Enrichment of engineered immune cells

Abstract

The present invention relates to fusion proteins and their use for obtaining and isolating enriched populations of immune cells. Exemplary fusion proteins include those comprising a dysfunctional purinergic P2X7 receptor (P2X7R) epitope portion and an antibody Fc region.

Description

[Technical Field]

[0001] The present invention relates to methods for enriching populations of immune cells, and compositions and molecules for carrying out the same.

[0002] Related Applications This application claims priority to Australian Provisional Patent Applications Nos. 2022902655 and 2023901949, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Cell therapy for treating cancer and other disease states is a rapidly growing field. The development of immune effector cells, such as T cells, that express chimeric antigen receptors (CARs) has revolutionized adoptive cell therapy.

[0004] The potential of this approach has been demonstrated in clinical trials in which CAR T cells were infused into adult and pediatric patients with B-cell malignancies, neuroblastoma, and sarcoma. To date, over 500 clinical trials designed to test the efficacy of CAR T cells targeted to bind to 64 different tumor-associated antigens have been enacted worldwide. Of these, three CD19-specific CAR T cell products have been approved for the treatment of acute lymphoblastic leukemia (ALL), large B-cell lymphoma, and mantle cell lymphoma. To date, most of the success of CAR T therapy has been observed in the context of so-called "liquid" tumors or when the CAR is directed against CD19, CD22, or B-cell maturation antigen (BCMA).

[0005] Human-derived T lymphocytes engineered to express CARs, expanded in in vitro culture and then infused into patients, exhibit robust cytotoxicity following tumor antigen recognition and subsequent activation. Various factors in the production and administration of these cells contribute to the in vivo persistence and sustained antitumor efficacy of CAR T cells.

[0006] To prepare CAR T cells for infusion into patients, it is first necessary to genetically modify a population of T cells to express the relevant CAR. This results in a mixed population of T cells, some of which express the CAR and some of which do not. To maximize the efficacy of CAR T therapy, it is desirable to obtain a population of T cells enriched for cells that express the CAR.

[0007] A key problem with CAR-engineered T cell therapy (and cell therapy in general) is the low transduction efficiency of T cells with CAR-expressing constructs. For example, peripheral blood T cells, which are often the target of CAR gene therapy, typically have a transduction efficiency of less than 50%, often 10-20%. As a result, producing sufficient numbers of cells for therapy requires an increase in the scale of patient cell collection and more extensive ex vivo T cell selection and expansion. This contributes to the high manufacturing costs of CAR T cell therapy.

[0008] Therefore, there is a need for improved and alternative approaches to preparing populations of immune cells prior to cell therapy.

[0009] 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 could reasonably be expected to be understood, considered relevant, and / or combined with other pieces of prior art by a person skilled in the art. Summary of the Invention

[0010] The present invention finds particular use in preparing a population of genetically modified immune cells that are engineered to bind to dysfunctional P2X7 receptors on cancer cells. Thus, in a first aspect, the present invention provides a fusion protein comprising: (i) a dysfunctional P2X7 receptor epitope portion; (ii) a fusion protein comprising an Fc region of an antibody.

[0011] The dysfunctional P2X7 receptor epitope portion may comprise a peptide comprising any amino acid sequence derived from the dysfunctional P2X7 receptor, but preferably comprises an epitope sequence that is found in the dysfunctional P2X7 receptor but not in the functional P2X7 receptor.

[0012] In a preferred embodiment, the amino acid sequence of the dysfunctional P2X7 receptor epitope portion comprises or at least consists of the amino acid sequence set forth in SEQ ID NO: 14. In a particularly preferred embodiment, the portion comprises at least the sequence set forth in SEQ ID NO: 7 or 9.

[0013] In any embodiment, the dysfunctional P2X7 receptor epitope portion comprises an amino acid sequence set forth in any of SEQ ID NOs: 7-69 or 122, or a sequence at least 80%, at least 81%, at least 82%, 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 thereto, provided that the sequence comprises at least the sequence set forth in SEQ ID NO: 14 or 7 or 9.

[0014] The present invention provides (i) a peptide; (ii) an Fc region of an antibody; Fusion proteins are provided in which the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 (preferably the amino acid sequence of SEQ ID NO: 14). Optionally, the peptide comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 7-69 or 122, or a sequence at least 80%, at least 81%, at least 82%, 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 thereto, provided that the sequence comprises at least the sequence set forth in SEQ ID NO: 14, 7, or 9.

[0015] In some embodiments, the Fc region of the antibody is an IgG, IgA, IgD, IgE, or IgM Fc region. Preferably, the Fc region is from an IgG antibody, such as an IgG1, IgG2, IgG2b, IgG3, or IgG4 antibody.

[0016] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chains.

[0017] In a preferred embodiment, the Fc region of the fusion protein comprises one or more amino acid substitutions, relative to a naturally occurring Fc sequence, that prevent or reduce the ability of the Fc region to homodimerize. Preferably, the amino acid substitutions comprise one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules. The cysteine ​​residues in the Fc region may be substituted with any other amino acid residue, optionally with glycine, serine, alanine, lysine, and glutamic acid, preferably with glycine or serine.

[0018] The cysteine ​​residues for substitution are preferably one or more of the cysteine ​​residues located in the region of the Fc region corresponding to the hinge region of an immunoglobulin. Examples of IgG1 hinge regions and variations thereof, including cysteine-to-serine substitutions, are provided in Table 3 herein. The hinge region of an immunoglobulin (e.g., IgG1) contains three cysteine ​​residues (numbered C220, C226, and C229 according to EU numbering). Thus, in any embodiment, at least one, at least two, or all three of the cysteine ​​residues in the immunoglobulin hinge region are substituted. Preferably, at least two or all three of the cysteine ​​residues are substituted. More preferably, all cysteine ​​residues in the Fc region, such as the hinge region, are substituted. In a particularly preferred embodiment, at least one of C226 and C229 is substituted, and preferably both C226 and C229 are substituted.

[0019] Therefore, in a preferred embodiment, the fusion protein comprises a hinge region for linking a peptide described herein (e.g., a dysfunctional P2X7 receptor epitope portion) and an Fc region of an antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences set forth in SEQ ID NOs: 76-113, or 136-137, or 141, or 142.

[0020] Furthermore, the Fc region preferably contains one or more amino acid substitutions to reduce affinity for an Fc receptor (FcR, including any of FcγRI, FcγRII, and FcγRIII), thereby reducing the ability of the fusion protein to elicit antibody-dependent cell-mediated toxicity (ADCC). Such substitutions are well known in the art and include, but are not limited to, "DANA" and "LALA" amino acid substitutions and variations thereof, as further defined herein. In a further embodiment, the Fc region may also contain substitutions that inhibit recruitment of complement C1q. Such mutations are also well known in the art and are further described herein. Furthermore, the Fc region may contain substitutions to reduce serum half-life (via attenuating or reducing the ability to bind to FcRN receptors). Relevant amino acid substitutions to alter effector function, serum half-life, and aggregation are well known to those of skill in the art and are further described herein, as exemplified in Table 1.

[0021] The present invention also provides heterodimeric asymmetric molecules comprising a fusion protein described herein (preferably comprising a peptide of SEQ ID NO: 7 or 14, or a variant thereof exemplified by any of SEQ ID NOs: 2 to 69) and an antibody Fc region, and further comprising an antibody Fc region that does not contain the peptide. Such asymmetric heterodimeric molecules can be obtained using knob-in-hole technology, as further described herein, to promote dimerization of non-identical Fc regions.

[0022] The present invention provides heterodimeric asymmetric molecules or monomeric fusion proteins for use in accordance with any of the methods further described herein, comprising an exogenous cell surface receptor for binding to a tumor-specific or tumor-associated antigen and comprising an intracellular signaling domain (e.g., a chimeric antigen receptor comprising expression on T cells), with application in methods for enriching immune cells. It will be understood that such molecules or fusion proteins comprise a single amino acid sequence capable of being bound by an exogenous immune cell surface receptor, so as to minimize the possibility that the molecule / fusion protein will cross-link multiple immune cells, thereby causing their undesired activation.

[0023] More specifically, a monomeric fusion protein is provided that comprises: i) an amino acid sequence capable of being recognized by or bound to an antigen recognition domain of an exogenous cell surface receptor, including an intracellular signaling domain (such as a chimeric antigen receptor); and ii) an Fc region of an antibody. Preferably, the amino acid sequence of the Fc region of the antibody is incapable of forming a homodimer with another Fc region of an antibody.

[0024] Further provided is a heterodimeric asymmetric molecule comprising: i) an amino acid sequence capable of being recognized by or bound by an antigen recognition domain of an exogenous cell surface receptor comprising an intracellular signaling domain (e.g., a chimeric antigen receptor, including one expressed on T cells), wherein the amino acid sequence is linked to a first Fc region of an antibody; and ii) a second Fc region of an antibody capable of forming a heterodimer with the first Fc region.

[0025] In preferred embodiments, the fusion protein has an affinity for an FcR that is less than about 250 nM, preferably less than 500 nM, less than 1000 nM, and most preferably less than 2000 nM.

[0026] In any embodiment, the fusion protein may comprise the amino acid sequence of a fusion protein set forth in any of SEQ ID NOs: 145-158, 160, and 161.

[0027] Preferably, the fusion protein or heterodimeric asymmetric molecule consists of or essentially consists of the peptide and the Fc region of an antibody, such that the fusion protein or heterodimeric asymmetric molecule does not contain the antigen-binding domain of an antibody (i.e., the fusion protein does not contain a VH, VL, Fab, Fv, or scFv derived from an antibody).

[0028] In a further embodiment, the fusion protein of the present invention may comprise one or more modifications to allow capture of the fusion protein, including when the protein binds to an immune cell expressing a receptor comprising an antigen binding domain for binding a dysfunctional P2X7 receptor (such as a chimeric antigen receptor (CAR) or modified TCR).

[0029] It will be understood that typically, the antigen-binding domain of the receptor will be capable of binding or recognizing the same epitope of the dysfunctional P2X7 receptor contained in the fusion protein. Furthermore, it is within the skill of the art, with knowledge of the specific epitope and binding target of a given anti-dysfunctional P2X7 receptor CAR, to be able to design a suitable fusion protein according to the present invention for use in binding to a CAR.

[0030] The one or more modifications to the fusion protein may be selected from a biotin moiety, fluorescein (FITC), a peptide tag (such as His, Myc, Flag, and related tags), and a magnetic label. Preferably, the modification is a biotin moiety or a magnetic moiety.

[0031] The magnetic moiety can be any commercially available magnetic moiety for use in isolating or capturing proteins or cells. For example, the magnetic moiety can include iron oxide microbeads (up to 50 nm diameter) or macrobeads (1-5 μm diameter). Preferably, the magnetic moiety includes microbeads.

[0032] In the case of biotin or a magnetic moiety, the moiety may be conjugated to the fusion protein using any method known to one of skill in the art. In one example, the moiety is conjugated to the fusion protein via one or more lysine residues of the protein and / or at the amino terminus of the protein.

[0033] In the case of peptide tag, the nucleic acid sequence encoding the tag can be included in the nucleic acid construct that encodes fusion protein, so that when expressed, fusion protein is expressed with the tag already linked to protein.Typically, tag is located at either N-terminus or C-terminus of fusion protein.Preferably, tag is located on fusion protein so as not to interfere with the binding of peptide by the receptor on immune cell (for example, dysfunctional P2X7 receptor epitope portion), which comprises antigen binding domain for binding peptide.

[0034] In a second aspect, the present invention also provides a method for obtaining a population of immune cells enriched for cells expressing an exogenous cell surface receptor comprising an antigen-binding domain and an intracellular signaling domain. Preferably, the receptor expressed by the immune cells is a chimeric antigen receptor (CAR) or, optionally, a modified T cell receptor (TCR). Optionally, the exogenous cell surface receptor comprises an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on a cancer cell, although it should be understood that the exogenous cell surface receptor may be a receptor for use in a "universal CAR" system.

[0035] In a preferred embodiment of the second aspect, there is provided a method for obtaining a population of immune cells or enriching a population of cells that express a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on a cancer cell, the method comprising: (i) providing a population of immune cells, preferably immune effector cells, which have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on cancer cells; (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises an epitope recognized by an antigen-binding domain of the receptor, and the polypeptide comprises a moiety to allow capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; Thereby, a method is provided for obtaining a population of immune cells that express a receptor having an antigen-binding domain for binding to a tumor-associated antigen or tumor-specific antigen on cancer cells.

[0036] Further provided is a method for obtaining a population of immune cells or enriching a population of cells that expresses a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on a cancer cell, comprising: (i) providing a mixed population of immune cells, preferably immune effector cells, wherein a subpopulation of the cells expresses a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on cancer cells; (ii) contacting the mixed population of cells with a polypeptide, wherein the polypeptide comprises an epitope recognized by an antigen-binding domain of the receptor, and the polypeptide comprises a moiety to allow capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; Thereby, a method is provided for obtaining a population of immune cells that express a receptor having an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on cancer cells.

