scFv, the variable regions of immunoglobulin heavy and light chains are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3-linker" and variations thereof, although one skilled in the art will appreciate that a variety of linker sequences and formats may be used.

[0155]

[0176] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species that the CAR is used in. For example, if it is planned to be used therapeutically in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise a human or humanized antibody or an antigen-binding fragment thereof. Human or humanized antibody or an antigen-binding fragment thereof can be produced by various methods that are well known in the art. The CAR disclosed herein has an extracellular linker / tag epitope binding domain as the antigen-binding domain, which allows it to indirectly bind to the antigen expressed on the target cell via the target cell binding molecule disclosed herein.

[0156]

[0177] As used herein, "spacer" or "hinge" refers to a hydrophilic region between the antigen-binding domain and the transmembrane domain. The CAR of the present invention may include an extracellular spacer domain, but such a spacer may be removed. The spacer may include, for example, an Fc fragment of an antibody or a fragment thereof, a hinge region of an antibody or a fragment thereof, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A prominent example of a spacer is the CD8 alpha hinge.

[0157]

[0178] The transmembrane domain of the CAR can be derived from any desired natural or synthetic source of such domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8 alpha or CD28. If the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides, the CAR can have two (or more) transmembrane domains. The division of the key signaling and antigen recognition modules allows small molecule-dependent, titratable and reversible control of CAR cell expression due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR (Wu et al., 2015, Science 350:293-303).

[0158]

[0179] The cytoplasmic domain (or intracellular signaling domain) of a CAR is involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. By "effector function" is meant a special function of a cell; for example, in T cells, the effector function can be cytolytic activity, or helper cell activity, including secretion of cytokines. An intracellular signaling domain refers to a portion of a protein that transmits an effector function signal and allows the cell expressing the CAR to carry out a special function. An intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein that is sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0159]

[0180] The function of the intracellular domain may be pro- or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0181] Prominent examples of intracellular signaling domains used in CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signaling following antigen receptor engagement.

[0160]

[0182] In general, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly, those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and secondly, those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0161]

[0183] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0184] Examples of ITAMs containing primary cytoplasmic signaling sequences that are frequently used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is the sequence derived from CD3 zeta.

[0162]

[0185] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0163]

[0186] The cytoplasmic domain of the CAR can be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, and are necessary for efficient lymphocyte response to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

[0164]

[0187] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order, with or without a linker. A short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A prominent linker is the glycine-serine doublet.

[0165]

[0188] As one example, the cytoplasmic domain may include the signaling domain of CD3-zeta and the signaling domain of CD28. In another example, the cytoplasmic domain may include the signaling domain of CD3-zeta and the signaling domain of CD27. In a further example, the cytoplasmic domain may include the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0166]

[0189] As mentioned above, the extracellular portion or transmembrane or cytoplasmic domain of the CAR may also contain a heterodimerization domain for the purpose of splitting the key signaling and antigen recognition modules of the CAR.

[0167]

[0190] The CARs of the present invention, i.e., CARs comprising an nfP2X7E200 binding domain, can be designed to contain any portion or part of the above-mentioned domains described herein in any order and / or combination that results in a functional CAR.

[0168]

[0191] The CAR disclosed herein, or a polypeptide(s) derived therefrom, a nucleic acid molecule or recombinant expression vector encoding said CAR, or a population of cells expressing said CAR, can be isolated and / or purified. The term "isolated" means modified or removed from a natural state. For example, an isolated cell population refers to the enrichment of such cells and their separation from other cells that are normally associated with said isolated cells in their naturally occurring state. An isolated cell population refers to a substantially purified cell population that is a more homogeneous cell population than found in nature. Preferably, an enriched cell population comprises at least about 90% of a selected cell type. In certain embodiments, the cell population comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of a selected cell type.

[0169] Target antigen epitope

[0192] The present invention provides for the use of molecules (e.g., peptides or polypeptides) that comprise or consist of an epitope of a target antigen that is recognized by a cellular immunotherapeutic agent, and thus the molecule or polypeptide competes with the target antigen on a cancer cell for binding to the cellular immunotherapeutic agent.

[0170]

[0193] As used herein, the terms peptide and polypeptide can be used interchangeably, particularly when the total length of the molecule is less than 50 amino acids.

[0194] Typically, the epitope contained in the molecule (e.g., peptide or polypeptide) comprises the same or substantially the same amino acid sequence of the epitope that the cellular immunotherapeutic agent intends to bind to on the target cell.For example, if the amino acid sequence of the epitope contained in the molecule is different from the amino acid sequence contained in the antigen on the target cell, the difference in amino acid sequence does not substantially affect the ability of the molecule to bind to the cellular immunotherapeutic agent (or still compete with the target antigen on the target cell for binding to the cellular immunotherapeutic agent).The amino acid sequence of the epitope contained in the molecule can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the epitope on the target antigen on the cell. In certain embodiments, the amino acid sequence of the epitope on the target antigen is the same sequence as an epitope contained on the molecule used to inhibit the activity of the cellular immunotherapeutic agent.

[0171]

[0195] In certain examples, the cellular immunotherapeutic agent is for binding to the extracellular domain of CD19 on target cells. In such examples, it is understood that the molecule for use according to the first or second aspect of the present invention comprises the same epitope of the ECD of CD19 that is bound by the cellular immunotherapeutic agent. The cellular immunotherapeutic agent that targets CD19 is known to those skilled in the art, as is the epitope that such immunotherapeutic agent binds to. For example, to inhibit the activity of CAR-T cells that comprise a CAR with an antigen recognition domain that consists of anti-CD19 scFv FMC683, those skilled in the art will understand that the molecule for use according to the first or second aspect of the present invention should comprise the epitope that is bound by scFv FMC683. Similarly, to inhibit the activity of CAR-T cells that comprise a CAR with an antigen recognition domain that consists of anti-CD19 scFv A3B1, those skilled in the art will understand that the molecule for use according to the first or second aspect of the present invention should comprise the epitope that is bound by scFv A3B1. The epitopes bound by the anti-CD19 antibodies FMC683, 3B10, and 4G7-2E3 used in various anti-CD19 cellular immunotherapeutics are described in Klesmith et al. (2019) Biochemistry 58:489-4881, which is incorporated herein by reference.

[0172]

[0196] In a similar example, the cellular immunotherapeutic agent is for binding CD20 on a target cell. In such an example, it will be understood that the molecule for use according to the first or second aspect of the invention comprises the same epitope of CD20 that is bound by the cellular immunotherapeutic agent.

[0173]

[0197] In other examples, the cellular immunotherapeutic agent is one that is intended to bind mesothelin, and thus the molecule or polypeptide includes an epitope of mesothelin.

[0198] In other examples, the cellular immunotherapeutic agent is one that is intended to bind EGFR, and thus the molecule or polypeptide includes an epitope of EGFR.

[0174]

[0199] In other examples, the cellular immunotherapeutic agent is one that is intended to bind GPC3, and thus the molecule or polypeptide comprises an epitope of GPC3.

[0200] In other examples, the cellular immunotherapeutic agent is one that is intended to bind MUC1, and thus the molecule or polypeptide includes an epitope of MUC1.

[0175]

[0201] In other examples, the cellular immunotherapeutic agent is directed to bind HER2, and thus the molecule or polypeptide includes an epitope of HER2.

[0202] In other examples, the cellular immunotherapeutic agent is directed against GD2, and thus the molecule or polypeptide includes an epitope of GD2.

