Enrichment of engineered immune cells

Abstract

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

Description

TECHNICAL FIELD

[0001] The present invention relates to methods for enriching populations of immune cells, and to compositions and molecules for performing such enrichment.

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of Australian Provisional Applications AU 2022902655 and AU 2023901949, the entire contents of which are incorporated herein by reference. BACKGROUND OF THE INVENTION

[0004] Cell therapies for treating cancer and other disease states are a rapidly evolving field. The development of immune effector cells such as T cells expressing chimeric antigen receptors (CARs) has revolutionized adoptive cell therapy.

[0005] The potential of this approach has been demonstrated in clinical trials in which CAR T cells have been infused into adult and pediatric patients with B cell malignancies, neuroblastoma, and sarcoma. To date, there have been over 500 clinical trials globally that have been designed to test the efficacy of CAR T cells targeting 64 different tumor-associated antigens. Among 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 where the CAR is directed against CD19, CD22, or B cell maturation antigen (BCMA).

[0006] Human T lymphocytes engineered to express a CAR are expanded in vitro culture and then infused into a patient, where they exert robust cytotoxicity following tumor antigen recognition and subsequent activation. Various factors in the manufacture and administration of these cells contribute to the in vivo persistence and durable anti-tumor effects of CAR T cells.

[0007] To prepare CAR T cells for infusion into a patient, the T cell population first needs to be genetically modified to express the relevant CAR. This results in a mixed T cell population, some of which express the CAR and some of which do not. To maximize the efficacy of CART therapy, a T cell population enriched for cells expressing the CAR is required.

[0008] A key problem with CAR-engineered T cell therapies (and cell therapies in general) is the low transduction efficiency of T cells with the CAR expression construct. For example, peripheral blood T cells, which are often the target of CAR gene therapy, typically have a transduction efficiency of less than 50% (usually 10% to 20%). Therefore, generating sufficient numbers of cells for treatment requires increasing the scale of patient cell collection as well as more extensive ex vivo T cell selection and expansion. This results in a high manufacturing cost for CAR T cell therapy.

[0009] Accordingly, there is a need for improved and alternative methods for preparing immune cell populations prior to cell therapy.

[0010] Any reference to prior art in this specification is not an admission or implication that such prior art constitutes a part of the common general knowledge in any jurisdiction or that such prior art can reasonably be expected to be understood, regarded as relevant, and / or combined with other prior art by a person skilled in the art. SUMMARY OF THE INVENTION

[0011] The present invention is particularly applicable to the preparation of genetically modified immune cell populations that are engineered to bind to a dysfunctional P2X7 receptor on cancer cells. Thus, in a first aspect, there is provided a fusion protein comprising:

[0012] (i) a dysfunctional P2X7 receptor epitope moiety; and

[0013] (ii) the Fc region of an antibody.

[0014] The dysfunctional P2X7 receptor epitope moiety may comprise a peptide that contains any amino acid sequence derived from the dysfunctional P2X7 receptor, but preferably comprises the sequence of an epitope found on the dysfunctional P2X7 receptor but not on the functional P2X7 receptor.

[0015] In a preferred embodiment, the amino acid sequence of the dysfunctional P2X7 receptor epitope moiety comprises or consists of at least the amino acid sequence shown in SEQ ID NO: 14. In a particularly preferred embodiment, the moiety comprises at least the sequence shown in SEQ ID NO: 7 or 9.

[0016] In any embodiment, the dysfunctional P2X7 receptor epitope portion comprises an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 69 or 122, or a sequence that is 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 to the amino acid sequence shown in any one of SEQ ID NOs: 7 to 69 or 122, provided that the sequence comprises at least the sequence shown in SEQ ID NO: 14 or 7 or 9.

[0017] The present invention provides a fusion protein comprising:

[0018] (i) a peptide; and

[0019] (ii) the Fc region of an antibody,

[0020] wherein 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 of any one of SEQ ID NOs: 7 to 69 or 122, or a sequence that is 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 to the amino acid sequence shown in any one of SEQ ID NOs: 7 to 69 or 122, provided that the sequence comprises at least the sequence shown in SEQ ID NO: 14 or 7 or 9.

[0021] In any embodiment, the Fc region of the antibody is the Fc region of IgG, IgA, IgD, IgE or IgM. Preferably, the Fc region is from an IgG antibody, such as an IgG1, IgG2, IgG2b, IgG3 or IgG4 antibody.

[0022] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 domain and the CH3 domain of the heavy chain.

[0023] In a preferred embodiment, compared to a naturally occurring Fc sequence, the Fc region of the fusion protein comprises one or more amino acid substitutions 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 the formation of disulfide bonds between Fc molecules. The cysteine residues of the Fc region can be substituted with any other amino acid residue, optionally glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

[0024] The cysteine residues to be substituted are preferably one or more of the cysteine residues located in the region of the Fc region corresponding to the hinge region of the immunoglobulin. Examples of IgG1 hinge regions and their variants, including cysteine to serine substitutions, are provided in Table 3 herein. The hinge region of an immunoglobulin (such as IgG1) contains three cysteine residues (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, preferably both C226 and C229 are substituted.

[0025] Thus, in a preferred embodiment, the fusion protein comprises a hinge region for linking a peptide as described herein (such as an epitope portion of a dysfunctional P2X7 receptor) to the Fc region of an antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences shown in SEQ ID NOs: 76 to 113, or 136 to 137, or 141 or 142.

[0026] In addition, the Fc region preferably comprises one or more amino acid substitutions for reducing the affinity for Fc receptors (FcRs, including any of FcγRI, FcγRII, and FcγRIII) and thereby reducing the ability of the fusion protein to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Such substitutions are well known in the art and include, but are not limited to, the "DANA" and "LALA" amino acid substitutions and their variants further defined herein. In a further embodiment, the Fc region can also comprise substitutions that eliminate the recruitment of complement C1q. Such mutations are also well known in the art and are further described herein. In addition, the Fc region can comprise substitutions for reducing the serum half-life (by attenuating or reducing the ability to bind to the FcRN receptor). The relevant amino acid substitutions for altering effector function, serum half-life, and aggregation are well known to those skilled in the art and are further described herein, including as exemplified in Table 1.

[0027] The present invention also provides a heterodimeric asymmetric molecule, which comprises a fusion protein as described herein (preferably comprising the peptide of SEQ ID NO: 7 or 14, or a variant thereof, as exemplified by any one of SEQ ID NOs: 2 to 69), and the Fc region of an antibody, and further comprises the Fc region of an antibody that does not contain the peptide. Such asymmetric heterodimeric molecules can be obtained using the knob-in-hole technology as further described herein to facilitate the dimerization of non-identical Fc regions.

[0028] The present invention provides a heterodimeric asymmetric molecule or a monomeric fusion protein for use in accordance with any method further described herein and applied to a method of enriching immune cells, which immune cells comprise an exogenous cell surface receptor that binds to a tumor-specific or tumor-associated antigen and comprises an intracellular signaling domain (such as a chimeric antigen receptor, including a chimeric antigen receptor expressed on a T cell). It will be understood that such a molecule or fusion protein comprises a single amino acid sequence that can be bound by the exogenous immune cell surface receptor to minimize the likelihood of cross-linking multiple immune cells by the molecule / fusion protein and thereby causing its unwanted activation.