[0037] Preferably, the cells are immune cells that express a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on cancer cells. Thus, there is provided a method for obtaining a population of immune cells that express a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on cancer cells, comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the population comprises cells that have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on cancer cells, or the cells express a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on cancer cells; (ii) contacting the cell population with a polypeptide, wherein the polypeptide comprises an epitope recognized by the CAR, and the polypeptide comprises a moiety to enable capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population; Also provided are methods whereby a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on cancer cells is obtained.

[0038] According to a further second aspect, there is provided a method for enriching a population of immune cells expressing a chimeric antigen receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on a cancer cell, the method comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on a cancer cell; (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises an epitope recognized by the CAR, and the polypeptide comprises a moiety to enable capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex of cells from the mixed population; Thereby, methods are provided to enrich a population of immune cells that express chimeric antigen receptors (CARs) for binding to tumor-associated or tumor-specific antigens on cancer cells.

[0039] Examples of tumor-associated or tumor-specific antigens that are typically targeted by cellular immunotherapeutics such as CARs 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.

[0040] It is well within the skill of one in the art to generate a polypeptide (or, in some cases, a nucleic acid encoding a polypeptide) comprising an epitope recognized by a CAR (or other receptor for binding to a tumor antigen). For example, with knowledge of the amino acid sequence recognized or bound by a given CAR, one skilled in the art can design a fusion protein (preferably a monomeric Fc fusion protein or an asymmetric heterodimeric Fc fusion protein described herein) for binding to the CAR and for use in the present methods. For example, in the context of a CAR for binding to CD19, the polypeptide would be recognized by the receptor and would comprise a similar epitope of the CD19 molecule fused to the Fc region of an antibody, preferably an Fc region defined herein in either SEQ ID NO: 159 or 162, or an Fc region incapable of forming homodimers.

[0041] In a preferred embodiment of the second aspect, the present invention also provides the use of the fusion protein according to the first aspect or the polypeptide as further described herein for obtaining a population of immune cells enriched for cells expressing a receptor comprising an antigen-binding domain for binding to dysfunctional P2X7 receptor.Preferably, the receptor expressed by the immune cells is a chimeric antigen receptor (CAR), or optionally a modified T cell receptor (TCR).

[0042] In a preferred embodiment of the second aspect, there is provided a method for obtaining a population of immune cells or enriching a population of cells that express a receptor comprising an antigen binding domain for binding to a dysfunctional P2X7 receptor, the method comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain for binding to a dysfunctional P2X7 receptor; (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises an epitope of the dysfunctional P2X7 receptor that is recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a moiety that allows capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; Thereby, a method is provided for obtaining a population of immune cells that express a receptor having an antigen binding domain for binding to a dysfunctional P2X7 receptor.

[0043] Further provided is a method for obtaining a population of immune cells or enriching a population of cells that expresses a receptor comprising an antigen binding domain for binding to a dysfunctional P2X7 receptor, the method comprising: (i) providing a mixed population of immune cells, preferably immune effector cells, wherein a subpopulation of the cells expresses a receptor comprising an antigen-binding domain for binding to the dysfunctional P2X7 receptor; (ii) contacting the mixed population of cells with a polypeptide, wherein the polypeptide comprises an epitope of the dysfunctional P2X7 receptor that is recognized by the antigen-binding domain of the receptor, and the polypeptide comprises a moiety to enable capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; Thereby, a method is provided for obtaining a population of immune cells that express a receptor having an antigen binding domain for binding to a dysfunctional P2X7 receptor.

[0044] Preferably, the cell is an immune cell expressing a chimeric antigen receptor (CAR) for binding to a linear epitope of a P2X7 receptor (such as a dysfunctional P2X7 receptor), wherein the epitope comprises or consists of the amino acids of SEQ ID NO: 7 (preferably the amino acids of SEQ ID NO: 14), or comprises or consists of the amino acids of any of SEQ ID NOs: 7-69 or 122, or a sequence at least 80%, at least 81%, at least 82%, 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 99% identical thereto, with the proviso that the sequence comprises at least the sequence set forth in SEQ ID NO: 14 or 7 or 9.

[0045] Thus, there is provided a method for obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a linear epitope of a P2X7 receptor (such as an epitope of a dysfunctional P2X7 receptor), comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the population comprises cells transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a linear epitope of a P2X7 receptor (such as an epitope of a dysfunctional P2X7 receptor), or wherein the cells express a chimeric antigen receptor (CAR) for binding to a linear epitope of a P2X7 receptor (such as an epitope of a dysfunctional P2X7 receptor); (ii) contacting the cell population with a polypeptide, wherein the polypeptide comprises an epitope of a linear epitope of a P2X7 receptor (such as an epitope of a dysfunctional P2X7 receptor) recognized by the CAR, and wherein the polypeptide comprises a moiety to enable capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population; Also provided is a method whereby a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a linear epitope of a P2X7 receptor (such as an epitope of a dysfunctional P2X7 receptor) is obtained.

[0046] According to a second aspect, there is provided a method for enriching a population of immune cells expressing a chimeric antigen receptor comprising an antigen binding domain for binding to a dysfunctional P2X7 receptor, comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor; (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor that is recognized by the CAR, and the polypeptide comprises a moiety to enable capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating from the mixed population a complex of cells expressing a chimeric antigen receptor (CAR) for binding to the dysfunctional P2X7 receptor bound to the fusion protein; Thereby, methods are provided to enrich a population of immune cells that express a chimeric antigen receptor (CAR) for binding to dysfunctional P2X7 receptors.

[0047] The present invention also finds use in methods for enriching immune cell populations for use in universal CAR systems. Accordingly, in a further embodiment of the second aspect, there is provided a method for obtaining a population of immune cells or enriching a population of cells that express an exogenous cell surface receptor comprising an antigen binding domain for binding to a peptide, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122); (i) providing a population of immune cells, preferably immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain for binding to a peptide; (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises a peptide recognized by the antigen-binding domain of the receptor and a moiety to enable capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; Thereby, a method is provided for enriching a population of immune cells expressing a receptor having an antigen-binding domain for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122).

[0048] Further provided is a method for obtaining a population of immune cells or enriching a population of cells that express an exogenous cell surface receptor comprising an antigen-binding domain for binding to a peptide, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 14 (or, optionally, the amino acid sequence of any of SEQ ID NOs: 2 to 69 and 122), comprising: (i) providing a mixed population of immune cells, preferably immune effector cells, wherein a subpopulation of the cells expresses a receptor comprising an antigen-binding domain for binding to a peptide; (ii) contacting the mixed population of cells with a polypeptide, wherein the polypeptide comprises a peptide recognized by an antigen-binding domain of a receptor, and the polypeptide comprises a moiety to allow capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; Thereby, a method is provided for obtaining a population of immune cells expressing a receptor having an antigen-binding domain for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2 to 69 and 122).

[0049] Preferably, the cells are immune cells that express a chimeric antigen receptor (CAR) for binding to the peptide. Thus, there is provided a method for obtaining a population of immune cells that express a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or, optionally, any of the amino acid sequences of SEQ ID NOs: 2 to 69 and 122), comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the population comprises cells that have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122), or the cells express a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122); (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises a peptide, and the polypeptide comprises a moiety to allow capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population; Also provided is a method, thereby obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122).

[0050] Further, according to a second aspect, there is provided a method for enriching a population of immune cells expressing a chimeric antigen receptor comprising an antigen-binding domain for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2 to 69 and 122), comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein the cells are transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or, optionally, the amino acid sequence of any of SEQ ID NOs: 2-69 and 122); (ii) contacting the cell population with a polypeptide, wherein the polypeptide comprises a peptide, and the polypeptide comprises a moiety to allow capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating from the mixed population a complex of cells expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122) bound to a polypeptide; Thereby, methods are provided for enriching a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122).

[0051] In any embodiment of the second aspect of the invention, the polypeptide comprises the amino acid sequence of the fusion protein of any embodiment of the first aspect of the invention (such that the polypeptide comprises a peptide as described herein, e.g., an epitope of a dysfunctional P2X7 receptor linked to an Fc region of an antibody).

[0052] In any embodiment of the second aspect of the present invention, the polypeptide comprises a first portion comprising a peptide as described herein, such as an epitope of a dysfunctional P2X7 receptor, linked to an additional amino acid sequence to promote solubility and stability of the first portion. The additional amino acid sequence (e.g., dysfunctional P2X7 receptor epitope) linked to the peptide may comprise any suitable linker or hinge region, such as those exemplified in Table 1 and Table 3. Such a linker or hinge region may comprise an amino acid sequence consisting of glycine and serine repeats (the so-called "GS" linker sequence, and its variants as further defined herein). The hinge region may also comprise a sequence derived from an immunoglobulin hinge region, such as those defined in Table 3. Optionally, the linker sequence may comprise a cleavable sequence.

[0053] In a further embodiment, the polypeptide may be in the form of a fusion protein comprising the peptide described herein (such as an epitope of a dysfunctional P2X7 receptor) linked to a further amino acid sequence. The further sequence may comprise serum albumin, transferrin, the carboxy-terminal peptide of chorionic gonadotropin (CG) beta chain, an imprecise repeat peptide sequence, a polypeptide sequence composed of proline-alanine-serine polymers, elastin-like peptide (ELP) repeat sequences, a homopolymer of glycine residues, or a gelatin-like protein. The polypeptide may comprise a linker or hinge region, as described above, for linking the epitope of a dysfunctional P2X7 receptor to the further amino acid sequence.

[0054] Additionally, the polypeptide may be in the form of a conjugate, including carbohydrates, lipids, liposomes, peptides, and aptamers conjugated to amino acid sequences comprising peptides (eg, epitopes of dysfunctional P2X7 receptors).

[0055] In any embodiment of the second aspect of the invention, the fusion protein or polypeptide may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 145-158, or 160, and 161, or a sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0056] According to a second aspect of the invention, the moiety for enabling capture of the polypeptide may be a detectable moiety selected from a biotin label, fluorescein (FITC), a peptide tag (such as His, Myc, Flag, and related tags), and a magnetic label. Preferably, the modification is a biotin moiety or a magnetic moiety.

[0057] The magnetic moiety can be any commercially available magnetic moiety for use in isolating or capturing proteins or cells, for example, the magnetic moiety can be a magnetic microbead or a magnetic macrobead.

[0058] In the case of biotin or a magnetic moiety, the moiety may be conjugated to the polypeptide using any method known to one of skill in the art, hi one example, the moiety is conjugated to the polypeptide via one or more lysine residues of the protein and / or at the amino terminus of the polypeptide.

[0059] In the case of peptide tag, the nucleic acid sequence encoding tag can be included in the nucleic acid construct encoding polypeptide, so that when expressed, polypeptide is expressed with the tag already linked to protein.Typically, tag is located at either N-terminus or C-terminus of polypeptide.Preferably, tag is located on polypeptide so as not to interfere with the binding of dysfunctional P2X7 receptor epitope part by receptor on immune cell, which comprises antigen binding domain for binding peptide part.

[0060] Optionally, the polypeptide is labeled with a biotin moiety, and the method may further comprise contacting the cells (after step ii) with an anti-biotin antigen-binding protein, preferably the anti-biotin antigen-binding protein comprising one or more moieties to allow capture of the complex. Optionally, the one or more moieties to allow capture of the complex comprise iron oxide particles (microbeads or macrobeads).

[0061] Optionally, the polypeptide comprises a magnetic label, and the isolating step comprises: i) applying a magnetic field to the population of cells; ii) removing or discarding cells that are not attracted to the magnetic field; and iii) removing the magnetic field, thereby providing a population of immune cells that express a chimeric antigen receptor (CAR) for binding to the dysfunctional P2X7 receptor.

[0062] Optionally, the method may further comprise expanding the isolated immune cells.

[0063] In any embodiment of the second aspect of the present invention, the method may further include treating the complex to release the cells from binding by the polypeptide. Such methods are known to those skilled in the art and depend on the nature of the polypeptide moiety to enable capture. For example, in the case of a biotinylated polypeptide, providing excess free biotin to the complex promotes dissociation of the bound polypeptide by a capture agent (e.g., anti-biotin beads, etc.).

[0064] In any aspect of the present invention, the fusion protein or polypeptide may comprise a cleavable linker that connects the epitope bound by the receptor (e.g., the epitope of a dysfunctional P2X7 receptor) with the Fc region and / or additional sequence, so as to enable capture of the fusion protein or polypeptide.Cleavable linkers are well known in the art and are further described herein.In any embodiment of the second aspect of the present invention, the method may further comprise treating the polypeptide with a protease or other agent to cleave the cleavable linker, thereby releasing the Fc region or additional amino acid sequence therefrom.

[0065] Optionally, the method further includes administering the isolated or enriched cells to a subject in need of immune cell therapy, such as for cancer. In certain embodiments, depending on the capture agent used, the immune cell / polypeptide complex may be administered directly to the subject.

[0066] In any embodiment of the second aspect, the population of immune cells is a population of effector immune cells, such as T cells, NK cells, or NKT cells. Optionally, the T cells are derived from stem cells, and optionally the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0067] Preferably, the population of immune cells is derived from a subject in need of treatment for cancer. Alternatively, the immune cells may be obtained from an allogeneic donor not in need of treatment.