[0176]

[0203] In other examples, the cellular immunotherapeutic agent is one that is intended to bind CEA, and thus the molecule or polypeptide includes an epitope of CEA.

[0204] In other examples, the cellular immunotherapeutic agent is intended to bind EpCAM, and thus the molecule or polypeptide includes an epitope of EpCAM.

[0177]

[0205] In another example, the cellular immunotherapeutic agent is one that binds LeY, and thus the molecule or polypeptide comprises an epitope of LeY.

[0206] In other examples, the cellular immunotherapeutic agent is directed against PSCA, and thus the molecule or polypeptide includes an epitope of PSCA.

[0178]

[0207] In other examples, the cellular immunotherapeutic agent is directed against CD276, and thus the molecule or polypeptide comprises an epitope of CD276.

[0208] In another example, the cellular immunotherapeutic agent is for binding to a dysfunctional P2X7 receptor, and thus the molecule or polypeptide comprises an epitope of the dysfunctional P2X7 receptor.

[0179]

[0209] Typically, the epitope of the dysfunctional P2X7 receptor comprises a peptide fragment of the dysfunctional P2X7 receptor, said fragment comprising an epitope not found on the functional P2X7 receptor.

[0180]

[0210] Therefore, dysfunctional P2X7 receptor epitope can be provided in the form of a fragment of dysfunctional P2X7 receptor that has at least one of the three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP.Such receptor cannot extend the opening of non-selective calcium channel to apoptotic pore.

[0181]

[0211] The scope of peptide fragments of dysfunctional P2X7 receptors is described in PCT / AU2002 / 000061 (as well as corresponding WO 2002 / 057306 and U.S. Pat. Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,450,380), PCT T / AU2008 / 001364 (as well as corresponding WO 2009 / 033233 and U.S. Pat. Nos. 8,440,186, 9,181,320, 9,944,701 or 10,597,45) and PCT / AU2009 / 000869 (as well as corresponding WO 2010 / 000041 and U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716). Exemplary peptides within these specifications that contain epitopes contemplated for use in the present invention are described below. PCT Application Published Peptide Sequence WO 2002 / 057306 GHNYTTRNILPGLNITC (SEQ ID NO:2) (also referred to herein as the "E200" epitope) WO 2009 / 033233 KYYKENNVEKRTLIKVF (SEQ ID NO: 3) (also referred to herein as the "E300" epitope) WO 2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 4) (also referred to herein as the "E200 / E300" or "composite" epitope).

[0182]

[0212] Further examples of E200, E300 and composite epitopes are described in Table 1 herein.As discussed elsewhere herein, peptides or polypeptides that include E200, E300 or composite epitopes can contain additional amino acids.In certain instances, additional amino acids can be directly derived from adjacent sequences present in nfP2X7 receptor sequence.Alternatively, additional amino acid sequences can be derived from one or more linker sequences, such as glycine / serine-rich linker sequences and / or hinge regions derived from immunoglobulins.Those skilled in the art will understand that such modifications can be made to E200, E300 or composite epitopes for the purpose of increasing the stability or solubility of peptides or polypeptides, or for the purpose of adjusting the degree of binding to CAR.

[0183]

[0213] In any embodiment of the first or second aspect, the polypeptide reduces the ability of cellular immunotherapeutic agent to bind to the dysfunctional P2X7 receptor on cell, preferably cancer cell.Preferably, the polypeptide blocks or destroys the interaction between cellular immunotherapeutic agent and the dysfunctional P2X7 receptor present on cancer cell.

[0184]

[0214] In any embodiment of the first or second aspect, the epitope on the polypeptide comprises the amino acid sequence that is substantially the same or homologous to the epitope on the dysfunctional P2X7 receptor that is bound by cellular immunotherapeutic agent.In other words, even if the amino acid sequences of the two epitopes are different, they are sufficiently homologous for the cellular immunotherapeutic agent to bind to both the polypeptide and the dysfunctional P2X7 receptor on the cell.In any embodiment, the epitope on the polypeptide comprises or consists of the amino acid sequence of the epitope on the dysfunctional P2X7 receptor that the cellular immunotherapeutic agent binds to.

[0185] Molecules for use according to the present invention

[0215] It will be understood that the architecture of the molecules of the invention will depend on whether the molecule is to be used according to the first or second aspect of the invention.

[0186]

[0216] In certain embodiments, the molecule may be in the form of a polypeptide. Such a polypeptide may be in the form of a fusion protein or a chimeric protein.

[0217] More specifically, the molecule is preferably in the form of a polypeptide that contains a sequence and architecture that promotes a transient and reversible effect in cellular immunotherapeutics.

[0187]

[0218] For example, to bind a target antigen to a CAR-T cell, the polypeptide may include an epitope for binding to the antigen recognition domain of the CAR, and may or may not include additional sequences to facilitate increased solubility and stability of the polypeptide.

[0188]

[0219] In the particular example of anti-nfP2X7 receptor CAR T cells, the molecule for use in the method of the present invention is preferably in the form of a peptide or polypeptide comprising the amino acid sequence of E200, E300, or composite epitope, as defined herein. In a particularly preferred embodiment, the molecule is a polypeptide or peptide comprising the amino acid sequence of the E200 epitope, preferably comprising at least any of the sequences set forth in SEQ ID NO: 3, 4, 5, or 6. Furthermore, the peptide or polypeptide may comprise additional amino acid residues derived from the nfP2X7 receptor sequence and occurring adjacent to the E200 epitope in the native receptor sequence. Furthermore, the polypeptide may comprise additional amino acid residues to improve the solubility and / or stability of the polypeptide, such as the addition of amino acid residues derived from the glycine / serine-rich linker region and / or IgG hinge region. Some examples of such additions to the "minimal" E200 epitope sequence are provided in Table 1 herein, although the skilled artisan will understand that any number of alternative modifications may be used.

[0189]

[0220] Also, it is well within the scope of a person skilled in the art to confirm the ability of a given molecule to be bound by related CAR.For example, in the context of nfP2X7 receptor binding CAR, a person skilled in the art can use routine techniques to confirm the binding of a polypeptide (or a series of polypeptides) by CAR, and thereby determine the suitability of the polypeptide for use in the method of the present invention.

[0190]

[0221] In the broader context of CARs other than those designed to bind to nfP2X7 receptor or E200 epitope, a person skilled in the art can similarly determine whether a designed molecule or polypeptide can bind to a CAR. For example, once the immunotherapeutic agent to be inhibited is determined, a person skilled in the art can easily determine the antigen that the immunotherapeutic agent is designed to bind to. In the simplest example, this can be done by referring to the product information on the immunotherapeutic agent and the knowledge of the target antigen. Then, a person skilled in the art can formulate the amino acid sequence of the molecule (preferably a peptide or polypeptide) to disrupt the interaction between the cellular immunotherapeutic agent and the target antigen. In the simplest example, a person skilled in the art can design a peptide or polypeptide that contains the same amino acid sequence as the target antigen of the cellular immunotherapeutic agent.

[0191]

[0222] Finally, those skilled in the art have identified the appropriate amino acid content of the molecule for disrupting the interaction between the cellular immunotherapeutic agent of the present invention and the target antigen, so that they can easily use routine techniques to determine whether the molecule a) binds to CAR T cells and b) can successfully inhibit the cell killing by CAR T cells.The method of determining the binding to target antigen and cytotoxicity is well known in the art.The non-limiting method and various experimental protocols for determining the binding to CAR T cells and the reduction of cell killing are described in detail in the examples herein.