[0029] More specifically, a monomeric fusion protein is provided, which comprises: i) an amino acid sequence recognized by or capable of being bound by the antigen recognition domain of an exogenous cell surface receptor, the exogenous cell surface receptor including an intracellular signaling domain (such as a chimeric antigen receptor); and ii) the Fc region of an antibody. Preferably, the amino acid sequence of the Fc region of the antibody cannot form a homodimer with another Fc region of the antibody.

[0030] In addition, a heterodimeric asymmetric molecule is provided, which comprises: i) an amino acid sequence recognized by or capable of being bound by the antigen recognition domain of an exogenous cell surface receptor, the exogenous cell surface receptor comprising an intracellular signaling domain (such as a chimeric antigen receptor, including a chimeric antigen receptor expressed on a T cell), wherein the amino acid sequence is linked to a first Fc region of an antibody; ii) a second Fc region of an antibody capable of forming a heterodimer with the first Fc region.

[0031] In a preferred embodiment, the affinity of the fusion protein for FcR is less than about 250 nM, preferably less than 500 nM, less than 1000 nM, and most preferably less than 2000 nM.

[0032] In any embodiment, the fusion protein may comprise the amino acid sequence of the fusion protein as shown in any one of SEQ ID NOs: 145 to 158, 160 and 161.

[0033] Preferably, the fusion protein or heterodimeric asymmetric molecule consists of or consists essentially of a 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 the antibody (i.e., such that the fusion protein does not contain VH, VL, Fab, Fv, or scFv derived from an antibody).

[0034] In a further embodiment, the fusion protein of the invention can comprise one or more modifications that allow for the capture of the fusion protein, including one or more modifications that allow for the capture of the fusion protein when the protein binds to an immune cell expressing a receptor that comprises an antigen-binding domain for binding a dysfunctional P2X7 receptor (such as a chimeric antigen receptor (CAR) or a modified TCR).

[0035] It will be understood that typically, the antigen-binding domain of the receptor will be an antigen-binding domain that is capable of binding or recognizing the same epitope of the dysfunctional P2X7 receptor contained in the fusion protein. Additionally, those skilled in the art of a given anti-dysfunctional P2X7 receptor CAR, knowing the specific epitope and binding target, will be able to design a suitable fusion protein that binds to the CAR according to the invention.

[0036] One or more modifications of the fusion protein can be selected from: a biotin moiety, fluorescein (FITC), peptide tags (such as His, Myc, Flag, and related tags), and magnetic labels. Preferably, the modification is a biotin moiety or a magnetic moiety.

[0037] The magnetic moiety can be any commercially available magnetic moiety for separating or capturing proteins or cells. For example, the magnetic moiety can include iron oxide microbeads (up to 50 nm in diameter) or large beads (1 - 5 μm in diameter). Preferably, the magnetic moiety includes microbeads.

[0038] In the case of a biotin or magnetic moiety, any method known to those skilled in the art can be used to conjugate these moieties to the fusion protein. In one instance, 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.

[0039] In the case of a peptide tag, the nucleic acid sequence encoding the tag can be included in the nucleic acid construct encoding the fusion protein such that when expressed, the fusion protein is expressed together with the tag already attached to the protein. Typically, the tag will be located at the N-terminus or C-terminus of the fusion protein. Preferably, the tag will be located on the fusion protein so as not to interfere with the binding of the receptor on the immune cell to the peptide (such as the dysfunctional P2X7 receptor epitope portion), the immune cell comprising an antigen-binding domain that binds to the peptide.

[0040] In a second aspect, the present invention also provides a method for obtaining a population of immune cells that is 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 that binds to a tumor-associated antigen or tumor-specific antigen on a cancer cell, but it will be understood that the exogenous cell surface receptor can be a receptor for a "universal CAR" system.

[0041] In a preferred embodiment of the second aspect, there is provided a method for obtaining a population of immune cells or for enriching a population of cells expressing a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on a cancer cell, the method comprising:

[0042] (i) providing a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on a cancer cell;

[0043] (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 that permits capture of the polypeptide;

[0044] thereby forming a complex of the polypeptide and the cells;

[0045] (iii) separating the complex from the population of cells,

[0046] thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on a cancer cell.

[0047] In addition, there is provided a method for obtaining a population of immune cells or for enriching a population of cells expressing a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on a cancer cell, the method comprising:

[0048] (i) providing a mixed population of immune cells, preferably a mixed population of immune effector cells, wherein a subpopulation of the cells expresses a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on a cancer cell;

[0049] (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 wherein the polypeptide comprises a moiety that permits capture of the polypeptide;

[0050] Thereby forming a complex of polypeptide and cells;

[0051] (iii) Separating the complex from the cell population,

[0052] Thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells.

[0053] Preferably, the cells are immune cells expressing a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells. Accordingly, there is also provided a method for obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells, the method comprising:

[0054] (i) Providing a population of immune cells, preferably a population of immune effector cells, wherein the population comprises cells that have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells; or wherein the cells express a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells;

[0055] (ii) Contacting the cell population with a polypeptide, wherein the polypeptide comprises an epitope recognized by the CAR, and wherein the polypeptide comprises a moiety that permits capture of the polypeptide;

[0056] Thereby forming a complex of polypeptide and cells;

[0057] (iii) Separating the complex from the population,

[0058] Thereby obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells.

[0059] Furthermore, according to a second aspect, there is provided a method for enriching a population of immune cells that express a chimeric antigen receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells, the method comprising:

[0060] (i) Providing a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells;

[0061] (ii) Contacting the cell population with a polypeptide, wherein the polypeptide comprises an epitope recognized by the CAR, and wherein the polypeptide comprises a moiety that permits capture of the polypeptide,

[0062] Thereby forming a complex of polypeptide and cells;

[0063] (iii) Separating the complex of cells from the mixed population,

[0064] thereby enriching a population of immune cells that express a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen on cancer cells.

[0065] Examples of tumor-associated antigens or tumor-specific antigens that are typically targeted by cellular immunotherapies 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.

[0066] Generating a polypeptide (or nucleic acid encoding the polypeptide, as appropriate) that contains an epitope recognized by a CAR (or other receptor that binds to a tumor antigen) is well within the capabilities of a person skilled in the art. For example, upon knowledge of the amino acid sequence recognized or bound by a given CAR, the person skilled in the art will be able to design a fusion protein (preferably a monomeric Fc fusion protein or an asymmetric heterodimeric Fc fusion protein as described herein) for binding to the CAR and for use in the method. For example, in the context of a CAR that binds to CD19, the polypeptide will contain a similar epitope of the CD19 molecule that is recognized by the receptor, fused to the Fc region of an antibody and preferably having an Fc region as defined in any one of SEQ ID NO:159 or 162 herein, or an Fc region that cannot form a homodimer.

[0067] In a preferred embodiment of the second aspect, the invention also provides the use of a fusion protein according to the first aspect or a polypeptide as further described herein for obtaining a population of immune cells that is enriched for cells that express a receptor comprising an antigen-binding domain that binds to a 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).

[0068] In a preferred embodiment of the second aspect, there is provided a method for obtaining a population of immune cells or for enriching a population of cells that express a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor, the method comprising:

[0069] (i) Providing a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid that encodes a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor;

[0070] (ii) contacting the cell population with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the antigen-binding domain of a receptor, and wherein the polypeptide comprises a moiety allowing the capture of the polypeptide;

[0071] thereby forming a complex of the polypeptide and the cells;

[0072] (iii) separating the complex from the cell population,

[0073] thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to the dysfunctional P2X7 receptor.