[0068] In particularly preferred embodiments of the second aspect of the invention, the exogenous cell surface receptor comprising an antigen-binding domain (e.g., a chimeric antigen receptor CAR) comprises an antigen-binding domain comprising the CDR amino acid sequence of PEP2-2-1 as described in PCT / AU2010 / 001070 (WO2011020155, or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508). More preferably, the antigen-binding domain of the receptor (e.g., CAR) comprises or consists of the amino acid sequence of the PEP2-2-1 antigen-binding protein as described in PCT / AU2010 / 001070 (WO2011020155, or the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), which are incorporated herein by reference.

[0069] The present invention also provides a composition comprising a population of immune cells that expresses chimeric antigen receptor (CAR) for binding to dysfunctional P2X7 receptor, wherein the population of cells is obtained by the method described herein.Preferably, the composition comprises more than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells that express chimeric antigen receptor (CAR) for binding to tumor-associated antigen or tumor-specific antigen (such as dysfunctional P2X7 receptor).

[0070] In a further embodiment, there is provided a kit for use in the methods described herein, comprising: a fusion protein that can be bound by a CAR to bind to a tumor-associated or tumor-specific antigen (such as a dysfunctional P2X7 receptor); - optionally one or more reagents to allow isolation of the fusion protein and its complexes.

[0071] Optionally, the kit comprises written instructions for use in the method of the second aspect of the invention.

[0072] As used herein, unless the context requires otherwise, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, ingredients, elements, or steps.

[0073] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and referring to the accompanying drawings, in which:

[0074] Sequence information [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Brief explanation of the drawings]

[0075] [Figure 1] Enrichment of Jurkat cells transduced and stabilized with a nucleic acid encoding a CAR for binding dysfunctional P2X7 receptors. A. Shown is the percentage of transduced cells enriched using a biotinylated protein containing the amino acid sequence of the E200 epitope. B. 48 hours after enrichment, the cells retain a high level of purity, demonstrating that the cells are not adversely affected by the enrichment procedure. Gray shading indicates before enrichment, and red shading indicates after enrichment. [Figure 2] Enrichment of primary donor cells (donor 12) transduced with a nucleic acid encoding a CAR to bind dysfunctional P2X7 receptors. Gray shading indicates before enrichment, red shading indicates after enrichment. [Figure 3] Enrichment of primary donor cells (donor 12) transduced with a nucleic acid encoding an alternative CAR to bind dysfunctional P2X7 receptors. Gray shading indicates before enrichment, and red shading indicates after enrichment. Enrichment was performed using a MACS column (Miltenyi Bioscience). [Figure 4] Enrichment of primary donor cells (donor 12) transduced with a nucleic acid encoding an alternative CAR to bind dysfunctional P2X7 receptors. Gray shading indicates before enrichment, red shading indicates after enrichment. Enrichment was performed using a Magnet Stand, Stem Cell Technologies. [Figure 5] EGFR staining for direct staining of CAR-expressing cells after enrichment using either monomeric or dimeric Fc-attenuated fusion proteins containing the nfP2X7 receptor epitope portion for binding by CAR. A = dimeric fusion protein (SEQ ID NO: 149), B = monomeric fusion protein (SEQ ID NO: 145), C = monomeric fusion protein (SEQ ID NO: 146). [Figure 6] Viability and purity of CAR-expressing cells using a monomeric Fc fusion containing the nfP2X7 receptor epitope portion. A. Staining with the dye 7AAD to determine the viability of enriched cells. B. Indirect CAR detection using tEGFR staining (AF647 primary labeled anti-EGFR mAb cetuximab, measured in the APC channel). C. Direct staining of nfP2X7CAR using biotinylated DetR2 (SEQ ID NO: 146) followed by staining with the anti-biotin antibody Vioblue (130-113-857 biotin antibody, VioBlue®, Miltenyi Biotech. [Figure 7] Percentage of CD25+ / CD69+ and PD-L1+ cells at 24 hours (A), 48 hours (B), and 72 hours (C) after enrichment with monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NOs: 145 and 149, respectively). [Figure 8] Transduction efficiency (%) on day 2 after MACS sorting using monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NOs: 145 and 149, respectively). D50, D53, and D71 = T cells from donors 50, 53, and 71, respectively. [Figure 9] Cell numbers (normalized to maximum expected cell numbers) after enrichment using monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NOs: 145 and 149, respectively). Cell numbers on days 1 and 2 after enrichment are shown for T cells from healthy donors 50 (D50) and 71 (D71). [Figure 10] Viability of enriched CAR T cells 2 days after MACS sorting. Percentage of 7AAD-negative cells is shown. D50, D53, and D71 = T cells from donors 50, 53, and 71, respectively. [Figure 11] Percentage of CD25+ / CD69+ and PD-L1+ cells 2 days after MACS enrichment with monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NOs: 145 and 149, respectively). Results are shown for T cells from healthy donor 50 (D50). DETAILED DESCRIPTION OF THE INVENTION

[0076] Reference will now be made in detail to 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.

[0077] 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. The present invention is in no way limited to the methods and materials described.

[0078] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0079] All patents and publications referenced herein are incorporated by reference in their entirety.

[0080] The present invention provides fusion proteins, compositions and kits comprising them, as well as their use in methods for enriching populations of immune cells, preferably cells expressing an exogenous cell surface receptor comprising an antigen-binding domain and an intracellular signaling domain.

[0081] Preferably, the fusion protein comprises (i) a linear peptide epitope portion derived from the P2X7 receptor (e.g., comprising amino acids from SEQ ID NO: 7 or 14) and capable of being recognized or bound by the antigen-recognition domain of the receptor expressed on immune cells, and (ii) an Fc region of an antibody, and optionally, iii) a portion for enabling capture of the fusion protein. The epitope portion enables specific binding of the fusion protein to target immune cells for enrichment, and the Fc region (preferably when comprising a portion for enabling capture) enables capture of the complex of the fusion protein and immune cells.

[0082] The inventors have demonstrated the utility of both homodimeric and monomeric proteins derived from the fusion proteins described herein for use in obtaining or enriching populations of immune cells.

[0083] In a particularly preferred embodiment, the Fc fusion protein is designed to contain only a single copy of a linear epitope derived from the P2X7 receptor. This can be achieved by introducing amino acid substitutions into the Fc region to prevent homodimerization, as described further herein, or alternatively, by using well-known knob-into-hole technology to ensure the formation of an asymmetric heterodimeric molecule (e.g., comprising an E200 peptide-Fc fusion protein and an Fc region that does not contain the E200 peptide). Such monomeric or asymmetric heterodimeric molecules have the advantage of reducing activation of target immune cells and preventing unnecessary exhaustion of target immune cells during the enrichment process (as further described herein in the Examples). Without wishing to be bound by theory, the inventors believe this is due to a reduced ability of the molecule to bridge either two different CAR receptors on one cell or two different CAR receptors on two separate CAR-expressing cells.

[0084] definition Unless otherwise defined, 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.

[0085] For purposes of interpreting this specification, the following definitions shall generally apply and, whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0086] As used herein, the term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings, or for either meaning.

[0087] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "a dysfunctional P2X7 receptor epitope portion" means one dysfunctional P2X7 receptor epitope portion or two or more dysfunctional P2X7 receptor epitope portions.

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

[0089] As used herein, "tumor-associated antigen" refers to an antigen expressed by cancer cells (the term "tumor antigen" can also be used to refer to the same). Tumor antigens are proteins produced by tumor cells that provoke 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, α-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.

[0090] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous 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-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate 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 with limited success. The type of tumor antigen referred to in this invention may be a tumor-specific antigen (TSA). TSAs are unique to tumor cells and do not occur on other cells in the body. Tumor-associated antigens (TAA) are not inherent to tumor cells and are also 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. TAAs may be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells. Tumor-associated antigens of most clinical interest are differentially expressed compared to corresponding non-tumor tissue, allowing preferential recognition of tumor cells by specific T cells or immunoglobulins.

[0091] 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 embryonic 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, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-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 tumor antigens according to the present 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 (Nectin-2), CD117 (c-kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (LI 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, PCSA CD276 and dysfunctional (nf) P2X7 receptor.

[0092] "Purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0093] "P2X7 receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, at least one of which has the amino acid sequence substantially as set forth in SEQ ID NO: 1 of Table 1 herein.

[0094] 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 monomers are isoforms (e.g., forms consisting of extracellular domain sequence or truncated forms thereof), naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants, including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of the monomers that form P2X7 receptor. 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 may have a modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO: 1 may be substituted, deleted, or residues may be inserted.

[0095] "Functional P2X7 receptor" generally refers to the form of P2X7 receptor that has three intact binding sites or clefts for binding to ATP.When bound to ATP, functional receptor forms a non-selective sodium / calcium channel that converts into a pore-like structure that allows calcium ions and molecules up to 900 Da to enter the cell matrix, one of the consequences of which may be the induction of programmed cell death.In normal homeostasis, the expression of functional P2X7 receptor is generally limited to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes.In addition, there may be some expression of functional P2X7 receptor on erythrocytes and other cell types.

[0096] "Dysfunctional P2X7 receptor" (also called "non-functional" or (nf)P2X7) is a P2X7 receptor that has a defective response to ATP, such that it cannot form an apoptotic pore under physiological conditions. Dysfunctional P2X7 receptor or (nfP2X7 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 leads to 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, or 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, receptor cannot form pore, which limits the degree that calcium ions can enter into the cytosol.Dysfunctional P2X7 receptor is expressed on a wide range of epithelial cancers and hematopoietic cancers.As used herein, term " dysfunctional P2X7 receptor " can be used interchangeably with term " non-functional P2X7 receptor " or " nfP2X7 " receptor.

[0097] A "cancer-associated P2X7 receptor" is a P2X7 receptor that is generally found on cancer cells (including pre-neoplastic cells, neoplastic cells, malignant cells, benign cells, or metastatic cells) but not on non-cancerous or normal cells.

[0098] "E200 epitope" generally refers to the epitope having the sequence GHNYTTRNILPGLNITC (SEQ ID NO: 2), variants of which are exemplified in Table 1, including any of SEQ ID NOs: 3, or 7-69, and 122.

[0099] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO: 4) or a variant thereof, as defined in SEQ ID NO: 5.

[0100] "Composite epitope" generally refers to an epitope formed from the juxtaposition of the E200 epitope and the E300 epitope, or portions of these epitopes. An example of a composite epitope comprising the E200 epitope and the E300 epitope is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 6).

[0101] As used herein, the term "antigen" is intended to include a substance that binds to or induces the production of one or more antibodies, and may include, but is 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 high affinity binding to a structure.

[0102] "Epitope" generally refers to the portion of an antigen 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., an epitope in which the hypervariable loops of the CDRs bind to residues as they appear in the tertiary or quaternary protein structure.

[0103] With respect to a receptor that refers to an antigen-binding domain that recognizes and binds to a dysfunctional P2X7 receptor, the terms "binds to," "specifically binds to," or "specific for" are intended to mean that the receptor does not substantially recognize or bind to other antigens in a sample.

[0104] "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 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 a molecule X for its partner Y can generally be represented by the 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 more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present disclosure.

[0105] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system and performs a specific effector function, e.g., α-β 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, γ-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic progenitor cell, such as an early progenitor cell subset that can mature or differentiate into pluripotent stem cells and somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (iPSCs and other artificial cell types). Preferred immune cells are cells with cytotoxic effector function, e.g., α-β T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or γ-delta T cells. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0106] As used herein, the term "autologous" refers to any material originating from the same subject into which it is subsequently reintroduced.

[0107] As used herein, the term "allogeneic" refers to any material derived from a different subject of the same species as the subject into which the material is being reintroduced.

[0108] A population of "enriched" or "purified" cells is an increase in the proportion of particular cells relative to other cells, e.g., compared to cells found in a subject's body or compared to the proportion before exposure to a peptide, nucleic acid, or vector of the invention. In some embodiments, in an enriched or purified population of cells, the particular cells comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% of the total cell population. A population of cells may be defined by one or more cell surface markers and / or characteristics.

[0109] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. These terms refer to the presence and / or expression of exogenous genes or nucleic acid sequences that, in turn, modify the genotype or phenotype of the cells or their progeny. The 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 not naturally expressed in these cells. For example, immune cells can be 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 variants thereof, or any other gene delivery mechanism such as electroporation or lipofection with CRISPR / Cas9, transposons (e.g., Sleeping Beauty), or variants thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0110] The amino acid structures and single and three letter abbreviations used throughout this specification are defined in Table 2, which lists the 20 naturally occurring proteinogenic amino acids that occur in proteins as the L-isomer. Table 2 [ka] [Table 2]

[0111] As used herein, the term "non-proteinogenic amino acid" refers to an amino acid having a side chain that does not occur in the naturally occurring L-α-amino acids listed in Table 2. Examples of non-proteinogenic amino acids and derivatives include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of the natural amino acids.