[0192]

[0223] Further examples of sequences to promote increased solubility and stability of a polypeptide may include sequences of serum albumin (preferably HSA), transferrin, sequences of the Fc region of an antibody, or sequences of the carboxy-terminal peptide of the chorionic gonadotropin (CG) β chain. Other sequences may include unstructured polypeptide sequences to increase the overall size and hydrodynamic radius of the polypeptide. Such approaches have previously been referred to as "XTEN-ylation" (inexact repeat peptide sequences), "PAS-ylation" (fusion of polypeptide sequences composed of proline-alanine-serine polymers, i.e., PAS), "ELP-ylation" (fusion to elastin-like peptide (ELP) repeat sequences), "HAP-ylation" (e.g., inclusion of homopolymers of glycine residues) or GLK (gelatin-like protein) fusions.

[0193]

[0224] It will be appreciated that a wide variety of other architectures may be adopted, such as, for example, the epitope-containing polypeptide may be conjugated or fused to any suitable carrier moiety, hi certain embodiments, the carrier moiety may be selected from carbohydrates, lipids, liposomes, peptides, and aptamers.

[0194]

[0225] In some embodiments, the polypeptide may be provided in the context of a liposome that includes the polypeptide on its surface by PEG coupling (e.g., a PEGylated liposome), providing the liposome with an epitope that is recognized and bound by a cellular immunotherapeutic agent.

[0195]

[0226] In the situation where the polypeptide comprises the sequence of an Fc region of an antibody, according to the first aspect of the invention, the Fc region preferably does not contain any other sequence which targets a cellular immunotherapeutic agent for cell killing when the polypeptide binds. In contrast, a polypeptide for use according to the second aspect of the invention preferably comprises an antibody Fc region which comprises such a sequence.

[0196]

[0227] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region contains two heavy chain fragments that contain the CH2 and CH3 domains of an antibody. In the context of the present invention, the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of said heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic interactions of the CH3 domain.

[0197]

[0228] According to the first aspect of the present invention, the Fc fusion protein preferably does not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to the biological activity attributable to the Fc region of an antibody and varies with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

[0198]

[0229] Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0199]

[0230] Fc regions of antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (US Pat. No. 7,332,581). For example, the antibody variant may include an Fc region with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, the substitutions are L234A and L235A (LALA) (see, for example, WO 2012 / 130831). Additionally, modifications can be made in the Fc region that result in altered (i.e., decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0200]

[0231] Other Fc modifications for use according to the first aspect of the invention include variants that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Such variants are also referred to herein as "knockout variants" or "KO variants." Variants that reduce binding to FcγR and complement are useful for reducing undesirable interactions mediated by the Fc region and tuning the selectivity of fusion proteins. Preferred knockout variants are described in U.S. Patent Application Publication No. 2008-0242845, entitled "Fc Variants with Optimized Properties," published October 2, 2008, which is expressly incorporated herein by reference. Preferred modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, numbering according to the EU index. Preferred substitutions include, but are not limited to, 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R. A preferred variant includes 236R / 328R. The variant can be used in conjunction with any IgG isotype or IgG isotype Fc region, including, but not limited to, human IgG1, IgG2, IgG3, and / or IgG4. Preferred IgG Fc regions for reducing FcγR and complement binding and reducing Fc-mediated effector functions are IgG2 and IgG4 Fc regions. Hybrid isotypes can be useful, such as the hybrid IgG1 / IgG2 isotype described in US Patent Application Serial No. 11 / 256,060. Other modifications to reduce FcγR and complement interactions include, but are not limited to, substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutation or enzymatic means, or by production in organisms such as bacteria that do not glycosylate proteins.These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691, which is incorporated by reference in its entirety.

[0201]

[0232] Fc modifications that improve binding to FcγR and / or complement can find use according to the second aspect of the invention. Such Fc variants can enhance Fc-mediated effector functions such as ADCC, ADCP, and / or CDC. Preferred modifications for improving FcγR and complement binding are described in US Patent Application Publication No. 2006-0024298, published February 2, 2006, and US Patent Application Publication No. 2006-0235208, published October 19, 2006, which are expressly incorporated herein by reference. Preferred modifications include substitutions at positions selected from the group consisting of 236, 239, 268, 324, and 332, numbering according to the EU index. Preferred substitutions include, but are not limited to, 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Preferred variants include, but are not limited to, 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other modifications to enhance FcγR and complement interactions include, but are not limited to, the substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl, 2009 (supra).

[0202]

[0233] In one embodiment, the fusions disclosed herein can incorporate Fc variants that enhance affinity to the inhibitory receptor FcγRIIb. Such variants can provide the fusions herein with immunomodulatory activity associated with FcγRIIb+ cells, including, for example, B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity for FcγRIIb to one or more activating receptors. Modifications for altering binding to FcγRIIb are described in U.S. Patent Application No. 12 / 156,183, entitled "Methods and Compositions for Inhibiting CD32b Expressing Cells," filed May 30, 2008, which is expressly incorporated herein by reference. In particular, Fc variants that improve binding to FcγRIIb may contain one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332 according to the EU index. Preferred substitutions for enhancing FcγRIIb affinity include, but are not limited to, 234D, 234E, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. More preferably, substitutions include, but are not limited to, 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Preferred Fc variants for enhancing binding to FcγRIIb include, but are not limited to, 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0203]

[0234] The fusion proteins described herein can incorporate Fc modifications in the context of any IgG isotype or IgG isotype Fc region, including but not limited to human IgG1, IgG2, IgG3, and / or IgG4. The IgG isotype can be selected to alter FcγR and / or complement-mediated effector function(s). Hybrid IgG isotypes are also useful. For example, US patent application Ser. No. 11 / 256,060 describes a number of hybrid IgG1 / IgG2 constant regions that can find use in certain inventions. In some embodiments of the invention, the fusion proteins can include a means for isotype modification, i.e., modification in the parent IgG to the amino acid type in the alternative IgG. For example, an IgG1 / IgG3 hybrid variant can be constructed by substitution means to replace IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that include one or more substitution means, for example, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments of the invention, IgG1 / IgG2 hybrid variants can be constructed by substitution means to replace IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that include one or more substitution means, for example, one or more of the following amino acid substitution means: 233E, 234L, 235L, -236G (referring to the insertion of glycine at position 236), and 327A.

[0204]

[0235] In an embodiment of the second aspect of the present invention, the fusion protein disclosed herein can incorporate Fc variants that improve FcRn binding. Such variants can enhance the in vivo pharmacokinetic properties of fusion protein. Preferred variants that increase FcRn binding and / or improve pharmacokinetic properties include, but are not limited to, substitutions at positions 259, 308, 428, and 434, including, but not limited to, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M (U.S. Patent Application No. 12 / 341,769, entitled "Fc Variants with Altered Binding to FcRn," filed December 22, 2008, which is incorporated by reference in its entirety). Other variants that increase Fc binding to FcRn include, but are not limited to, 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216; Hinton et al., 2006, Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Sciences, vol. 104(1):104-105, which is incorporated by reference in its entirety). Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall'Acqua et al., Journal of Immunology, 2002, 169:5171-5180; Dall'Acqua et al., 2006, Journal of Biological Chemistry, all of which are incorporated by reference in their entirety). 281:23514-23524).Other modifications to modulate FcRn binding are described in Yeung et al., 2010, J Immunol 182:7663-7671.

[0205]

[0236] The polypeptide may be in the form of a fusion protein, so that the region containing the epitope and any additional sequence is encoded by a single core construct, i.e., "gene fusion". Alternatively, the polypeptide may contain the dysfunctional P2X7 receptor epitope and any additional sequence that is connected by chemical conjugation or non-covalent attachment via peptide.