[0074] In addition, provided is a method for obtaining a population of immune cells or for enriching a population of cells expressing a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor, the method comprising:

[0075] (i) providing a mixed population of immune cells, preferably a population of immune effector cells, wherein a subset of the cells expresses a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor;

[0076] (ii) contacting the mixed cell population with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the antigen-binding domain of a receptor, and wherein the polypeptide comprises a moiety allowing the capture of the polypeptide;

[0077] thereby forming a complex of the polypeptide and the cells;

[0078] (iii) separating the complex from the cell population,

[0079] thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to the dysfunctional P2X7 receptor.

[0080] Preferably, the cell is an immune cell expressing a chimeric antigen receptor (CAR) that binds to a linear epitope of the P2X7 receptor, such as an epitope of a dysfunctional P2X7 receptor, wherein the epitope comprises or consists of the amino acid sequence of SEQ ID NO:7 (preferably the amino acid sequence of SEQ ID NO:14), or comprises or consists of any one of the amino acid sequences of SEQ ID NO:7 to 69 or 122, or a sequence that is 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 to any one of the amino acid sequences of SEQ ID NO:7 to 69 or 122, provided that the sequence comprises at least the sequence shown in SEQ ID NO:14 or 7 or 9.

[0081] Accordingly, there is also provided a method for obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a linear epitope of the P2X7 receptor, such as an epitope of a dysfunctional P2X7 receptor, the method comprising:

[0082] (i) providing a population of immune cells, preferably a population of immune effector cells, wherein the population comprises cells that have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a linear epitope of the P2X7 receptor, such as an epitope of a dysfunctional P2X7 receptor; or wherein the cells express a chimeric antigen receptor (CAR) that binds to a linear epitope of the P2X7 receptor, such as an epitope of a dysfunctional P2X7 receptor;

[0083] (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises an epitope of a linear epitope of the P2X7 receptor (such as an epitope of a dysfunctional P2X7 receptor) recognized by the CAR, and wherein the polypeptide comprises a moiety that allows capture of the polypeptide;

[0084] thereby forming a complex of the polypeptide and the cells;

[0085] (iii) separating the complex from the population,

[0086] thereby obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a linear epitope of the P2X7 receptor, such as an epitope of a dysfunctional P2X7 receptor.

[0087] In addition, 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 that binds to a dysfunctional P2X7 receptor, the method comprising:

[0088] (i) Provide a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor;

[0089] (ii) Contact the population of cells with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the CAR, and wherein the polypeptide comprises a moiety that allows capture of the polypeptide,

[0090] thereby forming a complex of the polypeptide and the cells;

[0091] (iii) Separate the complex of cells that bind to the fusion protein expressing the chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor from a mixed population,

[0092] thereby enriching a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor.

[0093] The present invention also finds an application in a method for enriching a population of immune cells for a universal CAR system. Thus, in another embodiment of the second aspect, there is provided a method for obtaining a population of immune cells or for enriching a population of cells expressing an exogenous cell surface receptor comprising an antigen-binding domain that binds 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 one of SEQ ID NOs: 2 to 69 and 122); the method comprises:

[0094] (i) Provide a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain that binds to the peptide;

[0095] (ii) Contact the population of cells with a polypeptide, wherein the polypeptide comprises the peptide recognized by the antigen-binding domain of the receptor and a moiety that allows capture of the polypeptide;

[0096] thereby forming a complex of the polypeptide and the cells;

[0097] (iii) Separate the complex from the population of cells,

[0098] thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds 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 one of SEQ ID NOs: 2 to 69 and 122).

[0099] In addition, a method is provided for obtaining a population of immune cells or for enriching a population of cells that express an exogenous cell surface receptor comprising an antigen-binding domain that binds 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 one of SEQ ID NOs: 2 to 69 and 122); the method comprises:

[0100] (i) providing a mixed population of immune cells, preferably a population of immune effector cells, wherein a subset of the cells expresses a receptor comprising an antigen-binding domain that binds to the peptide;

[0101] (ii) contacting the mixed population of cells with a polypeptide, wherein the polypeptide comprises the peptide recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a moiety that permits capture of the polypeptide;

[0102] thereby forming a complex of the polypeptide and the cells;

[0103] (iii) separating the complex from the population of cells,

[0104] thereby obtaining a population of immune cells that express a receptor having an antigen-binding domain that binds 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 one of SEQ ID NOs: 2 to 69 and 122).

[0105] Preferably, the cells are immune cells that express a chimeric antigen receptor (CAR) that binds to the peptide. Accordingly, the present invention also provides a method for obtaining a population of immune cells that express a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122), the method comprising:

[0106] (i) providing a population of immune cells, preferably a population of immune effector cells, wherein the population comprises cells that have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122); or wherein the cells express a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122),

[0107] (ii) contacting the population of cells with a polypeptide, wherein the polypeptide comprises the peptide, and wherein the polypeptide comprises a moiety that permits capture of the polypeptide;

[0108] Thereby forming a complex of the polypeptide and the cells;

[0109] (iii) Separating the complex from the population,

[0110] Thereby obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122).

[0111] Furthermore, 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 that binds to a peptide, the peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any one of SEQ ID NOs: 2 to 69 and 122), the method comprising:

[0112] (i) Providing a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122),

[0113] (ii) Contacting the population of cells with a polypeptide, wherein the polypeptide comprises the peptide and wherein the polypeptide comprises a moiety that allows capture of the polypeptide,

[0114] Thereby forming a complex of the polypeptide and the cells;

[0115] (iii) Separating the complex of cells expressing a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122) and bound to the polypeptide from the mixed population,

[0116] Thereby enriching a population of immune cells expressing a chimeric antigen receptor (CAR) that binds 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 one of SEQ ID NOs: 2 to 69 and 122).

[0117] In any embodiment of the second aspect of the present invention, the polypeptide comprises the amino acid sequence of the fusion protein of any embodiment of the first aspect of the present invention (such that the polypeptide comprises a peptide as described herein, such as an epitope of a dysfunctional P2X7 receptor linked to the Fc region of an antibody).

[0118] In any embodiment of the second aspect of the present invention, the polypeptide comprises a first part that comprises a peptide as described herein linked to an additional amino acid sequence for promoting the solubility and stability of the first part, such as an epitope of a dysfunctional P2X7 receptor. The additional amino acid sequence linked to the peptide (such as an epitope of a dysfunctional P2X7 receptor) may comprise any suitable linker or hinge region, such as those exemplified in Tables 1 and 3. Such linker or hinge regions 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 the hinge region of an immunoglobulin, such as the sequences defined in Table 3. Optionally, the linker sequence may comprise a cleavable sequence.

[0119] In a further embodiment, the polypeptide may be in the form of a fusion protein that comprises a peptide as described herein (such as an epitope of a dysfunctional P2X7 receptor) linked to an additional amino acid sequence. The additional sequence may include serum albumin, transferrin, the carboxy-terminal peptide of the beta chain of chorionic gonadotropin (CG), a non-exact repeat peptide sequence, a polypeptide sequence consisting of a proline-alanine-serine polymer, elastin-like peptide (ELP) repeats, 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 the dysfunctional P2X7 receptor to the additional amino acid sequence.