[0112] As used herein, the term "α-amino acid" refers to an amino acid having a single carbon atom (α-carbon atom) separating its carboxyl terminus (C-terminus) and amino terminus (N-terminus). α-Amino acids include naturally occurring and non-naturally occurring L-amino acids, including their D-isomers and derivatives thereof, such as salts or derivatives in which functional groups are protected by suitable protecting groups. Unless otherwise specified, the term "amino acid" as used herein refers to an α-amino acid.

[0113] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 6 carbon atoms. Where appropriate, alkyl groups include alkyl groups having the specified number of carbon atoms, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. 1-6 Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 5-methylpentyl.

[0114] 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. As used herein, the terms "subject" and "individual" may be used interchangeably.

[0115] Receptor epitope portion (dysfunctional P2X7 receptor epitope portion) The present invention relates to methods that utilize polypeptides that comprise an epitope recognized by the antigen-binding domain of a receptor on an immune cell (such as a CAR or TCR). The receptor is typically for binding to a tumor-associated or tumor-specific antigen on a cancer cell, and therefore the epitope portion of a polypeptide for use according to the present invention comprises a sequence derived from the tumor-associated or tumor-specific antigen bound by the receptor.

[0116] In certain embodiments, immune cells contain a receptor for binding the extracellular domain of CD19 on target cells. In such embodiments, it will be understood that a polypeptide for use according to the second aspect of the present invention will contain the same epitope of the ECD of CD19 bound by the receptor. Cellular immunotherapeutics having a receptor that targets CD19 are known to those of skill in the art, as are the epitopes to which such immunotherapeutics bind. For example, to enrich CAR-T cells comprising a CAR having an antigen-recognition domain consisting of anti-CD19 scFv FMC683, one skilled in the art will understand that a polypeptide for use according to the second aspect of the present invention should contain the epitope bound by scFv FMC683. Similarly, to obtain or enrich CAR-T cells comprising a CAR having an antigen-recognition domain consisting of anti-CD19 scFv A3B1, one skilled in the art will understand that a polypeptide for use according to the second aspect of the present invention should contain the epitope 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, incorporated herein by reference.

[0117] In a similar example, the immune cell may comprise a receptor for binding CD20 on the target cell, and in such an example, it will be understood that the polypeptide for use according to the second aspect of the invention will comprise the same epitope of CD20 that is bound by the receptor.

[0118] In other examples, the immune cell may contain a receptor (eg, a CAR) for binding mesothelin, and thus the polypeptide contains an epitope of mesothelin.

[0119] In other examples, the immune cell may contain a receptor (e.g., a CAR) for binding EGFR, and thus the polypeptide contains an epitope of EGFR.

[0120] In other examples, the immune cell may contain a receptor (e.g., a CAR) for binding to GPC3, and thus the polypeptide contains an epitope of GPC3.

[0121] In other examples, immune cells may contain receptors for binding MUC1, and thus the polypeptide comprises an epitope of MUC1.

[0122] In other examples, the immune cells contain receptors for binding HER2, and therefore the polypeptide contains an epitope of HER2.

[0123] In other examples, immune cells contain receptors for binding GD2, and therefore the polypeptide contains an epitope of GD2.

[0124] In other examples, immune cells contain receptors for binding CEA, and therefore the polypeptide contains an epitope of CEA.

[0125] In other examples, immune cells contain receptors for binding EpCAM, and thus the polypeptide contains an epitope of EpCAM.

[0126] In other examples, immune cells contain receptors for binding LeY, and therefore the polypeptide contains an epitope of LeY.

[0127] In other examples, immune cells contain receptors for binding PSCA, and thus the polypeptide contains an epitope of PSCA.

[0128] In other examples, immune cells contain receptors for binding CD276, and thus the polypeptide contains an epitope of CD276.

[0129] The present invention also provides a fusion protein comprising a dysfunctional P2X7 receptor epitope portion.

[0130] The dysfunctional P2X7 receptor epitope portion can be provided in the form of dysfunctional P2X7 receptor or a fragment of dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP. Such receptor cannot widen the opening of non-selective calcium channel to apoptotic pore.

[0131] According to the present invention, the dysfunctional P2X7 receptor epitope portion is typically in the form of a peptide fragment of the dysfunctional P2X7 receptor. Typically, the peptide comprises an epitope that is not found on or is not available for binding to the functional P2X7 receptor.

[0132] In some embodiments, the peptide comprises a proline at amino acid 210 of the dysfunctional P2X7 receptor. In some embodiments, the peptide comprises one or more amino acid residues ranging from glycine at amino acid 200 to cysteine ​​at amino acid 216 of the dysfunctional P2X7 receptor.

[0133] A range of peptide fragments of dysfunctional P2X7 receptors are disclosed in PCT / AU2002 / 000061 (and corresponding publications WO2002 / 057306, and US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2008 / 001364 (and corresponding publications and PCT / AU2009 / 000869 (and corresponding publications WO2010 / 000041, and US8,597,643, US9,328,155, or US10,238,716), the contents of all of which are incorporated in their entirety. Exemplary peptides within these specifications that contain epitopes contemplated for use in the present invention are described below.

[0134] PCT Publications Peptide Sequences WO2002 / 057306 GHNYTTRNILPGLNIT (SEQ ID NO: 3) WO2002 / 057306 GHNYTTRNILPGLNITC (SEQ ID NO: 2) (also referred to herein as the "E200" epitope) WO2009 / 033233 KYYKENNVEKRTLIKVF (SEQ ID NO: 4) (also referred to herein as the "E300" epitope) WO2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 6) (also referred to herein as the "E200 / E300" or "composite" epitope)

[0135] Non-limiting examples of variations of the E200 peptide sequence (including N-terminal and / or C-terminal extensions, and various linker, hinge, or spacer regions) are provided in Table 1.

[0136] The amino acid sequence of any one of SEQ ID NOs: 2 to 69 or 155 may comprise a portion of an epitope portion that can be recognized or bound by a receptor expressed on an immune cell (also referred to herein as the "recognition sequence" of the epitope portion).

[0137] In some embodiments, the epitope portion comprises or consists of an amino acid sequence selected from any of the peptide sequences listed in Table 1 above.

[0138] In some embodiments, the N-terminus of the epitope moiety is a free amine (-NH2).

[0139] In some embodiments, the C-terminus of the epitope moiety is a free acid (—COOH). In some embodiments, the C-terminus is a derivative or analog of the free acid group, such as an ester (—COOC alkyl) or a primary or secondary amide (—CONHR, where R is selected from H and C alkyl). Advantageously, having a C-terminus that is a derivative or analog of the free acid group may improve the biological stability of the peptide compared to the free acid. In some embodiments, the C-terminus is a derivative or analog of the free acid group that includes a functional moiety, such as biotin.

[0140] In an embodiment of either the first or second aspect, the epitope of the dysfunctional P2X7 receptor comprises or consists of an epitope that is found in the dysfunctional P2X7 receptor but not in the functional form of the P2X7 receptor.In other words, preferably, the polypeptide comprises or consists of an epitope that is specific for the dysfunctional P2X7 receptor.

[0141] In a further embodiment of the first or second aspect, the fusion protein comprises an epitope corresponding to E200, E300, or a 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. For example, one skilled in the art will understand that it is possible to include additional amino acids N-terminal or C-terminal to the region comprising the epitope bound by the anti-nfP2X7 receptor CAR. As a non-limiting example, and typically in the context of E200 defined as having an amino acid sequence substantially as defined in SEQ ID NO: 2 or 7 (and having a minimal sequence as defined in SEQ ID NO: 14), additional amino acids derived from the native sequence of P2X7 can be included within the polypeptide, for example, residues "DFP" N-terminal to the epitope in the P2X7 receptor sequence, and / or residues "TFHKT" C-terminal to 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 composite epitope.

[0142] 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 (e.g., to provide the sequence of SEQ ID NO: 7). One of skill in the art will understand that this can be done to reduce the possibility of any disulfide bonds between the polypeptide and another molecule.

[0143] It is also within the skill of the art to include additional amino acid residues in an E200, E300, or composite epitope (or extended epitope as discussed in the preceding paragraph) by, for example, 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 a linker sequence (such as a peptide 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 an E200, E300, or composite epitope as defined herein. Examples of such extended E200 epitope peptides are provided in Table 1.

[0144] Fc area In any embodiment, the amino acid sequence of the epitope of dysfunctional P2X7 receptor can be fused to the N-terminal region of the Fc region of antibody or its variant via its C-terminal region.In any embodiment, the amino acid sequence of the epitope of dysfunctional P2X7 receptor can be fused to the C-terminal region of the Fc region of antibody or its variant via its N-terminal region.

[0145] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chains.

[0146] The Fc region may contain one or more amino acid sequence modifications compared to a naturally occurring Fc sequence. The Fc region may contain one or more amino acid substitutions, such as substitutions of one or more cysteine ​​residues, to prevent dimerization of the molecule into an identical molecule. It will be understood that any amino acid substitution that prevents dimerization of the Fc region may be used. Thus, in vivo, the Fc fusion proteins described herein may be monomeric proteins. Preferably, the Fc region and hinge region derived from an immunoglobulin contain substitutions of at least one, at least two, or at least three cysteine ​​residues. Preferably, the substituted residues are at least C220, C226, and C229. In a preferred embodiment, the monomeric protein contains substitutions at all three of C220, C226, and C229 (numbered according to the EU system).

[0147] Thus, the Fc region of the fusion protein contains one or more amino acid substitutions, relative to a naturally occurring Fc sequence, that prevent or reduce the ability of the Fc region to homodimerize. Preferably, the amino acid substitutions include one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules. The cysteine ​​residues in the Fc region may optionally be substituted with any other amino acid residue, including glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

[0148] The cysteine ​​residues for substitution are preferably one or more of the cysteine ​​residues located in the region of the Fc region corresponding to the hinge region of an immunoglobulin. An example of an IgG1 hinge region and examples of variations thereof, including cysteine-to-serine substitutions, are provided in Table 3 herein. The hinge region of an immunoglobulin (e.g., of an IgG1) contains three cysteine ​​residues, numbered C220, C226, and C229 (according to EU numbering). Thus, in any embodiment, at least one, at least two, or all three of the cysteine ​​residues in an immunoglobulin hinge region are substituted. Preferably, at least two or all three of the cysteine ​​residues are substituted. More preferably, all cysteine ​​residues in the Fc region, such as the hinge region, are substituted. In a particularly preferred embodiment, at least one of C226 and C229 is substituted, and preferably both C226 and C229 are substituted.

[0149] Therefore, in a preferred embodiment, the fusion protein comprises a hinge region for linking the dysfunctional P2X7 receptor epitope portion and an Fc region of an antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences set forth in SEQ ID NOs: 76 to 113, or 136 to 137, or 141, or 142.

[0150] In further embodiments, the fusion protein region may comprise an Fc region corresponding to the Fc "hole" or "knob" for use in "knob-in-hole" heterodimers. The use of such Fc sequences is known in the art and provides asymmetric heterodimeric molecules that bind to an additional Fc region that does not contain the epitope moiety, including fusion proteins having a single copy of the epitope moiety and an Fc region described herein.

[0151] Those skilled in the art will be familiar with techniques and Fc sequences that allow for the formation of so-called monomeric fusion proteins, including, but not limited to, the use of the "knob-into-hole" IgG1 format (Ridgway et al., (1996), Protein Eng, 9:617-621). Such an approach in the context of the present invention allows for the expression and purification of heterodimeric fusion proteins with only one copy of a peptide epitope (e.g., an epitope portion derived from the E200 epitope described herein) per molecule. Examples of "knob-into-hole" Fc pairings are provided herein in SEQ ID NOS: 157 and 159 (knob and hole, respectively), 158 and 159, respectively, 160 and 162 (hole and knob, respectively), and 161 and 162, respectively. Thus, in any embodiment, the present invention provides a fusion protein comprising the amino acid sequence of any of SEQ ID NOs: 2-69 and 122 linked to an Fc region defined in SEQ ID NO: 160 or 162, wherein the fusion protein can form a heterodimer with an Fc region that does not include an E200 peptide portion.

[0152] Therefore, the fusion proteins of the present invention are preferably capable of forming heterodimeric molecules containing a single E200-containing amino acid sequence (in other words, the Fc portion of the fusion protein can heterodimerize with the Fc region of an antibody that does not contain an E200 peptide fused thereto).

[0153] In further embodiments, the Fc region may comprise one or more substitutions to eliminate or reduce effector function, such as reducing FcR-mediated binding and activation, as further described below.

[0154] 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 is the C-terminal region of an antibody. H 2 domain and C HIn the context of the present invention, the Fc region comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and by the C H The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively, and are held together by hydrophobic interactions of the three domains.

[0155] In some embodiments, the fusion protein does not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that an antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express FcγRIII only, 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).

[0156] 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, e.g., 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); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and the 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 additionally, ADCC activity of the molecule of interest can be measured in vivo as described, e.g., in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Also, a C1q binding assay can be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may 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 Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006), WO2013 / 120929A1).