[0206]

[0237] The fusion proteins described herein may include a linker region for linking the sequence comprising the dysfunctional P2X7 receptor epitope to any additional sequence described herein (e.g., an antibody Fc region, an HSA sequence, etc.).

[0207]

[0238] The term "linker" is used to refer to a polypeptide that comprises two or more amino acid residues connected by peptide bonds, and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2:1121-1123). A variety of linkers can find use in some embodiments described herein to covalently link two different polypeptides or peptide sequences.

[0208]

[0239] A "linker" herein is also referred to as a "linker sequence," "spacer," "tethering sequence," or grammatical equivalents thereof. Homo- or hetero-bifunctional linkers are well known (see Pierce Chemical Company catalogue, technical section on cross-linkers, 1994, pages 155-200, which is incorporated by reference in its entirety). Numerous strategies can be used to covalently link molecules to one another. These include, but are not limited to, polypeptide linkages between the N-terminus and C-terminus of a protein or protein domain, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant technology or peptide synthesis. The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of sufficient length to link two molecules such that they adopt the correct conformation relative to one another so that they retain the desired activity. In one embodiment, the linker is about 1-50 amino acids in length, preferably about 1-30 amino acids in length. In one embodiment, a linker of 1-20 amino acids in length can be used. Useful linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1. Alternatively, a variety of non-proteinaceous polymers can find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The fusion proteins of the invention can include a linker region (or spacer) located between the dysfunctional P2X7 receptor epitope and any of the additional sequences described herein.

[0209]

[0240] The linker is usually a peptide having a length of up to 20 amino acids. The term "linked" or "fused" refers to a covalent bond formed between two moieties, such as a peptide bond. Thus, in the context of the present invention, the linker can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

[0210]

[0241] In some aspects, the fusion protein of the present invention comprises a peptide linker. Those skilled in the art are familiar with the design and use of various peptide linkers composed of various amino acids and of various lengths, which are suitable for use as linkers according to the present invention. The linker may comprise various combinations of repeated amino acid sequences.

[0211]

[0242] Linker can be flexible linker (e.g., comprises repeats of glycine and serine residues), rigid linker (e.g., comprises glutamic acid and lysine residues, adjacent alanine repeats), and / or cleavable linker (e.g., sequence that is more susceptible to protease cleavage).Examples of such linkers are known to those skilled in the art and are described, for example, in Chen et al., (2013) Advanced Drug Delivery Reviews, vol. 65:1357-1369.

[0212]

[0243] In some embodiments, the peptide linker may comprise the amino acids glycine and serine of various lengths and combinations. In some embodiments, the peptide linker may comprise the sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS) or Gly-Gly-Gly-Gly-Ser (GGGGS) and variations or repeats thereof. In some embodiments, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer. The linker may comprise a series of repeating glycine and serine residues (GS) of different lengths, i.e. (GS) nwhere n is any number from 1 to 15 or more. For example, the linker can be (GS)3 (i.e., GSGSGS) or longer (GS) 11 or longer. It is understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins with linkers of such lengths are included within the scope of the present invention. Similarly, the linker can be a series of repeated glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker can include the amino acid sequence GGGSGGGGSGGGGS(G4S)3) and variations thereof.

[0213]

[0244] The peptide linker can consist of a series of repeats of Thr-Pro (TP) with one or more additional amino acids at the N-terminus and C-terminus of the repeat sequence. For example, the linker can comprise or consist of the sequence GTPTPTPTPTGEF (also known as the TP5 linker). In further embodiments, the linker can be a short and / or alpha-helical rigid linker (e.g., A(EAAAK)3A, a PAPAP linker or a dipeptide such as LE).

[0214]

[0245] In certain embodiments, the linker may be flexible and cleavable. Such linkers preferably contain one or more recognition sites for a protease that allows for cleavage.

[0215]

[0246] Preferred linkers of the present invention include sequences from antibody hinge regions. Hinge region sequences from any antibody isotype can be used, including, for example, hinge sequences from IgG1, IgG2, IgG3, and / or IgG4. The linker sequence can also include any sequence of the CL / CH1 domain of any length, rather than all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. The linker can be from an immunoglobulin heavy chain of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. The linker can be from an immunoglobulin light chain, for example, Cκ or Cλ. The linker sequence can also be from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences from hinge regions, and other naturally occurring sequences from other proteins.

[0216]

[0247] In an alternative embodiment of the second aspect of the present invention, the molecule may be in the form of a protein conjugate, comprising a first portion in the form of an epitope of a target antigen (i.e., to compete for binding to the cellular immunotherapeutic agent) and a second portion in the form of a toxin.Preferably, when such a molecule binds to the cellular immunotherapeutic agent, the cellular immunotherapeutic agent undergoes cell death mediated by the toxin.It is understood that any suitable toxin (such as, for example, a toxin that is typically conjugated to an antibody to induce cell death) can be utilized.

[0217]

[0248] In certain embodiments of the second aspect of the invention, the molecule may be in the form of an anti-idiotypic antibody for binding to a cellular immunotherapeutic agent and is conjugated to a cytotoxin or chemotherapeutic agent for inducing cell death of the cellular immunotherapeutic agent.

[0218]

[0249] One of skill in the art would be fully able to identify suitable anti-idiotypic antibodies for use in accordance with such embodiments of the invention, and would be familiar with suitable cytotoxic agents for conjugating to anti-idiotypic antibodies for use in this aspect of the invention. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents (e.g., maytansinoids, auristatins, drostatins, calicheamicin or derivatives thereof, taxanes, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, e.g., nucleases; antibiotics; toxins, e.g., small molecule toxins or enzymatically active toxins. In some embodiments, the antibody is conjugated to one or more cytotoxic agents, chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes. Also among the anti-idiotypic antibodies are immunoconjugates in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0219]

[0250] Conjugates of anti-idiotypic antibodies and cytotoxic agents can be made using any of a number of known protein coupling agents, such as linkers (see Vitetta et al., Science 238:1098 (1987); WO 94 / 11026). The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell, such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0220] nucleic acid

[0251] The present invention also provides nucleic acid molecules encoding the polypeptides described herein for use in inhibiting the activity of a cellular immunotherapeutic agent.

[0221]

[0252] The nucleic acid can be utilized to generate the polypeptide intended to be administered according to the present invention using in vitro methods. Alternatively, the nucleic acid can facilitate the in vivo expression of the polypeptide, such that the nucleic acid is administered to a subject in need of treatment. In such an embodiment, the nucleic acid can include a nucleic acid sequence encoding the polypeptide and a controllable promoter for inducing its expression. Alternatively, the promoter can provide for constitutive expression of the polypeptide. Furthermore, it is understood that, depending on the context, the nucleic acid can be for transient expression of the nucleic acid sequence encoding the polypeptide.

[0222]

[0253] A nucleic acid molecule can include any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified, RNA or DNA. For example, a nucleic acid molecule can include single-stranded and / or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that include DNA and RNA that can be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, a nucleic acid molecule can include triple-stranded regions that include RNA or DNA, or both RNA and DNA. A nucleic acid molecule can also include one or more modified bases, or DNA or RNA backbones that are modified for stability or for other reasons. A variety of modifications can be made to DNA and RNA, and thus the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0223]

[0254] In some embodiments of the second aspect of the invention, the nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid sequence of any one of SEQ ID NOs: 2, 3 and 4.