[0120] In addition, the polypeptide may be in the form of a conjugate that comprises a carbohydrate, a lipid, a liposome, a peptide, and an aptamer conjugated to an amino acid sequence comprising a peptide (such as an epitope of a dysfunctional P2X7 receptor).

[0121] In any embodiment of the second aspect of the present invention, the fusion protein or polypeptide may comprise: an amino acid sequence as shown in any one of SEQ ID NOs: 145 to 158 or 160 and 161, or a sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 145 to 158 or 160 and 161.

[0122] According to the second aspect of the present invention, the part for allowing the capture of the polypeptide may be any suitable part that can be bound by a capture agent or system, such as a biotin label, fluorescein (FITC), a peptide tag (such as His, Myc, Flag, and related tags), or a magnetic label. Preferably, the modification is a biotin moiety or a magnetic moiety.

[0123] The magnetic moiety can be any commercially available magnetic moiety for separating or capturing proteins or cells. For example, the magnetic moiety can be magnetic microbeads or macrobeads.

[0124] In the case of biotin or a magnetic moiety, any method known to those skilled in the art can be used to conjugate these moieties to the polypeptide. In 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.

[0125] In the case of a peptide tag, the nucleic acid sequence encoding the tag can be included in the nucleic acid construct encoding the polypeptide such that when expressed, the polypeptide is expressed with the tag already linked to the protein. Generally, the tag will be located at the N-terminus or C-terminus of the polypeptide. Preferably, the tag is positioned on the polypeptide so as not to interfere with the binding of a receptor on an immune cell to an epitope moiety of the dysfunctional P2X7 receptor, the immune cell comprising an antigen-binding domain that binds to the epitope moiety.

[0126] Optionally, in the case where the polypeptide is labeled with a biotin moiety, the method can further comprise (after step ii) the step of contacting the cells with an avidin antigen-binding protein, preferably wherein the avidin antigen-binding protein comprises one or more moieties that allow capture of the complex. Optionally, one or more moieties that allow capture of the complex include iron oxide particles (microbeads or macrobeads).

[0127] Optionally, where the polypeptide comprises a magnetic label, the separation step can include i) applying a magnetic field to a cell population; ii) removing or discarding cells not attracted by the magnetic field, and iii) removing the magnetic field, thereby providing a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to the dysfunctional P2X7 receptor.

[0128] Optionally, the method can further comprise the step of expanding the isolated immune cells.

[0129] In any embodiment of the second aspect of the present invention, the method can further comprise treating the complex to release the cells from the binding by the polypeptide. Such methods will be known to those skilled in the art and will depend on the nature of the polypeptide moiety that allows capture. For example, in the case of a biotinylated polypeptide, providing an excess of free biotin to the complex will promote the dissociation of the polypeptide bound by the capture agent (such as avidin beads, etc.).

[0130] In any aspect of the present invention, the fusion protein or polypeptide may comprise a cleavable linker that links an epitope bound by a receptor (such as an epitope of a dysfunctional P2X7 receptor) to the Fc region and / or other sequences that permit 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 reagent to cleave the cleavable linker, thereby releasing the Fc region or other amino acid sequence therefrom.

[0131] Optionally, the method further comprises the step of administering to a subject in need of treatment with immune cells (such as for treating cancer) the isolated or enriched cells. In certain embodiments, depending on the capture agent used, the immune cell / polypeptide complex may be administered directly to the subject.

[0132] In any embodiment of the second aspect, the immune cell population is an effector immune cell population, such as T cells, NK cells or NKT cells. Optionally, the T cells are derived from stem cells, optionally wherein the stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells.

[0133] Preferably, the immune cell population is derived from a subject in need of treatment for cancer. Alternatively, the immune cells may be obtained from an allogeneic donor who does not require treatment.

[0134] In a particularly preferred embodiment of the second aspect of the present invention, the exogenous cell surface receptor comprising an antigen-binding domain (such as a chimeric antigen receptor, CAR) comprises an antigen-binding domain comprising the CDR amino acid sequences of PEP2-2-1 as described in any one of PCT / AU2010 / 001070 (WO2011020155 or the corresponding US Patent Nos. US 9,127,059, US 9,688,771 or US10,053,508). More preferably, the antigen-binding domain of the receptor (such as a CAR) comprises or consists of the amino acid sequence of the PEP2-2-1 antigen-binding protein as described in any one of PCT / AU2010 / 001070 (WO2011020155 or the corresponding US Patent Nos. US 9,127,059, US 9,688,771 or US10,053,508) incorporated herein by reference.

[0135] The present invention also provides a composition comprising a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor, wherein the population of cells is obtained by the methods described herein. Preferably, the composition comprises greater than 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% cells expressing a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or tumor-specific antigen (such as a dysfunctional P2X7 receptor).

[0136] In a further embodiment, there is provided a kit for use in the methods described herein, the kit comprising:

[0137] - a fusion protein capable of being bound by a CAR that binds to a tumor-associated antigen or tumor-specific antigen (such as a dysfunctional P2X7 receptor);

[0138] - optionally, one or more reagents that allow for the isolation of the fusion protein and its complexes.

[0139] Optionally, the kit comprises written instructions for use in the methods of the second aspect of the present invention.

[0140] As used herein, unless the context requires otherwise, the term "comprising" and variations thereof such as "comprises" and "comprising" are not intended to exclude additional additives, components, integers or steps.

[0141] Other aspects of the invention and other embodiments of the aspects described in the preceding paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings.

[0142] Sequence information

[0143] Table 1: Exemplary sequences of dysfunctional P2X7 receptor and receptor epitope moieties

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] BRIEF DESCRIPTION OF THE DRAWINGS

[0154] Figure 1 : Enrichment of stabilized Jurkat cells transduced with nucleic acids encoding chimeric antigen receptors (CARs) that bind to dysfunctional P2X7 receptors. A. Shows the proportion of transduced cells enriched with a biotinylated protein using an amino acid sequence containing the E200 epitope. B. Shows that the cells maintain a high purity level 48 hours after enrichment and that the cells are not negatively affected by the enrichment process. The grey shading indicates before enrichment; the red shading indicates after enrichment.

[0155] Figure 2 : Enrichment of primary donor cells (donor 12) transduced with nucleic acids encoding CARs that bind to dysfunctional P2X7 receptors. The grey shading indicates before enrichment; the red shading indicates after enrichment.

[0156] Figure 3 : Enrichment of primary donor cells (donor 12) transduced with nucleic acids encoding alternative CARs that bind to dysfunctional P2X7 receptors. The grey shading indicates before enrichment; the red shading indicates after enrichment. Enrichment was performed using a MACS column (Miltenyi Bioscience).

[0157] Figure 4 : Enrichment of primary donor cells (donor 12) transduced with nucleic acids encoding alternative CARs that bind to dysfunctional P2X7 receptors. The grey shading indicates before enrichment; the red shading indicates after enrichment. Enrichment was performed using a magnet stand and stem cell technology.

[0158] Figure 5 : Direct staining of EGFR in CAR-expressing cells after enrichment using monomeric or dimeric Fc-attenuated fusion proteins containing the nfP2X7 receptor epitope moiety for binding by the 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).