[0157] In a preferred embodiment, the Fc fusion proteins of the present invention comprise an Fc region with reduced effector function. Fc regions with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants 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 mutation in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). For example, an antibody variant may comprise an Fc region with one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, substitutions are L234A and L235A (LALA) (see, e.g., WO2012 / 130831). Additionally, changes can be made in the Fc region that result in altered (i.e., reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J Immunol. 164:4178-4184 (2000) (e.g., G236R).

[0158] Further examples of modified Fc regions include those containing "LALALS" (amino acid substitutions L234A / L235A / M428L / N434S, as described in Zalevsky et al., (2010) Nat. Biotechnol. 28:157-159), LALAPG (amino acid substitutions L234A / L235A / P329G, as described in Gunn et al., (2021, Immunity 54:815)).

[0159] In some embodiments, the Fc region of the Fc fusion proteins of the invention may contain at least the "LALA" mutation (L234A and L235A) to reduce binding to FcR. The fusion protein may additionally or alternatively contain the mutation G346R to inhibit recruitment of complement C1q.

[0160] Other Fc modifications for use in the present invention include variants that reduce or eliminate binding to FcγRs 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 FcγRs and complement are useful for reducing undesired interactions mediated by the Fc region. Preferred knockout variants are those referred to as "Fc Variants with Optimized The present invention is described in US2008-0242845A1, published October 2, 2008, entitled "Properties," 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 variants include 234G, 235E, 235G, 236R, 237 Examples of variants include, but are not limited to, K, 267R, 269R, 325L, and 328R, where numbering is according to the EU index. A preferred variant includes 236R / 328R. The variants may be used in the context of 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, such as the hybrid IgG1 / IgG2 isotype described in U.S. Patent Application No. 11 / 256,060, may be useful.Other modifications to reduce FcγR and complement interactions include, but are not limited to, substitutions 297A, 297D, 234A, 235A, 237A, 318A, 228P, 236E, ΔG236, 265G, 268Q, 297Q, 309L, 330S, 331S, 327Q, 220S, 226S, 229S, 238S, 233P, 234A, and 234V, as well as removal of glycosylation at position 297 by mutation or enzymatic means, or by production in an organism such as a bacterium that does 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.

[0161] In some embodiments, the Fc region of the fusion protein comprises mutations to the complement (C1q) and / or Fcγ receptor (FcγR) binding sites, and in some embodiments, such mutations can render the fusion protein incapable of antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC).

[0162] The Fc region used in the context of the present invention preferably does not induce cytotoxicity, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0163] In some embodiments, the Fc region may contain one or more substitutions to reduce affinity for FcRn, thereby decreasing the serum or circulating half-life of the fusion protein. Substitutions to reduce affinity for FcRn are known in the art and are described, for example, in Ward et al., (2015), Mol. Immunol., 67:131-141, and Grevys et al., (2015), 194:5497-5508. Exemplary substitutions include substitutions at one or more of Ile253, His310, and His435, such as I253A, H310A, and H435A.

[0164] The term "Fc region" also includes native-sequence Fc regions and variant Fc regions. The Fc region may include the carboxyl terminus of the heavy chain. Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain the full-length heavy chain or a truncated variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to EU numbering, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the Fc region of an antibody may be contemplated. Amino acid sequence variants of the Fc region of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues in the amino acid sequence of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., inducing or supporting an anti-inflammatory response.

[0165] The Fc region of an antibody can be the Fc region of any of the antibody classes, such as IgA, IgD, IgE, IgG, and IgM. The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, when used in the context of the present invention, an antibody can be the Fc region of an IgG. For example, the Fc region of an antibody can be the Fc region of an IgG1, IgG2, IgG2b, IgG3, or IgG4. In some embodiments, the fusion protein of the present invention comprises the Fc region of an IgG. In the context of the present invention, the Fc region of an antibody is the Fc region of an IgG, preferably an IgG1.

[0166] Linker region between dysfunctional P2X7 receptor epitope and Fc region The dysfunctional P2X7 receptor epitope and the Fc region of the antibody (or serum albumin, transferrin, carboxy-terminal peptide of chorionic gonadotropin (CG) β chain, imprecise repeat peptide sequence, polypeptide sequence composed of proline-alanine-serine polymer, elastin-like peptide (ELP) repeat sequence), glycine residues or homopolymer of gelatin-like protein) may be linked directly or via a linker sequence. The linker sequence may be a spacer sequence as defined herein or as exemplified in Table 1 or Table 3. Alternatively, the linker sequence may be any amino acid-based linker sequence commonly used in the art.

[0167] The linker is usually a peptide having a length of up to 20 amino acids, but may be up to 50 amino acids in length. The term "linked to" or "fused to" refers to a covalent bond, such as a peptide bond, formed between two moieties. Thus, in the context of the present invention, the linker may 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 or more amino acids. For example, the fusion protein provided herein may comprise a linker between the epitope of a dysfunctional P2X7 receptor and the Fc region of an antibody, for example, between the N-terminus of the Fc region and the C-terminus of the dysfunctional P2X7 receptor epitope. As another example, the fusion protein provided herein may comprise a linker between the epitope of a dysfunctional P2X7 receptor and the Fc region of an antibody, for example, between the C-terminus of the Fc region and the N-terminus of the dysfunctional P2X7 receptor epitope moiety. In particular, dysfunctional P2X7 receptor epitope portion can be fused to the N-terminus of Fc region via a C-terminal linker.This linker has the advantage that different polypeptides of fusion protein can be independently folded and behave as expected.Therefore, in the context of the present invention, dysfunctional P2X7 receptor epitope portion and antibody Fc region can be comprised in a single chain multifunctional polypeptide.

[0168] In some embodiments, the fusion protein of the invention or the polypeptide for use in accordance with the invention comprises a peptide linker. In some embodiments, the peptide linker connects the dysfunctional P2X7 receptor epitope portion to the Fc region of the antibody. In some embodiments, the peptide linker can comprise the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Ser (GGGGS). In some embodiments, the peptide linker can comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or longer. The linker can be of different lengths, i.e., a (GS)n series of repeated glycine and serine residues (GS), where n is any number from 1 to 15 or more. For example, the linker can be (GS)3 (i.e., GSGSGS) or longer, or (GS)11 or longer. It will be 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. Preferably, n is 3 or less (i.e., when n is equal to 3, the linker is GSGSGS).

[0169] In further embodiments, the linker may include the inclusion of amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linker region may also include the sequence GSGK.

[0170] The peptide linker may consist of a repeating series of Thr-Pro (TP) with one or more additional amino acids N- and C-terminal to the repeat sequence. For example, the linker may comprise or consist of the sequence GTPTPTPTPTGEF (also known as a TP5 linker). In further embodiments, the linker may be a short and / or alpha-helical rigid linker (e.g., A(EAAAK)3A, PAPAP, or a dipeptide such as LE or CC).

[0171] In a further embodiment, instead of or in addition to the above-mentioned glycine-serine-based linker region, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety linked to the Fc region of an antibody via a hinge region. The link between the dysfunctional P2X7 receptor epitope moiety and the Fc region may comprise a combination of a hinge region and a linker region.

[0172] Examples of suitable hinge regions include those derived from immunoglobulins. The hinge region may be derived from IgG1, IgG2, IgG3, or IgG4 and may contain one or more amino acid substitutions (e.g., to prevent or reduce the possibility of disulfide bridge formation). Alternative hinge sequences may be derived from alternative immunoglobulin domains: CD8A, CD8B, CD4, or CD28, TRAC, TRBC, TRGC, TRDC.

[0173] Additional linker sequences can include any sequence of any length of the CL / CH1 domain, but do not include all residues of the CL / CH1 domain, e.g., the first 5-12 amino acid residues of the CL / CH1 domain. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linkers can also be derived from immunoglobulin light chains, e.g., Cκ or Cλ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences derived from hinge regions, and other naturally occurring sequences from other proteins.

[0174] Table 3 below provides non-limiting examples of suitable hinge regions for use in linking dysfunctional P2X7 receptor epitope moieties and Fc regions in the molecules of the invention.

[0175] It will be understood that the dysfunctional P2X7 receptor epitope portion can be linked to the Fc region (or other protein sequence defined herein) by more than one linker and / or more than one hinge region. For example, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion directly conjugated (N-terminal to C-terminal) to the Fc region. Alternatively, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, and then an Fc region. Still further, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, and then an Fc region. In yet another embodiment, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, another linker region, and then an Fc region. Of course, one skilled in the art will appreciate that alternative configurations are possible (i.e., the dysfunctional P2X7 receptor epitope portion is linked to the C-terminus of the Fc region via one or more linkers and / or hinge regions. [Table 3-1] [Table 3-2]

[0176] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is fused directly to the Fc region of an antibody (or 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) such that there is no linker between the two regions of the fusion protein or polypeptide.

[0177] In certain embodiments, the linker connecting the dysfunctional P2X7 receptor epitope moiety and the Fc region of the antibody (or serum albumin, transferrin, carboxy-terminal peptide of chorionic gonadotropin (CG) beta chain, imprecise repeat peptide sequence, polypeptide sequence composed of proline-alanine-serine polymer, elastin-like peptide (ELP) repeat sequence), homopolymer of glycine residues or gelatin-like protein) is a cleavable linker.

[0178] Cleavable linkers are well known in the art and include, for example, the sequence defined in SEQ ID NO: 144, which defines a cleavage site for the human rhinovirus 3C protease. Proteases for cleaving such cleavage sites are also readily available from commercial sources (e.g., Pierce HRV 3C Protease).

[0179] Other known cleavable linkers and other linkers that can be used in accordance with the present invention are disclosed in Chen et al., (2013) Adv. Drug. Deliv. Rev. 65:1357-1369, the contents of which are incorporated herein by reference.

[0180] Receptors and immune cells that express them The present invention finds use in the method for enriching the subpopulation of immune cells that express receptors that comprise antigen binding domains for binding to tumor-associated antigens and tumor-specific antigens, such as dysfunctional P2X7 receptors.Preferably, receptors are chimeric antigen receptors (CARs) or their variants.Receptors can also be modified TCRs.

[0181] The antigen recognition domain of the receptor preferably recognizes a target antigen expressed on a cancer cell. Any number of different immune cells expressing different antigen recognition domains to bind different target antigens can be utilized in accordance with the present invention, although it will be understood that it is necessary to utilize molecules that contain epitopes that compete for binding to the cellular immunotherapeutic agent.

[0182] For example, an immune cell can comprise a receptor having an antigen recognition domain for binding to any one of the following: 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 (Nectin-2), 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 EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, CD276, and PCSA.

[0183] In certain embodiments, the immune cells express a CAR (or a variant thereof) for binding dysfunctional P2X7 receptors. The extracellular portion of the CAR or a variant thereof may comprise an nfP2X7-binding domain that recognizes the E200 (or E300, or E200-300 complex) epitope disclosed herein.

[0184] Generally, CAR, its variant or TCR can comprise an extracellular domain (extracellular portion) comprising 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 comprise a signal peptide. Preferably, the extracellular portion of CAR, its variant or TCR comprises the nfP2X7 binding domain that recognizes the E200 (or E300, or E200-300 complex) epitope disclosed herein.

[0185] Typically, the antigen recognition domain of the CAR or TCR comprises a binding polypeptide comprising amino acid sequence homology to 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 V of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L The CAR preferably comprises amino acid sequence homology to the CDR1, CDR2, and CDR3 domains of the chain. As can be seen, the CAR will preferably be able to recognize the same dysfunctional P2X7 receptor epitope portion as present on the fusion protein of the present invention.

[0186] While it will be appreciated that the methods of the present invention find use in enriching cells expressing any CAR for binding to a dysfunctional P2X7 receptor, in a preferred embodiment, the binding polypeptide of the CAR is a CAR-binding polypeptide 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, Nos. 8,399,617, 8,709,425, 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. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent No. 8,440,18 Nos. 6, 9,181,320, 9,944,701, or 10,597,451), PCT / AU2008 / 001365 (or corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Pat. Nos. 8,597,643, 9,328,155, or 10,23 8,716), PCT / AU2010 / 001070 (or any one of the corresponding publications WO / 2011 / 020155, US9,127,059, US9,688,771, or US10,053,508), and PCT / AU2010 / 001741 (or any one of the corresponding publications WO2011 / 075789 or US8,835,609). H and / or V LThe antibody comprises the amino acid sequences of the CDRs of the respective chains, the entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequences of PEP2-2-1, 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 BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010 / 0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0187] In further embodiments, the CAR binding polypeptide is a polypeptide of interest 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 10,450). ,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or No. 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Patents Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Patents Nos. 8,597,643, 9,328,155 or 10,238,716). and the V of antibodies described in PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US9,127,059, US9,688,771, or US10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO2011 / 075789 or US8,835,609). H and / or V LThe entire contents of which are incorporated herein by reference, include the amino acid sequences of the CDRs of PEP2-2-1, 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 BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010 / 0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0188] In further embodiments, the CAR binding polypeptide is a polypeptide of interest 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 10,450,380). any one of PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010 / 0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or 10,597,451) Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Patent Nos. 8,597,643, 9,328,155, or 10,238,716), PCT / AU2010 / 001070 (or corresponding publications and PCT / AU2010 / 001741 (or any one of the corresponding publications WO2011 / 075789 or US8,835,609), the entire contents of which are incorporated herein by reference.Preferably, the antibody comprises the CDR amino acid sequences of PEP2-2-1, 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 BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010 / 0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0189] CARs also typically include a signal peptide. "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a specific organelle (such as the endothelial endoplasmic reticulum) and / or to the cell surface.