[0255] The present invention further provides a nucleic acid construct comprising a nucleic acid molecule encoding a polypeptide of the present invention or a portion thereof. The nucleic acid construct may further comprise one or more of the following: an origin of replication for one or more hosts; a selectable marker gene active in one or more hosts; and / or one or more transcription control sequences.

[0224]

[0256] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on the cell in which it is expressed, facilitating the identification and / or selection of cells that have been transfected or transformed with a construct.

[0225]

[0257] A "selection marker gene" includes any nucleotide sequence that, when expressed by cells transformed with a construct, confers a phenotype to the cells that facilitates the identification and / or selection of these transformed cells. A range of nucleotide sequences encoding suitable selection markers are known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selection markers include an adenosine deaminase (ADA) gene; a cytosine deaminase (CDA) gene; a dihydrofolate reductase (DHFR) gene; a histidinol dehydrogenase (hisD) gene; a puromycin-N-acetyltransferase (PAC) gene; a thymidine kinase (TK) gene; a xanthine-guanine phosphoribosyltransferase (XGPRT) gene, or an antibiotic resistance gene, such as an ampicillin resistance gene, a puromycin resistance gene, a bleomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, and an ampicillin resistance gene; fluorescent reporter genes, such as genes encoding green, red, yellow, or blue fluorescent proteins; and luminescence-based reporter genes, such as a luciferase gene, that allow optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS), among others.

[0226]

[0258] Furthermore, it should be noted that the selectable marker gene can be a separate open reading frame in the construct, or can be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0227]

[0259] As mentioned above, the nucleic acid construct may also include one or more transcription control sequences. The term "transcription control sequence" should be understood to include any nucleic acid sequence that acts on the transcription of an operably connected nucleic acid. Transcription control sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer or upstream activation sequence, and a transcription terminator. Typically, a transcription control sequence includes at least a promoter. The term "promoter" as used herein describes any nucleic acid that confers, activates, or enhances the expression of a nucleic acid in a cell.

[0228]

[0260] In some embodiments, at least one transcription control sequence is operably connected to the nucleic acid molecule of the second aspect of the present invention.For the purposes of this specification, a transcription control sequence is considered to be "operably connected" to a given nucleic acid molecule if the transcription control sequence can promote, inhibit or otherwise regulate the transcription of the nucleic acid molecule.Thus, in some embodiments, the nucleic acid molecule is under the control of a transcription control sequence, such as a constitutive promoter or an inducible promoter.

[0229]

[0261] "Nucleic acid construct" can be in any suitable form, such as a plasmid, phage, transposon, cosmid, chromosome, vector, etc., and can replicate when associated with appropriate control elements, and can introduce the genetic sequence contained within the construct between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, the nucleic acid construct is a vector. In some embodiments, the vector is a viral vector.

[0230]

[0262] A promoter can regulate the expression of an operably linked nucleic acid molecule constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs. Thus, a promoter can include, for example, a constitutive promoter or an inducible promoter. A "constitutive promoter" is a promoter that is active under most environmental and physiological conditions. An "inducible promoter" is a promoter that is active under a specific environmental or physiological condition. The present invention contemplates the use of any promoter that is active in the cell of interest. Thus, a wide range of promoters can be easily ascertained by one skilled in the art.

[0231]

[0263] Mammalian constitutive promoters can include, but are not limited to, Simian Virus 40 (SV40), Cytomegalovirus (CMV), P-actin, Ubiquitin C (UBC), Elongation factor-1 alpha (EF1A), Phosphoglycerate kinase (PGK) and CMV early enhancer / chicken beta actin (CAGG).

[0232]

[0264] Inducible promoters can include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters whose activity is regulated by compounds such as alcohol, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include, among others, tetracycline-regulated promoters (see, for example, U.S. Pat. No. 5,851,796 and U.S. Pat. No. 5,464,758); steroid-responsive promoters, such as glucocorticoid receptor promoters (see, for example, U.S. Pat. No. 5,512,483), ecdysone receptor promoters (see, for example, U.S. Pat. No. 6,379,945), and the like; and genera-responsive promoters, such as metallothionein promoters (see, for example, U.S. Pat. No. 4,940,661, U.S. Pat. No. 4,579,821 and U.S. Pat. No. 4,601,978).

[0233]

[0265] As mentioned above, the control sequence may also include a terminator. The term "terminator" refers to the DNA sequence at the end of a transcription unit that signals the end of transcription. A terminator is generally a 3' non-translated DNA sequence that contains a polyadenylation signal, and promotes the addition of a polyadenylation sequence to the 3' end of a primary transcript. Similar to a promoter sequence, a terminator may be any terminator sequence that is operable in the cell, tissue or organ in which it is intended to be used. Suitable terminators are known to those skilled in the art.

[0234]

[0266] As will be appreciated, the nucleic acid constructs of the present invention can further include additional sequences, such as sequences that allow for enhanced expression, cytoplasmic or membrane transport, and location signals. Specific non-limiting examples include an internal ribosome entry site (IRES) or a cleavage site (e.g., P2A, T2A).

[0235]

[0267] The present invention extends to all genetic constructs essentially as described herein. These constructs may further comprise nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotic organisms and / or for the integration of the genetic construct or a part thereof into the genome of a eukaryotic cell.

[0236]

[0268] Methods for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the third aspect of the present invention, into eukaryotic cells are known in the art.As will be understood, the most suitable method for introducing a nucleic acid construct into a desired host cell depends on many factors, such as the size of the nucleic acid construct, the type of host cell, the desired efficiency of transfection / transduction, and the final desired or required survival rate of the transfected / transduced cells.Non-limiting examples of such methods include chemical transfection by chemicals, such as cationic polymers, calcium phosphate, or structures, such as liposomes and dendrimers; non-chemical methods, such as electroporation, sonoporation, heat shock or optical transfection; particle-based methods, such as "gene gun" delivery, magnetofection, or impalefection or viral transduction.

[0237]

[0269] The nucleic acid construct is selected depending on the desired method of transfection / transduction. In some embodiments of the third aspect of the invention, the nucleic acid construct is a viral vector and the method of introducing the nucleic acid construct into the host cell is viral transduction. Methods utilizing viral transduction to induce expression of CAR in PBMCs (Parker, LL. et al., Hum Gene Ther. 2000; vol. 11: 2377-87), and more generally utilizing retroviral systems for transduction of mammalian cells (Cepko, C. and Pear, W. Curr Protoc Mol Biol. 2001, unit 9.9), are known in the art. In other embodiments, the nucleic acid construct is a plasmid, cosmid, artificial chromosome, etc., and can be transfected into cells by any suitable method known in the art.

[0238] Treatment and Administration Methods

[0270] As discussed further herein, the present invention finds application in the treatment of a variety of conditions, preferably in the treatment of cancer.

[0239]

[0271] More specifically, the present invention finds particular application in fine-tuning or switching off various cellular immunotherapeutic agents that target dysfunctional P2X7 receptor.Such application can find use in treating, minimizing or reducing the risk of abnormal immune response that arises from such cellular immunotherapeutic agents.

[0240]

[0272] Inappropriate or uncontrolled activation of the immune system leads to potentially lethal immune responses consisting of a positive feedback loop between cytokines and white blood cells, resulting in highly elevated levels of various cytokines. Such uncontrolled immune activation, often referred to as cytokine cascade, cytokine-associated toxicity, or cytokine release syndrome, can be induced by many physical conditions or medical therapies, most notably immunotherapy, which specifically harnesses the recipient's immune system to fight disease.