[0159] Figure 6: Viability and purity of CAR-expressing cells using monomeric Fc fusions containing nfP2X7 receptor epitope moieties. A. Dye 7AAD staining was used to determine the viability of enriched cells. B. Indirect CAR detection using tEGFR staining (Cetuximab, an AF647 primary-labeled anti-EGFR monoclonal antibody measured in the APC channel). C. Direct staining of nfP2X7 CAR using biotinylated DetR2 (SEQ ID NO:146), followed by staining with antibiotin antibody Vioblue (130-113-857 biotin antibody, Miltenyi Biotech).

[0160] Figure 7 : Ratio of CD25+ / CD69+ to PD-L1+ cells at 24 hours (A), 48 hours (B), and 72 hours (C) after enrichment using monomeric or dimeric fusion proteins (having amino acid sequences of SEQ ID NO:145 and 149, respectively).

[0161] Figure 8 : Transduction efficiency (%) on day 2 after MACS sorting using monomeric or dimeric fusion proteins (having amino acid sequences of SEQ ID NO:145 and 149, respectively). D50, D53, and D71 = T cells from donors 50, 53, and 71, respectively.

[0162] Figure 9 : Cell counts (normalized to the maximum expected cell count) after enrichment using monomeric or dimeric fusion proteins (having amino acid sequences of SEQ ID NO:145 and 149, respectively). Shown are cell counts of T cells from healthy donors 50 (D50) and 71 (D71) on day 1 and day 2 after enrichment.

[0163] Figure 10 : Viability of CAR T cells enriched on day 2 after MACS sorting. Shown is the percentage of 7AAD-negative cells. D50, D53, and D71 = T cells from donors 50, 53, and 71, respectively.

[0164] Figure 11 : Ratio of CD25+ / CD69+ to PD-L1+ cells 2 days after MACS enrichment using monomeric or dimeric fusion proteins (having amino acid sequences of SEQ ID NO:145 and 149, respectively). Shown are the results for T cells from healthy donor 50 (D50). Detailed Description

[0165] Reference will now be made in detail to certain embodiments of the present invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the invention as defined by the claims.

[0166] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. The present invention is in no way limited to the methods and materials described.

[0167] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All such different combinations constitute various alternative aspects of the invention.

[0168] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0169] The present invention provides fusion proteins, compositions and kits comprising said fusion proteins, and the use of said fusion proteins, compositions and kits in methods for enriching immune cell populations (preferably cells expressing an exogenous cell surface receptor comprising an antigen-binding domain and an intracellular signaling domain).

[0170] Preferably, the fusion protein comprises: (i) a linear peptide epitope portion derived from the P2X7 receptor (e.g., comprising an amino acid sequence derived from SEQ ID NO: 7 or 14) and recognized or capable of being bound by an antigen recognition structure of a receptor expressed on an immune cell; and (ii) the Fc region of an antibody; and optionally (iii) a moiety that allows capture of the fusion protein. The epitope portion allows the fusion protein to specifically bind to target immune cells for enrichment, and the Fc region (preferably when comprising a capture-permitting moiety) allows capture of the complex of the fusion protein and the immune cell.

[0171] The inventors have demonstrated the use of homodimeric and monomeric proteins derived from the fusion proteins described herein for obtaining or enriching immune cell populations.

[0172] In a particularly preferred embodiment, Fc fusion protein is designed to include only a single copy of a linear epitope derived from P2X7 receptors. As further described herein, this can be achieved by introducing amino acid substitutions into the Fc region to prevent homodimerization, or alternatively, using known knob-and-mortar techniques to ensure the formation of asymmetric heterodimer molecules (e.g., comprising E200 peptide-Fc fusion proteins and not comprising E200 peptide Fc regions). Such monomers or asymmetric heterodimer molecules have the advantages of reducing the activation of target immune cells and preventing the undesirable loss of target immune cells during enrichment process c (as further described herein in the examples). Not wishing to be bound by theory, the inventors believe that this is due to the ability of two different CAR receptors on a cell or two independent CAR expressing cells to reduce molecular cross-linking.

[0173] definition

[0174] Unless defined otherwise, 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.

[0175] For the purposes of interpreting this specification, the following definitions will generally apply and, where appropriate, terms used in the singular will also include the plural and vice versa.

[0176] As used herein, the term "and / or", such as "X and / or Y", will be understood to mean "X and Y" or "X or Y", and should be understood to provide clear support for both meanings or either meaning.

[0177] The articles "a" and "an" used herein refer to one or more than one (i.e., at least one) grammatical object of the article. As an example, "dysfunctional P2X7 receptor epitope portion" means a dysfunctional P2X7 receptor epitope portion or more than one dysfunctional P2X7 receptor epitope portion.

[0178] As used herein, the term "comprise" and variations of the term, such as "comprising", "including" and "comprising", are not intended to exclude additional additives, components, integers or steps, unless the context requires otherwise.

[0179] As used herein, "tumor-associated antigen" refers to an antigen expressed by cancer cells (the term "tumor-antigen" may also be used to refer to the same antigen). Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, hK4 prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, P501S prostate protein, 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.

[0180] In one embodiment, a tumor antigen comprises one or more antigenic cancer epitopes associated with a malignancy. Malignancies express a number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma and prostate 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. Another group of target antigens are carcinoembryonic antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen that is unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for B-cell lymphoma target antigens. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for monoclonal antibody passive immunotherapy with limited success. The types of tumor antigens mentioned in the present invention can also be tumor-specific antigens (TSA). TSA are unique to tumor cells and are not produced on other cells in the body. Tumor-associated antigens (TAA) are not unique to tumor cells; rather, they are also expressed on normal cells under conditions where an immune tolerance state to the antigen cannot be induced. The expression of the antigen on the tumor can occur under conditions that enable the immune system to respond to the antigen. TAA may be antigens that are 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. Those tumor-associated antigens that are of greatest clinical interest are differentially expressed compared to the corresponding non-tumor tissues and allow for preferential recognition of tumor cells by specific T cells or immunoglobulins.

[0181] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-1), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multi-lineage 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 generated by 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, p180erbB3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\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, MOV 18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.Particularly preferred examples of target cell antigens according to the 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 (Connexin 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, in particular EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSACD276 and the dysfunctional (nf) P2X7 receptor.

[0182] A "purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0183] A "P2X7 receptor" generally refers to a purinergic receptor formed by three protein subunits or monomers, at least one of which has an amino acid sequence substantially as shown in SEQ ID NO:1 in Table 1 herein.

[0184] Insofar as the P2X7 receptor is formed by three monomers, it is "trimeric" or "trimer". "P2X7 receptor" encompasses naturally occurring variants of the P2X7 receptor, for example, where the P2X7 monomer is a splice variant, allelic variant, SNP and isotype, including naturally occurring truncated or secreted forms of the monomers forming the P2X7 receptor (e.g., forms consisting of extracellular domain sequences or truncated forms thereof), naturally occurring variant forms (e.g., alternative splicing forms) and naturally occurring allelic variants. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the 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 inserted with residues.

[0185] "Functional P2X7 receptor" generally refers to a form of the P2X7 receptor that has three intact binding sites or clefts for binding ATP. When bound to ATP, the functional receptor forms a non-selective sodium / calcium channel that transforms into a pore-like structure, allowing calcium ions and molecules up to 900 Da to enter the cytoplasm, one of the consequences of which may be the induction of programmed cell death. Under normal steady state conditions, the expression of functional P2X7 receptors is generally limited to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. There may also be some expression of functional P2X7 receptors on red blood cells and other cell types.