[0190] Generally, the "antigen-binding domain" (or antigen-recognition domain) refers to the region of a CAR that specifically binds to an antigen (and thus can target cells containing the antigen). A CAR may contain 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 bispecific antibody. Any molecule that specifically binds to a given antigen, such as an affibody or ligand-binding domain from a naturally occurring receptor, may be used as the antigen-binding domain. Often, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of an immunoglobulin heavy chain and a light chain 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 understand that a variety of linker sequences and formats may be used.

[0191] CARs may also include a "hinge" region (sometimes called a spacer region or linker region) that links the antigen-binding domain to the transmembrane domain. This is typically a hydrophilic region located between the antigen-binding domain and the transmembrane domain. CARs may include an extracellular hinge domain, but it is also possible to exclude such a hinge. The hinge region may include, for example, an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial hinge sequence, or a combination thereof. One example of a hinge region is the CD8α hinge.

[0192] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source for such a 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, CD8α or CD28. If the main signaling and antigen recognition modules (domains) are located on two (or more) polypeptides, the CAR can have two (or more) transmembrane domains. The division of the main signaling and antigen recognition modules allows for small molecule-dependent, titratable, and reversible control of CAR cell expression (Wu et al., 2015, Science 350:293-303), which is due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR.

[0193] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to perform a specific function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector function.

[0194] The function of the intracellular domain may be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0195] Examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequence of the T cell receptor (TCR) and co-receptors that initiate signal transduction after antigen receptor binding.

[0196] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0197] Examples of ITAMs containing primary cytoplasmic signaling sequences commonly 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 notably, the sequence derived from CD3 zeta.

[0198] The cytoplasmic domain of the CAR can be designed to contain a CD3-zeta signaling domain, either alone or in combination with any 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 contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for efficient and sufficient 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.

[0199] In some embodiments, the activating receptor (from which a portion of the signaling domain is derived) is a CD3 co-receptor complex or an Fc receptor.

[0200] 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, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0201] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0202] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified 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 a glycine-serine duplex.

[0203] As another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0204] As mentioned above, either the extracellular portion of the CAR, or the transmembrane domain, or the cytoplasmic domain may also contain a heterodimerization domain for the purpose of separating the main signaling and antigen recognition modules of the CAR.

[0205] Fusion proteins of the invention or polypeptides for use in accordance with the invention, e.g., CARs that bind to a CAR comprising an nfP2X7E200 binding domain, may be designed to include any portion or part of the above domains described herein in any order and / or combination that results in a functional CAR.

[0206] The affinity that dysfunctional P2X7 receptor binding domain of CAR binds to nfP2X7 recognition site E200 of fusion protein or polypeptide of the present invention for use according to the present invention can vary, but generally, binding affinity can be within the range of about 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM or about 1 nM, preferably at least about 10 pM or 1 pM.In preferred embodiments, binding affinity is at least about 1 nM or at least about 10 nM.

[0207] The receptor (e.g., a CAR, a variant thereof, or a TCR, or a variant thereof) is typically expressed by an immune cell.

[0208] The immune cells may be "engineered cells," "genetically modified cells," or "immune effector cells" as described herein. Furthermore, the immune cells may be immune cell precursors that can differentiate into immune cells. Cells that can differentiate into immune cells (e.g., T cells expressing a dysfunctional P2X7CAR) may be stem cells, multilineage progenitor cells, or induced pluripotent stem cells.

[0209] The immune cells can be leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells (including CD4+ T cells or CD8+ T cells), natural killer cells, natural killer T cells, or γδ T cells.

[0210] In any embodiment, the immune 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).

[0211] In any embodiment, the immune cells optionally do not express accessory molecules, which may be checkpoint, exhaustion, or apoptosis-related signaling receptors, and 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).

[0212] In some embodiments, immune cells comprise two or more different receptors (for example, two or more CARs, or variants thereof). CARs can bind to different epitopes on the same target molecule (for example, different epitopes on dysfunctional P2X7 receptor). Alternatively, CARs can bind to different target molecules, so that only one of CARs binds to dysfunctional P2X7 receptor.

[0213] 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 and / or non-identical signal transduction domains.In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize dysfunctional P2X7 receptors), but have different signal transduction domains, such as one CAR with a signal transduction domain that has a part of an activating receptor and the other CAR with a signal transduction domain that has 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 dysfunctional P2X7 receptors, and the other CAR can take any suitable form and be directed against any suitable antigen.

[0214] Methods for Enriching Immune Cells It is well within the skill of a person skilled in the art to confirm the ability of a given fusion protein or polypeptide as defined herein to be bound by relevant receptors.For example, in the context of nfP2X7 receptor-binding CAR, a person skilled in the art can use conventional techniques to confirm the binding of a polypeptide (or a series of polypeptides) by CAR, thereby determining the suitability of the polypeptide for use in the method of the present invention.

[0215] It will be appreciated that the methods of the present invention can be used to isolate or enrich any population of immune cells that express a receptor (including a CAR) that comprises an antigen binding domain that recognizes an epitope of the dysfunctional P2X7 receptor contained in the fusion protein.

[0216] Furthermore, once the fusion protein or polypeptide of the present invention is contacted with any population of cells, including target cells (i.e., cells capable of binding to the dysfunctional P2X7 receptor epitope on the fusion protein), one skilled in the art can use routine laboratory techniques to release the fusion protein / cell complexes from the capture agent, thereby obtaining an enriched cell sample.

[0217] The use of magnetic microbeads or macrobeads for isolating cell and protein populations is well known to those skilled in the art. The average diameter of the beads can range from 10 nm to 10 μm. Biocompatible magnetic particles are commercially available and consist, for example, of magnetic iron oxide forms coated with dextran molecules or a silica shell. The solid support can also be a polymer containing magnetic material. Commercially available forms of such beads, including MicroBeads (Miltenyi Biotec) for use in conjunction with MACS® columns and Dynabeads magnetic beads (Applied Biosystems), are readily available and well known.

[0218] The use of microbeads (50 nm diameter) has advantages over the use of macrobeads (1-5 μm diameter) because they can be directly injected into patients requiring immune cell therapy. Thus, in certain embodiments, it is not necessary to isolate immune cells from the polypeptide / cell complexes according to the second aspect of the invention; the complexes can be administered directly. In alternative embodiments, in which microbeads or macrobeads are linked to the fusion protein or polypeptide via a biotin moiety, it may be possible to separate the beads from the fusion protein or polypeptide by adding excess free biotin.

[0219] Furthermore, one skilled in the art will appreciate that more than one round of enrichment may be performed to increase the purity of the final cell composition.

[0220] In some embodiments, the present invention provides a method for enriching cells that express a chimeric antigen receptor (CAR) over cells that do not express a CAR in a composition, the method comprising contacting the cells with a fusion protein described herein. In some embodiments, the CAR-expressing cells are cells transduced with a nucleic acid molecule encoding the CAR. In some embodiments, the CAR-expressing cells are clones of cells transduced with a nucleic acid molecule encoding the CAR that express the CAR.

[0221] Compositions and Uses Thereof The present invention also provides compositions comprising, consisting essentially of, or consisting of one or more cells expressing one or more CARs for binding to dysfunctional P2X7 receptors that have been enriched according to one or more of the methods of the present invention.

[0222] As used herein, a composition "comprising" one or more cells expressing one or more CARs that have been enriched according to one or more of the methods of the invention may include other compounds and cells. As used herein, a composition "consisting essentially of" one or more cells expressing one or more CARs that have been enriched according to one or more of the methods of the invention may include other compounds and cells, so long as they do not substantially alter the activity or function of the cells expressing one or more CARs in the composition. As used herein, a composition "consisting of" one or more cells expressing one or more CARs that have been enriched according to one or more of the methods of the invention means that the composition does not contain other functional cells in addition to the one or more cells expressing one or more CARs.

[0223] A composition comprised of one or more cells expressing one or more CARs that have been enriched according to one or more of the methods of the invention may contain components other than cells, such as compounds, proteins, pharmaceutically acceptable carriers, surfactants, preservatives, etc. In some embodiments, a composition comprised of one or more cells expressing one or more CARs that have been enriched according to one or more of the methods of the invention may contain insignificant amounts of contaminants.

[0224] In some embodiments, the amount of one or more cells expressing one or more CARs for binding to dysfunctional P2X7 receptors is at least about 50% of the total cells in the composition.In some embodiments, the amount of one or more cells expressing one or more CARs is at least about 60% of the total cells in the composition.In some embodiments, the amount of one or more cells expressing one or more CARs is at least about 70% of the total cells in the composition.In some embodiments, the amount of one or more cells expressing one or more CARs is at least about 80% of the total cells in the composition.In some embodiments, the amount of one or more cells expressing one or more CARs is at least about 90% of the total cells in the composition.In some embodiments, the amount of one or more cells expressing one or more CARs is at least about 95% of the total cells in the composition.In some embodiments, the composition according to the present invention comprises a therapeutically effective amount of one or more cells expressing one or more CARs.

[0225] The compositions or cell populations obtained by the methods of the present invention may be used in the treatment of diseases or conditions characterized by the expression of dysfunctional P2X7 receptors.

[0226] In one embodiment, the subject in need of treatment includes a subject with a benign tumor, a precancerous tumor, or a non-metastatic tumor. In one embodiment, the cancer is precancerous or preneoplastic.

[0227] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer may be located in any organ or tissue, particularly an organ or tissue with relatively high hemodynamic pressure, such as the lung, liver, kidney, pancreas, intestine, and brain. The secondary cancer may be detected in the ascites and / or lymph nodes.

[0228] In one embodiment, the cancer may be substantially undetectable.

[0229] "Precancerous" or "preneoplastic" generally refers to a condition or growth that typically precedes or develops into cancer. A "precancerous" growth can have cells characterized by aberrant cell cycle regulation, proliferation, or differentiation, as can be determined by cell cycle markers.

[0230] Cancers can be solid or "liquid" tumors. In other words, cancers can be growths in tissue (carcinomas, sarcomas, adenomas, etc.) or cancers present in body fluids such as blood or bone marrow (e.g., lymphomas and leukemias).

[0231] In certain preferred embodiments, the cancer in need of treatment may be a cancer characterized by low expression of dysfunctional P2X7 receptor. An example of such a cancer is Burkitt's lymphoma. However, immunohistochemical analysis of the surface expression of dysfunctional P2X7 (nfP2X7) receptor in patient tumor biopsies showed IHC scores ranging from 1+ to 3+. Thus, samples with low expression may be found in a wide range of tumor types. Examples include, but are not limited to, various types of solid tumors, including neuroblastoma, colorectal cancer, lung cancer, kidney cancer, skin cancer, breast cancer, brain cancer, and prostate cancer. Such differences in expression levels in different tissues may be due to tumor formation from cells in an early transformation state (tissues with the highest receptor expression may be those with the highest proliferation rates).

[0232] Other examples of cancers that can be treated according to the methods of the invention include blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinomatous tumors, gastric cancer, and pancreatic islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, lung cancer, including small cell lung cancer (SCKC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, and lung squamous cell carcinoma, These include peritoneal cancer, hepatocellular carcinoma, stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, testicular cancer, tumors, esophageal cancer, tumors of the biliary tract, and head and neck cancer. [Example]

[0233] Example 1: Enrichment of nfP2X7 receptor-binding CAR T cells using biotin-labeled fusion proteins Jurkat cells and / or primary CD4+ and CD8+ T cells (mixed at a 1:1 ratio after enrichment) from healthy volunteer donors (donors 12 and 57) were stably transduced with lentivirus (third-generation LV system) to express anti-nfP2X7 chimeric antigen receptors (CARs), which contained an antigen-binding domain for binding to the E200 epitope of the P2X7 receptor.

[0234] The Jurkat nfP2X7 reporter cell line was grown as a single cell clone expressing nfP2X7-CAR by FACS sorting. The CAR construct also contained a cleaved EGFR (tEGFR) downstream of a ribosomal skip site (T2A) as a marker gene for use in allowing detection of transduced cells after enrichment while the CAR receptor was still occupied by the enrichment reagent (DetR1 or DetR2 as shown in Table 1, as an exemplary dimeric or monomeric Fc-attenuated fusion protein).

[0235] To mimic the enrichment process, Jurkat wild-type cells (CAR-negative, tEGFR-negative cells) were mixed with Jurkat nfP2X7-CAR-tEGFR-expressing cells at a 1:1 ratio, or the primary generated nfP2X7CAR T cell product was used. One round of positive selection was performed as outlined below.