[0241]

[0273] Although different terms may be used to describe cytokine storm, cytokine cascade, or cytokine release syndrome, these conditions all have in common the uncontrolled activation of the immune system, which can have fatal consequences.

[0242]

[0274] As used herein, cytokine-associated toxicity refers to harmful cytokine responses to aberrant immune system activation, e.g., caused by disease, but also including immunomodulatory therapies, that can be life-threatening.

[0243]

[0275] Cytokine-associated toxicity is also described in the art using the term cytokine release syndrome (CRS). When severe enough, this syndrome can be called hypercytokinemia or "cytokine storm". As used herein, CRS defines a systemic inflammatory response in patients that is characterized by, among other things, hypotension, fever and / or rigors, potentially leading to death. CRS is believed to be caused by an uncontrolled positive feedback loop between cytokines and immune cells, resulting in highly elevated levels of various cytokines. CRS also involves systemic expression of immune system mediators, including increased levels of pro-inflammatory and anti-inflammatory cytokines.

[0244]

[0276] Those skilled in the art are familiar with the clinical symptoms of cytokine-associated toxicity or hypercytokinemia, and therefore, are also familiar with various methods for identifying individuals in need of treatment for cytokine-associated toxicity, including hypercytokinemia.

[0245]

[0277] The table below lists some of the recognized clinical signs and symptoms of cytokine-associated toxicity.

[0246] [Table 2]

[0247]

[0278] Additionally, the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE v4.0) includes a scoring system designed for cytokine response syndromes associated with antibody therapy, as shown below: Grade 1 - mild reaction; infusion interruption not indicated; intervention not indicated Grade 2 - Treatment or infusion interruption required but responds rapidly to symptomatic treatment (e.g., antihistamines, NSAIDs, narcotics, IV fluids); prophylaxis indicated for <24 hours Grade 3 - prolonged (e.g., not responding promptly to symptomatic treatment and / or briefly interrupting infusion); recurrence of symptoms after initial improvement; hospitalization indicated for clinical sequelae (e.g., renal dysfunction, pulmonary infiltrates) Grade 4 - life-threatening outcome; vasopressor or ventilatory support indicated

[0279] Hypercytokinemia as used herein is a potentially lethal immune response resulting from inappropriate positive signaling between cytokines and immune cells, and ultimately cytokine release. In patients, this leads to high fever, swelling and redness, extreme fatigue, nausea, and in some cases, even fatality. Although over 150 known inflammatory mediators are believed to be released during a cytokine storm, generally, in the method of the present invention, a person skilled in the art can determine the level of cytokine-associated toxicity by measuring the serum level of one or more suitable cytokines, for example, measuring 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cytokines, for example, the cytokines are independently selected from IL-Iβ, TNFα, IL-6, IL-8 (CXCL8), IL-2, IL-10, IFNy, IL-12p70, and GM-CSF (e.g., IL-6, TNFα, IFNy, IL-2, and IL-8).

[0248]

[0280] Test-based methods for measuring serum levels of cytokines indicative of cytokine-associated toxicity are known to those of skill in the art and are useful in the methods of the invention, for example, to determine whether an individual is suffering from cytokine-associated toxicity and to determine whether treatment according to the invention has been successful.

[0249]

[0281] Various methods for determining cytokine levels in biological samples, including serum and plasma samples, are known to those skilled in the art.In brief, the level of inflammatory cytokines can be determined in biological samples by enzyme-linked immunosorbent assay (ELISA) using ELISA kit according to manufacturer's protocol.

[0250]

[0282] Alternatively, serum cytokine levels can be determined using a multiplex bead array kit according to the manufacturer's instructions (eg, Bio-Plex Human Cytokine Assay).

[0251]

[0283] Those skilled in the art will also appreciate that baseline inflammatory cytokine levels may be very high in certain individuals due to underlying disease (receiving immunotherapeutic agents). Thus, those skilled in the art will also appreciate that determining the fold increase, net increase, or rate of change increase in cytokine levels, rather than absolute levels, may provide a better indication of the likelihood of cytokine-related toxicity in an individual.

[0252]

[0284] Other methods for determining the possibility of cytokine-associated toxicity include monitoring proteins that indicate increased cytokine levels.For example, C-reactive protein (CRP) is an acute phase protein produced by the liver, and can often serve as a reliable surrogate for IL-6 bioactivity.Therefore, those skilled in the art also understand the usefulness of measuring CRP levels as a means of identifying individuals who need treatment for cytokine-associated toxicity.

[0253]

[0285] The methods of the invention may also be used to attenuate or permanently switch off the activity of cellular immunotherapeutics in situations where tumor lysis syndrome is suspected or at risk.

[0254]

[0286] Thus, the present invention provides a method for minimizing or reducing the risk of tumor lysis syndrome in a subject undergoing or receiving treatment with a cellular immunotherapeutic agent for binding to a target antigen on a cell (such as, but not limited to, a dysfunctional P2X7 receptor), the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent to bind to a target antigen on a cell; - administering to a subject a molecule, optionally a polypeptide for binding to a cellular immunotherapeutic agent or a nucleic acid encoding said polypeptide, wherein the molecule or polypeptide comprises or consists of an epitope of a target antigen, the epitope on the molecule or polypeptide competes with an epitope on the target antigen for binding to the cellular immunotherapeutic agent, whereby the molecule or polypeptide disrupts an interaction between the cellular immunotherapeutic agent and the target antigen on the cell; Thereby, the risk of tumor lysis syndrome in a subject can be minimized or reduced. Preferably, the method does not reduce the viability of the cells of the cellular immunotherapeutic agent.

[0255]

[0287] In a further embodiment, the present invention provides a method of treating tumor lysis syndrome in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen (such as, but not limited to, a dysfunctional P2X7 receptor), the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent to bind to a target antigen on a cell; - administering to a subject a molecule, optionally a polypeptide for binding to a cellular immunotherapeutic agent or a nucleic acid encoding said polypeptide, wherein the molecule or polypeptide comprises or consists of an epitope of a target antigen, The epitope on the molecule polypeptide competes with an epitope on the target antigen for binding to the cellular immunotherapeutic agent, whereby the molecule or polypeptide disrupts the interaction between the cellular immunotherapeutic agent and the target antigen on the cell; The present invention provides a method for treating tumor lysis syndrome in a subject, the method preferably not decreasing the viability of cells of the cellular immunotherapeutic agent.

[0256]

[0288] In a further embodiment, the invention provides a method of minimizing or reducing the risk of tumor lysis syndrome in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent to bind to a target antigen on a cell; - administering to a subject a polypeptide for binding to a cellular immunotherapeutic agent, or a nucleic acid encoding said polypeptide, wherein the polypeptide i) an epitope of a target antigen that competes for binding to a cellular immunotherapeutic agent, whereby the polypeptide disrupts the interaction between the cellular immunotherapeutic agent and the target antigen on the cell; ii) Amino acid sequence for inducing cell-mediated killing wherein when the polypeptide binds to the cellular therapeutic agent, the cellular immunotherapeutic agent is targeted for cell-mediated killing; Thereby, methods are provided that minimize or reduce the risk of tumor lysis syndrome in a subject.

[0257]

[0289] In a further embodiment, the invention provides a method of treating tumor lysis syndrome in a subject who has undergone or is undergoing treatment with a cellular immunotherapeutic agent for binding to a target antigen, the method comprising: - providing a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent to bind to a target antigen on a cell; - administering to a subject a polypeptide for binding to a cellular immunotherapeutic agent, or a nucleic acid encoding said polypeptide; said polypeptide i) an epitope of the target antigen that competes for binding to the cellular immunotherapeutic agent, whereby the polypeptide disrupts the interaction between the cellular immunotherapeutic agent and the target antigen on the cell; ii) Amino acid sequence for inducing cell-mediated killing wherein when the polypeptide binds to the cellular therapeutic agent, the cellular immunotherapeutic agent is targeted for cell-mediated killing; Thereby, a method is provided for treating tumor lysis syndrome in a subject.