[0186] "Dysfunctional P2X7 receptor" (also referred to as "non-functional" or (nf)P2X7) is a P2X7 receptor that is impaired in its response to ATP such that it cannot form an apoptotic pore under physiological conditions. A dysfunctional P2X7 receptor or (nfP2X7 receptor) generally refers to a form of the P2X7 receptor that has a conformation different from that of the functional P2X7, whereby the receptor cannot form an apoptotic pore, but it can still function as a non-selective channel by maintaining a single functional ATP binding site located between adjacent monomers. One example is cis-isomerization of one or more monomers at Pro210 (according to SEQ ID NO:1). The isomerization may be caused by any molecular event that results in misfolding of the monomers, which events include, for example, mutations in the primary sequence of the monomers or abnormal post-translational processing. One consequence of the isomerization is that the receptor cannot bind ATP at one, or more specifically two, ATP binding sites on the trimer, and thus cannot extend the opening of the channel. In this case, the receptor cannot form a pore, which limits the extent of calcium ion entry into the cytoplasm. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial and hematopoietic cancers. As used herein, the term "dysfunctional P2X7 receptor" may be used interchangeably with the terms "non-functional P2X7 receptor" or "nfP2X7 receptor".

[0187] "Cancer-associated P2X7 receptor" generally refers to a P2X7 receptor that is found on cancer cells (including pre-neoplastic, neoplastic, malignant, benign, or metastatic cells), but is absent from non-cancerous or normal cells.

[0188] "E200 epitope" generally refers to an epitope having the sequence GHNYTTRNILPGLNITC (SEQ ID NO:2). Its variants are exemplified in Table 1, including any one of SEQ ID NO:3 or 7 to 69 and 122.

[0189] The "E300 epitope" generally refers to an epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO:4) or a variant thereof, as defined in SEQ ID NO:5.

[0190] A "composite epitope" generally refers to an epitope formed by the juxtaposition of E200 and E300 epitopes or portions of these epitopes. An example of a composite epitope containing E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6).

[0191] As used herein, the term "antigen" is intended to include substances that bind to one or more antibodies or elicit one or more antibodies, and can include, but are not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and is recognizable 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 their multimers, Fab fragments or their multimers, antibodies or their multimers, single-chain antibodies or their multimers, or any other molecule that can bind to a structure with high affinity.

[0192] An "epitope" generally refers to the part of an antigen that binds to the antigen-binding site of an antibody. An epitope can be "linear", i.e., the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to an amino acid sequence 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 present in the tertiary or quaternary protein structure.

[0193] With respect to the terms "bind", "specifically bind" or "specific for" referring to a receptor that recognizes and binds to an antigen-binding domain of a dysfunctional P2X7 receptor, it means that the receptor substantially does not recognize or bind to other antigens in the sample.

[0194] "Binding affinity" generally refers to the total strength 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 specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the methods described herein. Low-affinity antibodies generally bind to antigens slowly and dissociate easily, while high-affinity antibodies generally bind to antigens more quickly and remain bound for a longer time. There are various methods known in the art for measuring binding affinity, and any of them can be used for the purposes of the present invention.

[0195] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system and perform specific effector functions, such as α-β T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-δ T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages or any hematopoietic progenitor cells, such as pluripotent stem cells and early progenitor cell subsets that can mature or differentiate into somatic cells. These cells can be naturally occurring or generated by cytokine exposure, artificially / genetically modified cells (such as iPSCs and other artificial cell types). Preferred immune cells are cells with cytotoxic effector functions, such as α-β T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or γ-δ T cells. "Effector function" refers to a specific function of a cell, for example, in T cells, the effector function can be cytolytic activity or helper cell activity, including the secretion of cytokines.

[0196] As used herein, the term "autologous" refers to any material that is derived from the same subject and subsequently reintroduced into that subject.

[0197] As used herein, the term "allogeneic" refers to any material that is derived from a different subject of the same species as the subject into which the material is reintroduced.

[0198] An "enriched" or "purified" cell population is an increase in the ratio of a specific cell to other cells, for example, compared to the cells found in a subject or compared to the ratio before exposure to a peptide, nucleic acid or vector of the present invention. In some embodiments, in an enriched or purified cell population, the specific cell comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% of the total cell population. The cell population can be defined by one or more cell surface markers and / or characteristics.

[0199] As used herein, the terms "engineered cell" and "genetically modified cell" are used interchangeably. The term means a cell that contains and / or expresses a foreign gene or nucleic acid sequence, which in turn modifies the genotype or phenotype of the cell or its progeny. In particular, the term refers to the fact that cells (preferably immune cells) can be engineered by recombinant methods known in the art to stably or transiently express a peptide or protein that is not expressed in these cells in their native state. For example, immune cells are engineered to express an artificial construct, such as a chimeric antigen receptor, on their cell surface. For example, the CAR sequence can be delivered into the cells using an adenovirus, an adeno-associated virus (AAV)-based, retroviral or lentiviral vector or any other pseudotyped variant thereof, or any other gene delivery mechanism such as electroporation or liposome transfection using 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).

[0200] The amino acid structures, as well as the single-letter and three-letter abbreviations, used throughout the specification are defined in Table 2, which lists the 20 proteinogenic naturally occurring amino acids that exist in the L-isomer form in proteins.

[0201] Table 2

[0202]

[0203] Amino acid Three-letter abbreviation One-letter symbol Side chain structure (R) Alanine Ala A <![CDATA[-CH3]]> Arginine Arg R <![CDATA[-(CH2)3NHC(=N)NH2]]> Asparagine Asn N <![CDATA[-CH2CONH2]]> Aspartic acid Asp D <![CDATA[-CH2CO2H]]> Cysteine Cys C <![CDATA[-CH2SH]]> Glutamine Gln Q <![CDATA[-(CH2)2CONH2]]> Glutamic acid Glu E <![CDATA[-(CH2)2CO2H]]> Glycine Gly G -H Histidine His H <![CDATA[-CH2(4-imidazolyl)]]> Isoleucine Ile I <![CDATA[-CH(CH3)CH2CH3]]> Leucine Leu L <![CDATA[-CH2CH(CH3)2]]> Lysine Lys K <![CDATA[-(CH2)4NH2]]> Methionine Met M <![CDATA[-(CH2)2SCH3]]> Phenylalanine Phe F <![CDATA[-CH2Ph]]> Proline Pro P The structure of the amino acid is shown in the above formula (2). Serine Ser S <![CDATA[-CH2OH]]> Threonine Thr T <![CDATA[-CH(CH3)OH]]> Tryptophan Trp W <![CDATA[-CH2(3-indolyl)]]> Tyrosine Tyr Y <![CDATA[-CH2(4-hydroxyphenyl)]]> Valine Val V <![CDATA[-CH(CH3)2]]>

[0204] As used herein, the term "non-proteinogenic amino acid" refers to an amino acid having a side chain that is not present in the naturally occurring L-α-amino acids described 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-phenylvaleric acid, 6-aminohexanoic acid, tert-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of natural amino acids.