[0236] A fusion protein containing the sequence of SEQ ID NO: 149 was labeled with a biotin label. The Fc fusion protein was conjugated to biotin using EZ-Link™ NHS-LC-LC-Biotin (ThermoFisher, catalog number 21343) according to the manufacturer's instructions. The labeled Fc fusion protein was added to a mixture of untransduced and transduced cells at the indicated concentrations of 1 or 2 μg / ml, and the cell / fusion protein mixture was incubated at room temperature for 10 minutes and washed with magnetic-activated cell sorting (MACS) cell separation buffer (Miltenyi Biotec) according to the manufacturer's instructions.

[0237] Method 1: Either anti-biotin microbeads (Miltenyi Biotec, 130-090-485) were then added to the mixture, which was then incubated for another 15 minutes at 4°C. The anti-biotin microbeads are magnetic particles coated with anti-biotin antibodies to allow for the separation of cells bound by the anti-biotin antibody-coated microbeads. Separation was then performed according to the manufacturer's instructions.

[0238] The cell suspension is loaded onto an MS MACS® column (Miltenyi Biotec, 130-042-201), which is placed in the magnetic field of the MACS separator. The magnetically labeled material (i.e., containing the fusion protein and anti-biotin bound to the CAR T cells) is retained within the column. The flow-through (containing cells that do not bind to the fusion protein) is discarded.

[0239] Method 2: Cell enrichment was performed using EasySep technology from STEMCELL Technologies according to the manufacturer's instructions as indicated, after primary incubation with the indicated Fc-fusion protein, as compared to MACS technology from Miltenyi Biotec.

[0240] In both MACS and EasySep technologies, the final step is to remove the probe from the magnetic field and then elute the magnetically retained material, which includes those Jurkat or primary T cells that have been successfully transduced with the lentiviral vector, express the nfP2X7-CAR, and thus can be bound by a biotin-labeled fusion protein containing the E200 epitope of the P2X7 receptor.

[0241] Figure 1 shows the results of the enrichment protocol for Jurkat nfP2X7CAR T cells, pre- and post-enrichment, 0 and 48 hours post-enrichment, whereby tEGFR was used as a marker gene to identify CAR-expressing cells. Direct detection of nfP2X7CAR-positive cells was performed by indirect staining using the same LC-LC-biotinylated Fc-attenuated fusion protein DetR1 or DetR2 (as defined in Table 1) and a secondary anti-biotin antibody. After a single round of positive selection, the purity of CAR-expressing cells was >90%. 48 hours post-enrichment (Figure 1B) shows that viability and purity were still >90%.

[0242] Similar experiments were performed using primary cells obtained from two separate donors ("Donor 12" and "Donor 57"). Briefly, buffy coats (PBMCs) were obtained from the donors using standard techniques. Cells were enriched for CD4+ and CD8+ T cells using CD4+ / CD8+ microbeads (MACS). After enrichment, the cells were mixed at a 1:1 ratio and then transduced with a lentivirus expressing an anti-nfP2X7CAR construct.

[0243] Figure 2 shows the results of the enrichment protocol for nfP2X7CAR T cells from "donor 12," whereby tEGFR was used as a marker gene to identify CAR-expressing cells, pre- and post-enrichment, 0 and 48 hours post-enrichment. Direct detection of nfP2X7CAR-positive cells was performed by indirect staining using the same LC-LC-biotinylated Fc-attenuated fusion proteins DetR1 or DetR2 and a secondary anti-biotin antibody. After a single round of positive selection, the purity of CAR-expressing cells was >90%. 48 hours post-enrichment (Figure 2B) shows that viability and purity were still >90%.

[0244] Figure 3 shows the results of the enrichment protocol (using a CAR with an alternative nfP2X7 receptor antigen-binding domain to the one in the paragraph "CAR2" above) for nfP2X7 CAR T cells from "Donor 12," whereby tEGFR was used as a marker gene to identify CAR-expressing cells, pre- and post-enrichment, 0 and 48 hours post-enrichment. Direct detection of nfP2X7 CAR-positive cells was performed by indirect staining using the same LC-LC-biotinylated Fc-attenuated fusion protein DetR1 or DetR2 and a secondary anti-biotin antibody. After a single round of positive selection, the purity of CAR-expressing cells was >90%. 48 hours post-enrichment (Figure 3B) shows that viability and purity were still >90%.

[0245] Figure 4 shows the results of the enrichment protocol for nfP2X7CAR T cells from "donor 12" pre- and post-enrichment using EasySep technology as a comparison with the MACS technology in Figure 3, whereby tEGFR was used as a marker gene to identify CAR-expressing cells, at 0 and 48 hours post-enrichment. Direct detection of nfP2X7CAR-positive cells was performed by indirect staining using the same LC-LC-biotinylated Fc-attenuated fusion proteins DetR1 or DetR2 and a secondary anti-biotin antibody. After a single round of positive selection, the purity of CAR-expressing cells was >90%. 48 hours post-enrichment (Figure 4B) shows that viability and purity were still >90%.

[0246] The table below shows a summary of the enrichments performed in Figures 1-4. [Table 4]

[0247] Figure 5 shows the results of the enrichment protocol for nfP2X7 CAR T cells from "donor 57," whereby tEGFR was used as a marker gene to identify CAR-expressing cells, pre- and post-enrichment, 0 and 48 hours post-enrichment. Direct detection of nfP2X7CAR-positive cells was performed by indirect staining using the same LC-LC-biotinylated Fc-attenuated fusion proteins DetR1 or DetR2 and a secondary anti-biotin antibody.

[0248] Enrichment was performed by MACS technology. The data show enrichment with the dimeric Fc-attenuated fusion protein DetR1 (SEQ ID NO: 149) compared to the monomeric Fc-attenuated fusion protein DetR1 (SEQ ID NO: 145) and the monomeric Fc-attenuated fusion protein DetR2 (SEQ ID NO: 146).

[0249] conditions: 1. LC-LC-biotinylated dimeric DetR1 Fc attenuation 2. LC-LC-biotinylated Monomeric DetR1 Fc Attenuation 3. LC-LC-biotinylated Monomeric DetR1 Fc Attenuation

[0250] Prior to isolation, CAR expression was 55% via DetR1 and 60% via tEGFR. A total of 1x10E 7 T cells were labeled at a concentration of 2ug / mL for 10 minutes at room temperature, and then separation continued using the MS column according to the manufacturer's instructions for MACS separation (see above).

[0251] After a single round of positive selection, the purity of CAR-expressing cells was >90%. 48 hours after enrichment, viability and purity were still >90%.

[0252] Cell numbers (=yield) and EGFR expression before and after isolation indicate CAR-positive and CAR-negative fractions. The use of marker genes facilitates direct detection of CAR.

[0253] The results are summarized in the table below. [Table 5]

[0254] Although these data indicate that the use of the dimeric form of the fusion protein resulted in higher yields, in practice it would be preferable to utilize the monomeric protein to avoid enrichment for immune cell activation. Furthermore, the above results may be a reflection of the superior biotin labeling using the dimeric protein compared to the monomeric protein, which provides increased yields.

[0255] Figure 6 shows the purity and viability of CAR T cells after isolation, as measured by indirect CAR detection using a viability dye (7AAD, measured in channel PerCP) and the marker gene tEGFR (AF647 primary labeled anti-EGFR mAb cetuximab, measured in APC channel). Direct detection of the CAR receptor shows the still partially occupied situation of isolated CAR-positive cells. T cells expressing the anti-nfP2X7 CAR were detected by staining the CAR T cells with biotinylated DetR2 (SEQ ID NO: 146) followed by a secondary anti-biotin antibody in Vioblue (130-113-857 biotin antibody, VioBlue®, Miltenyi Biotech).

[0256] Example 2: Enrichment using monomeric fusion proteins leads to less activation and exhaustion compared to homodimeric fusion proteins. A series of enrichment experiments were carried out similar to those described in Example 1. Briefly, Jurkat cells and / or primary CD4+ T cells and CD8+ T cells (mixed at a 1:1 ratio after enrichment) from a healthy volunteer donor (donor 24) were stably transduced by lentivirus (third generation LV system) to express E200-targeting chimeric antigen receptor (CAR), which contained an antigen-binding domain for binding to the E200 epitope of the P2X7 receptor.

[0257] CAR T cells were enriched using either a monomeric or dimeric fusion protein, each containing a peptide moiety that can be bound by the CAR or protein. The fusion proteins used in this experiment contain the amino acid sequences of SEQ ID NOs: 158 (monomer) and 149 (capable of forming homodimers in vitro).

[0258] Figure 7 shows the levels of CD25+ / CD69+ expression (measures of T cell activation, respectively) and PD-1 expression (measures of T cell exhaustion) at 24, 48, and 72 hours after enrichment with various concentrations of fusion protein (10 ng / ml to 400 ng / ml).

[0259] The results show that enrichment using monomeric fusion proteins leads to significantly less T cell activation and significantly less T cell exhaustion in a concentration-dependent manner compared to using dimeric fusion proteins.

[0260] Example 3: Efficiency, viability and yield after CAR T enrichment using biotin beads and MS columns Enrichment of CAR T cells using MACS technology was performed according to "Method 1" in Example 1. T cells were obtained from three healthy donors (donors 50, 53, and 71, D50, D53, and D71).

[0261] Figure 8 shows that enrichment using monomeric or dimeric fusion proteins is approximately equal, indicating that there is no significant loss of enrichment potential using monomeric fusion proteins compared to dimeric fusion proteins.

[0262] Results shown represent two replicate experiments using T cells from donors 50 and 71.

[0263] Figure 9 shows cell numbers on days 1 and 2 after MACS sorting, normalized to the maximum expected cell number. The results show that significantly higher cell numbers are obtained when using monomeric fusion proteins for the enrichment process compared to using homodimeric fusion proteins. The results shown are for T cells obtained from two healthy donors (donor 50 and donor 71).

[0264] Figure 10 shows cell viability two days after MACS sorting, as measured by the percentage of 7AAD-negative cells in the cell population. The results demonstrate a decrease in overall cell viability when enrichment is performed using dimeric fusion proteins compared to when using monomeric fusion proteins. The results shown represent two replicate experiments using T cells from donors 50 and 71.

[0265] Figure 11 shows CD25+ / CD69+ and PD-1 expression of enriched CAR T cells 2 days after MACS sorting.

[0266] The results show that cells enriched using the dimeric fusion protein have significantly higher levels of the activating markers CD25 and CD69 compared to cells enriched using the monomeric fusion protein. Additionally, cells enriched using the dimeric fusion protein have significantly higher levels of the exhaustion marker PD-1 compared to cells enriched using the monomeric fusion protein.

[0267] Overall, the results show that enrichment of CAR T cells using a monomeric fusion protein (such as having the amino acid sequence of SEQ ID NO: 158) provides: - Enrichment of CAR-positive T cells at levels comparable to those obtained when using dimeric fusion proteins - Improved recovery of cells, with greater cell numbers and higher cell viability compared to enrichment using homodimeric fusion proteins (e.g., containing two copies of an E200-derived peptide). - Less activation and less exhaustion of the enriched cells compared to cells obtained using dimeric fusion proteins.

[0268] These results indicate that using the enrichment methods described herein involving the use of monomeric fusion proteins, superior cell products are obtained compared to using homodimeric fusion proteins that contain two moieties (peptide sequences) that can be bound by the CAR and likely lead to cross-activation of CAR T cells.

[0269] Example 4: Enrichment using asymmetric heterodimeric molecules Similar experiments are performed using the heterodimeric asymmetric molecules described herein (e.g., such that the molecules comprise dimerization between an E200 peptide-Fc fusion protein and a non-identical Fc region of an antibody using KIH technology). The results similarly show that enriching CAR T cells using heterodimeric asymmetric molecules comprising a single copy of the E200 peptide sequence leads to significantly reduced T cell activation and T cell exhaustion in a concentration-dependent manner compared to using a homodimeric fusion protein comprising two copies of the E200 peptide (e.g., the dimer is a homodimer of the E200-Fc fusion protein). These results indicate that it is preferable to use asymmetric heterodimeric molecules or monomeric fusion proteins (e.g., comprising a single amino acid sequence that can be recognized by the antigen-binding domain of the CAR) for the purpose of enriching CAR T cells in order to minimize undesired activation and exhaustion of CAR T cells in patients.

[0270] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

1. A fusion protein comprising: (i) Dysfunctional P2X ７ a receptor epitope portion; (ii) A fusion protein comprising an Fc region of an antibody.

2. Dysfunctional P2X ７ The receptor epitope portion is a dysfunctional P2X ７ Receptors are found, but functional P2X ７ The fusion protein of claim 1, comprising an epitope sequence not found in the receptor.

3. Dysfunctional P2X ７ 3. The fusion protein according to claim 1, wherein the amino acid sequence of the receptor epitope portion comprises or at least consists of the amino acid sequence shown in SEQ ID NO: 7, preferably the amino acid sequence of SEQ ID NO:

14.

4. Dysfunctional P2X ７ The fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the receptor epitope portion comprises or consists of the sequence shown in SEQ ID NO: 9 or 122.

5. Dysfunctional P2X ７ 4. The fusion protein of claim 1, wherein the amino acid sequence of the receptor epitope portion comprises an amino acid sequence set forth in any of SEQ ID NOs: 7 to 69 or 122, or a sequence at least 80%, at least 81%, at least 82%, 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 thereto, provided that the sequence comprises at least the sequence set forth in SEQ ID NO: 14 or 7.