[0258]

[0290] As used herein, tumor lysis syndrome refers to a group of metabolic abnormalities that can occur as a complication during cancer treatment, where a large number of tumor cells are dissolved at the same time by the treatment, releasing their contents into the bloodstream.This most often occurs after lymphoma and leukemia treatment.In oncology and hematology, this is a potentially fatal complication, and patients with increased risk of TLS should be closely monitored before, during, and after a course of chemotherapy.

[0259]

[0291] Tumor lysis syndrome is characterized by high blood potassium (hyperkalemia), high blood phosphorus (hyperphosphatemia), low blood calcium (hypocalcemia), high blood uric acid (hyperuricemia), and higher than normal levels of blood urea nitrogen (BUN) and other nitrogen-containing compounds (azotemia). These changes in blood electrolytes and metabolites are the result of the cellular contents of dying cells being released into the bloodstream from the breakdown of cells. In this regard, TLS is similar to rhabdomyolysis, with comparable mechanisms and blood chemistry, but different causes. In TLS, breakdown occurs after cytotoxic therapy or from cancers with high cell turnover and tumor proliferation rates. The metabolic abnormalities seen in tumor lysis syndrome ultimately cause nausea and vomiting, but can lead to more severe acute urate nephropathy, acute renal failure, seizures, cardiac arrhythmias, and death.

[0260]

[0292] It is understood that the method of the present invention provides flexibility with respect to the timing of administration of the cellular immunotherapeutic agent and molecule described herein.In certain embodiments of the first aspect of the present invention, the timing of administration of the molecule (e.g., polypeptide) that comprises the epitope of the target antigen can be such that the molecule is administered only at the first sign or symptom that requires reducing the activity of the cellular immunotherapeutic agent (e.g., evidence of the increased presence of circulating cytokines that may predict impending cytokine storm).

[0261]

[0293] Alternatively, according to the first aspect of the present invention, the molecule can be administered after such time that the cellular immunotherapeutic agent has already successfully reduced a significant proportion of the tumor burden, but the cellular immunotherapeutic agent is preferably given a chance to repopulate / regain efficacy. In this way, the timing of administration of the molecule allows the clinician to temporarily turn off the cellular immunotherapeutic agent to prevent cell exhaustion. Furthermore, it is within the skill of the art to determine the appropriate dose of the molecule to be administered to allow a mild, moderate or significant reduction in the activity of the cells. Furthermore, the polypeptide can be administered according to a dosage gradient to allow a gradual attenuation of the activity of the cells.

[0262]

[0294] Sustained activation of CAR T cells can result in overactivation and activation-induced cell death. In the case of effector cell survival, sustained activation of CAR T cells via the CAR receptor leads to maturation into Teff cells, which highly express markers PD-1, TIGIT, TIM-3 and other markers defined as markers of activation, resulting in exhaustion of immune cells. Exhausted cells cannot undergo clonal expansion and lose effector function. Chronic activation of T cells leads to their impaired function, and the same is true for CAR T cells. One of the major obstacles in cancer immunotherapy that mobilizes T cells is chronic activation, which can lead to acute overactivation and activation-induced cell death; as well as chronic sustained activation (without any re-recovery period) that causes functional impairment of T cells and CAR T cells. By providing the cells with a resting or recovery period, the level of T cell function or CAR T cell function required for long-term effector function of T cells and CAR T cells can be restored.

[0263]

[0295] In contrast to so-called "liquid cancers", the clearance of solid tumor tumor cells by CAR T cells requires a longer time interval. In B-cell lineage malignancies, such as B-cell precursor ALL treated with Kymriah (anti-CD19 CAR T cell therapy), tumor clearance is observed after 2 weeks in most patients, and complete responses (full MRD response) are found after 4 weeks at the latest. This means that all tumor cells are eliminated and the CAR T cells have the opportunity to restore their function. In lymphomas, the time to complete remission can take several months, making tumor clearance more difficult, since the T cells already show signs of dysfunction. The time of activation is unphysiologically prolonged. In solid tumors, tumor clearance takes even longer (more than 6 months) and, in combination with the tumor microenvironment, CAR T cells show functional impairments, driven in part by chronic persistent activation. Their survival is promoted by providing the cells with cyclic activation phases (when the CAR is functional) and recovery phases (when the CAR does not recognize the cancer cells).

[0264]

[0296] Thus, the method of the first aspect of the present invention may include a cycle of treatment with cellular immunotherapeutic agents (e.g., CAR T cells) for several weeks before administering a molecule to attenuate the activity of the cells. The cycle may be an activation period of about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks before administering a molecule that promotes reversible switching off or attenuation of activity. The period during which activity is attenuated or switched off may be about 1 week, about 2 weeks, or longer. In certain embodiments, according to the first aspect of the present invention, the period of CAR T activity is about 2 weeks, followed by about 2 weeks of reversible inhibition of CAR T activity.

[0265]

[0297] According to the second aspect of the present invention, in certain circumstances, it may be necessary to quickly remove and permanently switch off the activity of cellular immunotherapeutic agents.Again, the timing of administration of molecules can be easily determined by clinicians and can be based on the indication of potentially harmful immune response induced by immunotherapy, so that the activity of cellular immunotherapeutic agents needs to be permanently switched off for the benefit of the subject undergoing treatment. EXAMPLES

[0266]

[0298] Example 1: Anti-nfP2X 7 Blockade of CAR T cell killing

[0299] T cells were transduced with a lentiviral vector encoding an anti-nfP2X7 CAR. The CAR used contains an antigen-binding domain for binding the E200 epitope of the receptor (described elsewhere herein).

[0267]

[0300] The CAR T cell product showed 41% CAR expression. The effector to target ratio was 5:1 (T cells vs. MOLM-13), corresponding to an ET ratio of 2:1 (nfP2X7-CAR T vs. MOLM-13).

[0268]

[0301] Cells derived from the MOLM-13 cell line constitutively expressing firefly luciferase and an eGFP reporter (MOLM-13_LUC_eGFP) were seeded with 25,000 cells per well + CAR T cells of the indicated ET.

[0269]

[0302] Four different peptide variants were used to reduce the killing of MOLM-13 cancer cells by nfP2X7CAR T cells (Figure 1A). All four peptides contain the sequence of the E200 epitope of nfP2X7, which is also the epitope bound to the nfP2X7CAR used in this study. The peptides contain various linker regions at the N- and C-terminal regions. The sequences of the peptides are provided in Table 1.

[0270]

[0303] As shown in Figure 1A, all four peptides were able to reduce the engagement of direct antigen-binding CAR T cells and decrease the number of cancer cells as indicated by the percentage of MOLM-13 cells that were still viable after 24 hours, indicating significant blockade of direct CAR function using peptides that contain the sequence of the epitope recognized by the CAR antigen-binding domain.

[0271]

[0304] Similar experiments were performed using an "indirect" CAR T system. Briefly, MOLM-13 cells were co-cultured with anti-nfP2X7 CAR T cells together with a polypeptide that contains a) an epitope bound by the CAR T cells, and b) an antigen-binding domain for binding to CD33. In this way, the polypeptide allows the binding of anti-nfP2X7 CAR T cells to MOLM-13 cells via a different antigen (that is CD33). The further addition of peptide variants, as used in the first experiment, also significantly reduced the killing of MOLM-13 cells compared to when no peptide was added (Figure 1B).