[0205] As used herein, the term "α-amino acid" refers to an amino acid having a single carbon atom (α-carbon atom) that separates the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). α-Amino acids include naturally occurring and non-naturally occurring L-amino acids and their D-isomers and derivatives, such as salts or derivatives in which the functional groups are protected by suitable protecting groups. Unless otherwise specified, the term "amino acid" as used herein refers to α-amino acids.

[0206] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms. Where appropriate, the alkyl group may have a specific number of carbon atoms, such as C 1-6 alkyl, which includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight-chain or branched-chain arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 5-methylpentyl.

[0207] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, dairy cow, pig, goat, chicken, dog, monkey, or human. Preferably, the subject is a human. The subject can be a subject (patient) suffering from a disease such as cancer. As used herein, the terms "subject" and "individual" can be used interchangeably.

[0208] Receptor epitope moiety (dysfunctional P2X7 receptor epitope moiety)

[0209] The present invention relates to methods of using polypeptides comprising epitopes recognized by the antigen-binding domains of receptors (such as CAR or TCR) on immune cells. The receptors are typically used to bind to tumor-associated antigens or tumor-specific antigens on cancer cells, and thus, the epitope moieties of the polypeptides used according to the present invention comprise sequences derived from the tumor-associated antigens or tumor-specific antigens that bind to the receptors.

[0210] In certain instances, the immune cells comprise a receptor for binding the extracellular domain of CD19 to a target cell. It will be understood that in these instances, the polypeptides used according to the second aspect of the present invention will comprise the same epitopes as the ECD of CD19 that binds to the receptor. Cellular immunotherapeutic agents having a receptor that targets CD19 are known to those skilled in the art, and the epitopes bound by such immunotherapeutic agents are also known. For example, in order to enrich CAR-T cells comprising a CAR having an antigen recognition domain consisting of anti-CD19 scFv FMC683, those skilled in the art will understand that the polypeptides used according to the second aspect of the present invention should comprise the epitopes bound by scFv FMC683. Similarly, in order to obtain or enrich CAR-T cells comprising a CAR having an antigen recognition domain consisting of anti-CD19 scFv A3B1, those skilled in the art will understand that the polypeptides used according to the second aspect of the present invention should comprise the epitopes bound by scFv A3B1. The epitopes bound by the anti-CD19 antibodies FMC683, 3B10, and 4G7-2E3 used in various anti-CD19 cellular immunotherapies are described in the following references, which are incorporated herein by reference: Klesmith et al., (2019) Biochemical, 58:489-4881.

[0211] In similar instances, the immune cells can include receptors for binding CD20 on target cells. It will be understood that in such instances, the polypeptide used according to the second aspect of the present invention will comprise the same epitope as the CD20 bound by the receptor.

[0212] In other instances, the immune cells can comprise a receptor for binding mesothelin (e.g., a CAR), and thus the polypeptide comprises an epitope of mesothelin.

[0213] In other instances, the immune cells can comprise a receptor for binding EGFR (e.g., a CAR), and thus the polypeptide comprises an epitope of EGFR.

[0214] In other instances, the immune cells can comprise a receptor for binding GPC3 (e.g., a CAR), and thus the polypeptide comprises an epitope of GPC3.

[0215] In other instances, the immune cells can comprise a receptor for binding MUC1, and thus the polypeptide comprises an epitope of MUC1.

[0216] In other instances, the immune cells comprise a receptor for binding HER2, and thus the polypeptide comprises an epitope of HER2.

[0217] In other instances, the immune cells comprise a receptor for binding GD2, and thus the polypeptide comprises an epitope of GD2.

[0218] In other instances, the immune cells comprise a receptor for binding CEA, and thus the polypeptide comprises an epitope of CEA.

[0219] In other instances, the immune cells comprise a receptor for binding EpCAM, and thus the polypeptide comprises an epitope of EpCAM.

[0220] In other instances, the immune cells comprise a receptor for binding LeY, and thus the polypeptide comprises an epitope of LeY.

[0221] In other instances, the immune cells comprise a receptor for binding PSCA, and thus the polypeptide comprises an epitope of PCSA.

[0222] In other instances, the immune cells comprise a receptor for binding CD276, and thus the polypeptide comprises an epitope of CD276.

[0223] The present invention also provides a fusion protein comprising an epitope portion of a dysfunctional P2X7 receptor.

[0224] The dysfunctional P2X7 receptor epitope moiety can be provided in the form of a dysfunctional P2X7 receptor or a fragment of a dysfunctional P2X7 receptor that has at least one of three ATP binding sites formed at the interface between adjacent properly packed monomers that cannot bind ATP. Such receptors cannot extend the opening of non-selective calcium channels to apoptotic pores.

[0225] According to the present invention, the dysfunctional P2X7 receptor epitope moiety is typically in the form of a peptide fragment of a dysfunctional P2X7 receptor. Generally, the peptide contains an epitope that is not found or not available for binding on a functional P2X7 receptor.

[0226] In some embodiments, the peptide contains a proline at amino acid position 210 of the dysfunctional P2X7 receptor. In some embodiments, the peptide contains one or more amino acid residues that span from glycine at amino acid position 200 to cysteine at amino acid position 216 (including the endpoints) of the dysfunctional P2X7 receptor.

[0227] In PCT / AU2002 / 000061 (and corresponding publications WO 2002 / 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 WO2009 / 033233 and US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,45) and PCT / AU2009 / 000869 (and corresponding publications WO 2010 / 000041 and US 8,597,643, US 9,328,155 or US10,238,716), which are incorporated herein by reference in their entirety, a series of peptide fragments of the dysfunctional P2X7 receptor are known and discussed. Exemplary peptides containing epitopes intended for use in the present invention from these specifications are described below.

[0228] PCT Publication Peptide Sequence

[0229] WO 2002 / 057306 GHNYTTRNILPGLNIT(SEQ ID NO:3)

[0230] WO 2002 / 057306 GHNYTTRNILPGLNITC(SEQ ID NO:2) (also referred to herein as the "E200" epitope)

[0231] WO 2009 / 033233 KYYKENNVEKRTLIKVF (SEQ ID NO:4) (also referred to herein as the "E300" epitope)

[0232] WO 2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6) (also referred to herein as the "E200 / E300" or "composite" epitope)

[0233] Non-limiting examples of variants of the E200 peptide sequence are provided in Table 1 (including those with N-terminal and / or C-terminal extensions, as well as various linkers, hinges or spacers).

[0234] The amino acid sequence of any one of SEQ ID NOs: 2 to 69 or 155 may comprise a part of an epitope moiety that is recognized by or capable of binding to a receptor expressed on an immune cell (also referred to herein as the "recognition sequence" of the epitope moiety).

[0235] In some embodiments, the epitope moiety comprises or consists of an amino acid sequence selected from any of the peptide sequences listed in Table 1 above.

[0236] In some embodiments, the N-terminus of the epitope moiety is a free amine (-NH2).

[0237] 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 analogue of the free acid group, such as an ester (-COOC1-6 alkyl) or a primary or secondary amide (-CONHR4, where R4 is selected from H and C1-6 alkyl). Advantageously, compared with the free acid, a derivative or analogue of the free acid group at the C-terminus can improve the biological stability of the peptide. In some embodiments, the C-terminus is a derivative or analogue of the free acid group that contains a functional moiety, such as biotin.