6. The fusion protein according to any one of claims 1 to 5, wherein the Fc region of an antibody is an Fc region of IgG, IgA, IgD, IgE, or IgM.

7. The fusion protein of claim 6, wherein the Fc region is from an IgG antibody, preferably IgG1.

8. The fusion protein according to any one of claims 1 to 7, wherein the Fc region of the fusion protein comprises the CH2 and CH3 domains of an antibody.

9. 9. The fusion protein of any one of claims 1 to 8, wherein the Fc region of the fusion protein comprises one or more amino acid substitutions relative to a naturally occurring Fc sequence that prevent or reduce the ability of the Fc region to homodimerize.

10. 10. The fusion protein of claim 9, wherein the amino acid substitutions comprise one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules, and optionally, the cysteine ​​residues in the Fc region are substituted with any of glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

11. 11. The fusion protein of any one of claims 1 to 10, wherein the Fc region comprises one or more amino acid substitutions to reduce affinity for an Fc receptor (FcR including any of FcγRI, FcγRII, and FcγRIII), and / or to reduce recruitment of complement C1q, and / or to reduce affinity for FcRn.

12. 12. The fusion protein of claim 11, wherein the one or more amino acid substitutions reduce the ability of the fusion protein to elicit antibody-dependent cell-mediated toxicity (ADCC).

13. The fusion protein of any one of claims 1 to 12, wherein the fusion protein has an affinity for FcR of less than about 250 nM, preferably less than 500 nM, less than 1000 nM, most preferably less than 2000 nM.

14. the protein further comprises a linker region, optionally the dysfunctional P2X ７ The fusion protein according to any one of claims 1 to 13, comprising a cleavable linker region for linking the receptor epitope portion and the Fc region of an antibody.

15. 15. The fusion protein of any one of claims 1 to 14, wherein the fusion protein comprises one or more modifications to allow for detection of the fusion protein, including when the fusion protein is bound to a cell or a binding partner.

16. The one or more modifications are ７ A receptor (such as a dysfunctional P2X receptor) containing an antigen-binding domain for binding to the receptor ７ 16. The fusion protein of claim 15, wherein the fusion protein allows capture of the fusion protein upon binding to an immune cell expressing a receptor (such as a chimeric antigen receptor (CAR) for binding to a receptor).

17. 17. The fusion protein of claim 16, wherein the one or more modifications are selected from a biotin moiety, fluorescein (FITC), a peptide tag (such as His, Myc, Flag, and related tags), and a magnetic label.

18. An asymmetric heterodimeric molecule comprising the fusion protein of any one of claims 1 to 17.

19. Dysfunctional P2X ７ 20. Use of a fusion protein or molecule according to any one of claims 1 to 18 for obtaining a population of immune cells enriched for cells expressing a receptor comprising an antigen binding domain for binding to the receptor.

20. the immune cells have a dysfunctional P2X ７ 20. The use of claim 19, wherein the antibody expresses a chimeric antigen receptor for binding the receptor.

21. 1. A method for obtaining a population of immune cells or enriching a population of cells that express a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on a cancer cell, comprising: (i) providing a population of immune cells, preferably immune effector cells, which have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on cancer cells; (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises an epitope recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a moiety to allow capture of the polypeptide; contacting, thereby forming a complex between said polypeptide and said cell; (iii) isolating the complex from the population of cells; This method results in obtaining a population of immune cells that express a receptor having an antigen-binding domain for binding to a tumor-associated antigen or tumor-specific antigen on cancer cells.

22. 1. A method for obtaining a population of immune cells or enriching a population of cells that express a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on a cancer cell, comprising: (i) providing a mixed population of immune cells, preferably immune effector cells, wherein a subpopulation of said cells expresses a receptor comprising an antigen-binding domain for binding to a tumor-associated or tumor-specific antigen on cancer cells; (ii) contacting the mixed population of cells with a polypeptide, wherein the polypeptide comprises an epitope recognized by the antigen-binding domain of the receptor, and the polypeptide comprises a moiety to allow capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; This method results in obtaining a population of immune cells that express a receptor having an antigen-binding domain for binding to a tumor-associated antigen or tumor-specific antigen on cancer cells.

23. 23. The method of claim 21 or 22, wherein the cells are immune cells that express a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen on a cancer cell, and the polypeptide comprises an epitope recognized by the CAR, thereby obtaining or enriching a population of immune cells that express the CAR.

24. The immune cells are ７ 24. The method of any one of claims 21 to 23, wherein the tumor expresses a receptor for binding to a tumor-associated or tumor-specific antigen selected from the group consisting of mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSA, CD19, CD20, Clec9a, CD276, PD-L1, and PD-L2.

25. Dysfunctional P2X ７ 1. A method for obtaining a population of immune cells or enriching a population of cells that express a receptor comprising an antigen binding domain for binding to a receptor, comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein said cells contain a dysfunctional P2X ７ providing a nucleic acid encoding a receptor comprising an antigen-binding domain for binding to the receptor; (ii) contacting said population of cells with a polypeptide, wherein said polypeptide is a dysfunctional P2X receptor that is recognized by the antigen-binding domain of said receptor. ７ a polypeptide comprising an epitope of a receptor, the polypeptide comprising a moiety for enabling capture of the polypeptide; contacting, thereby forming a complex between said polypeptide and said cell; (iii) isolating the complex from the population of cells; This leads to dysfunctional P2X ７ A method of obtaining a population of immune cells that express a receptor having an antigen-binding domain for binding to the receptor.

26. Dysfunctional P2X ７ 1. A method for obtaining a population of immune cells or enriching a population of cells that express a receptor comprising an antigen binding domain for binding to a receptor, comprising: (i) providing a mixed population of immune cells, preferably immune effector cells, wherein a subpopulation of said cells contains a dysfunctional P2X ７ providing a receptor comprising an antigen-binding domain for binding to the receptor; (ii) contacting the mixed population of cells with a polypeptide, wherein the polypeptide is a dysfunctional P2X receptor recognized by the antigen-binding domain of the receptor; ７ a polypeptide comprising an epitope of a receptor, the polypeptide comprising a moiety for enabling capture of the polypeptide; contacting the polypeptide with the cell, thereby forming a complex; (iii) isolating the complex from the population of cells; This leads to dysfunctional P2X ７ A method of obtaining a population of immune cells that express a receptor having an antigen-binding domain for binding to the receptor.

27. The cells obtained or enriched contain a dysfunctional P2X ７ 27. The method of claim 25 or 26, wherein the receptor is an immune cell expressing a chimeric antigen receptor (CAR) for binding to the receptor.

28. Dysfunctional P2X ７ 1. A method for obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a receptor, comprising: (i) providing a mixed population of immune cells, preferably immune effector cells, said population comprising a dysfunctional P2X ７ or the cells are transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) for binding to a P2X receptor, or the cells are transduced with a nucleic acid encoding a P2X receptor for binding to a P2X receptor. ７ providing a chimeric antigen receptor (CAR) for binding to a receptor; (ii) contacting said population of cells with a polypeptide, wherein said polypeptide is a dysfunctional P2X polypeptide recognized by said CAR. ７ a polypeptide comprising an epitope of a receptor, the polypeptide comprising a moiety for enabling capture of the polypeptide; contacting, thereby forming a complex between said polypeptide and said cell; (iii) isolating the complex from the population; This leads to dysfunctional P2X ７ A method of obtaining a population of immune cells that express a chimeric antigen receptor (CAR) for binding to a receptor.

29. Dysfunctional P2X ７ 1. A method for enriching a population of immune cells that express a chimeric antigen receptor comprising an antigen binding domain for binding to the receptor, comprising: (i) providing a population of immune cells, preferably immune effector cells, wherein said cells contain a dysfunctional P2X ７ providing a chimeric antigen receptor (CAR) for binding to a chimeric antigen receptor; (ii) contacting said population of cells with a polypeptide, wherein said polypeptide is a dysfunctional P2X polypeptide recognized by said CAR. ７ a polypeptide comprising an epitope of a receptor, the polypeptide comprising a moiety for enabling capture of the polypeptide; contacting, thereby forming a complex between said polypeptide and said cell; (iii) dysfunctional P2X bound to the fusion protein ７ and isolating said complex of cells expressing a chimeric antigen receptor (CAR) for binding to a receptor from the mixed population; This leads to dysfunctional P2X ７ A method for enriching a population of immune cells that express a chimeric antigen receptor (CAR) for receptor binding.

30. The polypeptide comprises a first portion, a tumor-associated or tumor-specific antigen (e.g., dysfunctional P2X) linked to an additional amino acid sequence to promote solubility and stability of the first portion. ７ 30. The method of any one of claims 21 to 29, comprising a first portion comprising an epitope of a target molecule (such as a target molecule of a receptor).

31. 31. The method of claim 30, wherein the further amino acid sequence linked to the epitope of the tumor-associated or tumor-specific antigen comprises a linker or hinge region.

32. 32. The method of claim 31 , wherein the linker comprises a cleavable sequence.

33. The polypeptide may comprise a dysfunctional P2X linked to a further amino acid sequence. ７ 33. The method of claim 31 or 32, in the form of a fusion protein comprising an epitope of a receptor, optionally with a further sequence selected from 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.

34. The polypeptide may be a carbohydrate, a lipid, a liposome, a peptide, or the dysfunctional P2X. ７ 30. The method of any one of claims 21 to 29, in the form of a conjugate comprising an aptamer conjugated to said amino acid sequence comprising an epitope of a receptor.

35. 35. The method of any one of claims 21 to 34, wherein the moiety for enabling capture of the polypeptide is a detectable moiety selected from a biotin moiety, fluorescein (FITC), a peptide tag (such as His, Myc, Flag and related tags), and a magnetic label.

36. 36. The method of any one of claims 20 to 35, wherein the polypeptide is labeled with a biotin moiety, and wherein the method further comprises (after step ii) contacting the cell with an anti-biotin antigen binding protein, preferably wherein the anti-biotin antigen binding protein comprises one or more moieties to enable its capture.

37. 37. The method of claim 36, wherein the one or more moieties for enabling capture of the anti-biotin antigen-binding protein comprise iron oxide particles (microbeads or macrobeads).

38. 36. The method of any one of claims 21 to 35, wherein the polypeptide comprises a magnetic label, and the isolating step comprises: i) applying a magnetic field to the population of cells; ii) removing or discarding the cells that are not attracted to the magnetic field; and iii) removing the magnetic field, thereby providing a population of immune cells that express the receptor.

39. 39. The method of any one of claims 21 to 38, wherein the method further comprises expanding the isolated immune cells.

40. 40. The method of any one of claims 21 to 39, wherein the method further comprises administering the isolated cells to a subject in need of treatment for cancer.

41. 41. The method of any one of claims 21 to 40, wherein said population of immune cells is a population of effector immune cells such as T cells, NK cells, or NKT cells.

42. 42. The method of claim 41, wherein the immune cell is a T cell.

43. 43. The method of claim 41 or 42, wherein the cells are derived from stem cells, optionally wherein the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells.

44. 44. The method of claim 41 or 43, wherein the cells are derived from a subject in need of treatment for cancer.

45. 43. The method of claim 41 or 42, wherein the cells are obtained or derived from an allogeneic donor not in need of treatment.

46. 46. ​​A composition comprising a population of immune cells expressing a chimeric antigen receptor (CAR) for binding to a tumor-associated antigen or a tumor-specific antigen, wherein said population of cells is obtained by the method of any one of claims 21 to 45.

47. 47. The composition of claim 46, wherein the composition comprises more than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of cells expressing a chimeric antigen receptor (CAR) for binding to a tumor-associated or tumor-specific antigen.

48. 48. The composition of claim 46 or 47, wherein the composition is for use in treating cancer in a subject in need thereof.

49. 48. A method of treating cancer in a subject in need thereof, comprising administering to said subject a composition of claim 46 or 47.

50. 48. Use of the composition of claim 46 or 47 in the manufacture of a medicament for treating cancer in a subject.

51. A kit for use in the method of any one of claims 21 to 45, comprising: a fusion protein according to any one of claims 1 to 17, a heterodimer according to claim 18 or a polypeptide comprising an epitope of a tumor-associated or tumor-specific antigen, and a moiety enabling capture of said polypeptide, - optionally one or more reagents allowing the isolation of said fusion protein and its complexes.

52. A fusion protein according to any one of claims 1 to 17, or a heterodimer according to claim 18, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 145 to 158, or 160 and 161.

53. The method of any one of claims 21 to 29, wherein the polypeptide comprises the amino acid sequence of the fusion protein of any one of claims 1 to 17.

54. 46. ​​The method of any one of claims 21 to 45, wherein the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 145 to 158, or 160, or 161.

55. The method of any one of claims 21 to 45, wherein the polypeptide comprises an asymmetric heterodimer according to claim 18.

56. 56. The method of claim 55, wherein the asymmetric heterodimer comprises a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 157, 157, 160, or 161.

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