[0272]

[0305] The kinetics of reduced killing of MOLM-13 cancer cells by nfP2X7CAR T cells are shown in Figure 2. After 24 hours, the peptide continues to block cell killing by CAR T cells, whereas in the absence of peptide, CAR T cells continue to reduce MOLM-13 cell viability.

[0273]

[0306] Example 2: Anti-nfP2X 7 Blockade of CAR T cell killing by peptides and nfP2X 7 CAR interaction

[0307] Figure 3 shows that the reduction in cell killing by T cells is specifically due to the interaction of the peptide with nfP2X7CAR. More specifically, T cells (unmodified or expressing nfP2X7CAR) were cultured with JeKo-1 cells at an ET ratio of 5 to 1. Blockade was performed using peptide variant 2 at 5ug / mL.

[0274]

[0308] On day 5 of co-culture, cell counts were performed to assess total JeKo-1 cell numbers. The total number of JeKo-1 cells added in the experiment was 20,000 on days 1 and 3 of the experiment (thus the total amount of cells added was 40,000 JeKo-1 per condition). In the first group of experiments, i.e., when T cells not expressing nfP2X7CAR were used, JeKo-1 cells expanded to a total of 60,000 cells after 5 days in co-culture. This result was not altered by the addition of blocking peptides.

[0275]

[0309] In contrast, in the second group of experiments (i.e., using T cells expressing the nfP2X7CAR), no JeKo-1 cells were observed on day 5 of co-culture when no blocking peptide was added. Addition of 5ug / mL of blocking peptide variant 2 reversed this, such that after 5 days, 40,000 JeKo-1 cells were present in culture. These results indicate that cancer cell killing by CAR T cells can be reduced (i.e., attenuated) by the use of a peptide that contains an epitope recognized by the CAR expressed by the T cells.

[0276]

[0310] Figure 4 also shows that the blockade of CAR T function by peptides can be controlled by decreasing the amount of peptide used, and by using a larger amount of peptide, a greater blockade of cell death can be achieved. In other words, the effect is dose-dependent.

[0277]

[0311] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings, all of which different combinations constitute various alternative embodiments of the invention.

Claims

1. A molecule for binding to a cellular immunotherapeutic agent that binds to a target antigen, the molecule comprising or consisting of an epitope of the target antigen, for use in inhibiting the activity of the cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent, A molecule whose epitope competes with an epitope on the target antigen, thereby disrupting the interaction between the cellular immunotherapeutic agent and the target antigen.

2. The molecule of claim 1, wherein the inhibition is reversible, such that once the molecule is removed from the subject's circulation, activity of the cellular immunotherapeutic agent is restored.

3. The molecule described in claim 2, wherein inhibition does not reduce cell viability of a cellular immunotherapeutic agent.

4. A molecule for binding to a cellular immunotherapeutic agent that binds to a target antigen, the molecule comprising or consisting of an epitope of the target antigen, for use in minimizing or reducing the risk of an abnormal inflammatory response in a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent, A molecule whose epitope competes with an epitope on the target antigen, thereby disrupting the interaction between the cellular immunotherapeutic agent and the target antigen.

5. A molecule for binding to a cellular immunotherapeutic agent that binds to a target antigen, the molecule comprising or consisting of an epitope of the target antigen, for use in treating an abnormal inflammatory response in a subject that has undergone or is undergoing treatment with a cellular immunotherapeutic agent, A molecule whose epitope competes with an epitope on the target antigen, thereby disrupting the interaction between the cellular immunotherapeutic agent and the target antigen.

6. The molecule of claim 4 or 5, wherein the abnormal inflammatory response comprises cytokine-associated toxicity.

7. The molecule of claim 6, wherein the abnormal inflammatory response comprises "cytokine release syndrome" (CRS) or hypercytokinemia.

8. A molecule for binding to a cellular immunotherapeutic agent that binds to a target antigen, the molecule comprising or consisting of an epitope of the target antigen, for use in increasing the persistence of the cellular immunotherapeutic agent in a subject that has undergone or is undergoing treatment with the cellular immunotherapeutic agent, A molecule whose epitope competes with an epitope on the target antigen, thereby disrupting the interaction between the cellular immunotherapeutic agent and the target antigen.

9. The molecule of any one of claims 1, 4, 5 and 8, wherein the target antigen is expressed or present on the surface of a cancer cell.

10. A molecule described in any one of claims 1, 4, 5 and 8, comprising a fusion protein comprising an epitope and further sequences to promote improved solubility and stability of the polypeptide.

11. A molecule described in any one of claims 1, 4, 5 and 8, comprising a fusion protein comprising an epitope and a further sequence to promote improved solubility and stability of the polypeptide, wherein the further sequence comprises an Fc region of an antibody, and wherein the Fc region of the antibody does not induce ADCC or CDC.

12. A molecule described in any one of claims 1, 4, 5 and 8, further comprising a compound for inducing cell death.

13. A molecule described in any one of claims 1, 4, 5 and 8, further comprising a compound for inducing cell death, which compound reduces the viability of cells of a cellular immunotherapeutic agent, preferably targeting the cells for degradation / death.

14. The molecule of any one of claims 1, 4, 5 and 8 in the form of a fusion protein comprising an epitope and an additional sequence to facilitate targeting of the cellular immunotherapeutic agent for cell-mediated killing by a cytotoxic immune cell (e.g., a cytotoxic T lymphocyte or NK cell) when the cellular immunotherapeutic agent is bound to the polypeptide.

15. A molecule according to any one of claims 1, 4, 5 and 8, in the form of a fusion protein comprising an epitope and a further sequence to facilitate targeting of the cellular immunotherapeutic agent for cell-mediated killing by a cytotoxic immune cell (e.g., a cytotoxic T lymphocyte or NK cell) when the cellular immunotherapeutic agent is bound to the polypeptide, the further sequence comprising an Fc region of an antibody, preferably the Fc region exhibits effector function and binds to an Fcγ receptor (FCγR) which induces ADCC or CDC.

16. The molecule of any one of claims 1, 4, 5 and 8, wherein the cellular immunotherapeutic agent comprises an immune cell or a precursor thereof, the immune cell expressing a receptor comprising an antigen recognition domain and a signal transduction domain.

17. A molecule described in any one of claims 1, 4, 5 and 8, wherein the cellular immunotherapeutic agent comprises an immune cell or a precursor thereof, the immune cell expressing a receptor comprising an antigen recognition domain and a signaling domain, and the antigen recognition domain binds to a tumor-specific or tumor-associated antigen expressed on the cell surface.

18. A cellular immunotherapeutic agent comprising an immune cell or a precursor thereof, the immune cell expressing a receptor comprising an antigen recognition domain and a signal transduction domain, the antigen recognition domain binding to a tumor-specific or tumor-associated antigen expressed on the cell surface, the antigen recognition domain binding to any one of CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 ( ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LICAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and in particular EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSA, CD276, TROP-2, DLL1, PSMA, and dysfunctional (nf)P2X. ７ 9. The molecule according to any one of claims 1, 4, 5 and 8, which binds to a tumor-associated antigen selected from the group consisting of receptors.

19. Dysfunctional P2X antigen recognition domain of cellular immunotherapy agent ７ 10. A molecule according to any one of claims 1, 4, 5 and 8, which binds to a receptor.

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