[0238] In any embodiment of the first or second aspect, the epitope that dysfunctions the P2X7 receptor comprises or consists of an epitope that is found only on the dysfunctional P2X7 receptor but not on the functional form of the P2X7 receptor. In other words, preferably, the polypeptide comprises or consists of an epitope that is specific to the dysfunctional P2X7 receptor.

[0239] In a further embodiment of the first or second aspect, the fusion protein comprises an epitope corresponding to the E200, E300 or composite E200 / E300 epitope as defined herein. Obtaining the various polypeptides for use according to the invention will be within the capabilities of the person skilled in the art. For example, the person skilled in the art will understand that additional amino acids may be included at the N-terminus or C-terminus of the region containing the epitope bound by the anti-nfP2X7 receptor CAR. In a non-limiting example, and in the context of E200, E200 is generally defined as having an amino acid sequence substantially as defined in SEQ ID NO: 2 or 7 (and having a minimum sequence as defined in SEQ ID NO: 14), and other amino acids from the native sequence of the P2X7 receptor may be included in the polypeptide, such as the residues "DFP" N-terminal to the epitope in the P2X7 receptor sequence and / or the residues "TFHKT" C-terminal to the epitope in the P2X7 receptor sequence. In any embodiment, in addition to the sequence of E200 or E300 or the composite epitope, 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.

[0240] In a preferred embodiment, the sequence of the E200 epitope is further modified to replace the cysteine residue (residue 17 in SEQ ID NO: 2) with a serine residue (such as providing the sequence of SEQ ID NO: 7). The person skilled in the art will understand that doing so can reduce the likelihood of any disulfide bond binding between the polypeptide and another molecule.

[0241] Including additional amino acid residues in the E200, E300 or composite epitope (or the extended epitope as discussed in the above paragraphs) is also within the capabilities of the person skilled in the art, for example by adding at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 additional amino acid residues to the N-terminal and C-terminal regions of the peptide consisting of the amino acid sequence of the relevant epitope. Generally, such additional amino acids may be derived from a linker sequence (such as a peptide containing glycine and serine residues); or from the hinge region of an immunoglobulin. Generally, no more than 30, no more than 25 or no more than 20 amino acid residues are added to the N-terminal and / or C-terminal residues of the E200, E300 or composite epitope as defined herein. Examples of such extended E200 epitope peptides are provided in Table 1.

[0242] Fc region

[0243] In any embodiment, the amino acid sequence of the epitope of the dysfunctional P2X7 receptor can be fused to the N-terminal region of the Fc region of an antibody or a variant thereof via its C-terminal region. In any embodiment, the amino acid sequence of the epitope of the dysfunctional P2X7 receptor can be fused to the C-terminal region of the Fc region of an antibody or a variant thereof via its N-terminal region.

[0244] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 domain and the CH3 domain of the heavy chain.

[0245] Compared to the naturally occurring Fc sequence, the Fc region may comprise one or more amino acid sequence modifications. The Fc region may comprise one or more amino acid substitutions, such as substitutions of one or more cysteine residues, to prevent the molecule from dimerizing into identical molecules. It will be understood that any amino acid substitution that prevents dimerization of the Fc region can be used. Thus, in vivo, the Fc fusion proteins described herein can be monomeric proteins. Preferably, the Fc region and hinge region derived from an immunoglobulin comprise substitutions of at least one, at least two or at least three cysteine residues. Preferably, the residues substituted are at least C220, C226 and C229. In a preferred embodiment, the monomeric protein comprises substitutions at all three positions of C220, C226 and C229 (according to EU system numbering).

[0246] Thus, compared to the naturally occurring Fc sequence, the Fc region of the fusion protein may comprise one or more amino acid substitutions 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 the formation of disulfide bonds between Fc molecules. The cysteine residues in the Fc region can be substituted with any other amino acid residue, optionally glycine, serine, alanine, lysine and glutamate, preferably glycine or serine.

[0247] The cysteine residues to be substituted are preferably one or more cysteine residues located in the region of the Fc region corresponding to the hinge region of the immunoglobulin. Examples of IgG1 hinge regions and their variants, including cysteine to serine substitutions, are provided in Table 3 herein. The hinge region of an immunoglobulin (such as IgG1) comprises three cysteine residues (C220, C226 and C229 according to EU numbering). Thus, in any embodiment, at least one, at least two or all three cysteine residues in the immunoglobulin hinge region are substituted. Preferably, at least two or all three 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, preferably both C226 and C229 are substituted.

[0248] Thus, in a preferred embodiment, the fusion protein comprises a hinge region for linking the epitope portion of the dysfunctional P2X7 receptor to the 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.

[0249] In a further embodiment, the fusion protein region may comprise an Fc region corresponding to the Fc “mortar” or “pestle” for a “mortar and pestle” heterodimer. The use of such Fc sequences is known in the art and provides an asymmetric heterodimeric molecule comprising a fusion protein with a single copy of the epitope portion described herein and an Fc region that binds to another Fc region that does not contain the epitope portion.

[0250] Those skilled in the art will be familiar with the techniques and Fc sequences that allow for the formation of so-called monomeric fusion proteins, including but not limited to the use of the “mortar and pestle” IgG1 form (Ridgway et al., (1996), Protein Eng, 9:617 - 621). In the context of the present invention, this approach enables the expression and purification of a heterodimeric fusion protein having only one copy of the peptide epitope per molecule (e.g., an epitope portion derived from the E200 epitope described herein). Examples of “mortar and pestle” Fc pairings are provided herein as SEQ ID NOs: 157 and 159 (pestle and mortar, respectively), 158 and 159, 160 and 162 (pestle and mortar, respectively), and 161 and 162. Thus, in any embodiment, the present invention provides a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 2 to 69 and 122 linked to an Fc region as defined by SEQ ID NO: 160 or 162, wherein the fusion protein is capable of forming a heterodimer with an Fc region that does not contain the E200 peptide portion.

[0251] Thus, the fusion protein of the present invention is preferably a fusion protein capable of forming a heterodimeric molecule comprising a single E200-containing amino acid sequence. (In other words, the Fc portion of the fusion protein can form a heterodimer with the Fc region of an antibody that does not contain the E200 peptide fused thereto).

[0252] In a further embodiment, the Fc region may include one or more substitutions for eliminating or reducing effector functions, such as reduced binding and activation via FcR, as further described below.

[0253] The term “Fc region” herein is used 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 comprises two heavy chain segments that contain the C H 2 domain and the C H3 domains. In the context of the present invention, the Fc region comprises two heavy chain fragments, preferably the CH2 domain and the CH3 domain of the heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the C H 3 domains. The heavy chain constant regions corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0254] In some aspects, the fusion protein does not have any effector function or any detectable effector function. "Effector function" or "effector activity" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains the ability to bind FcRn. The main cells mediating ADCC - NK cells - express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of the following reference: Ravetch and Kinet, Annu. Rev. Immunol. 9:457 - 492 (1991).

[0255] Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example: 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)), No. 5,821,337 (see: Bruggemann, M. et al., J. Exp. Med. 166:1351 - 1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, for example: ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example: Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example: Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929Al).

[0256] In a preferred embodiment, the Fc fusion protein of the present invention comprises an Fc region with reduced effector function. Fc regions with reduced effector function include those having substitutions at one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are replaced by alanine (U.S. Patent No. 7,332,581). For example, an antibody variant may comprise one or more Fc regions having amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, the substitutions are L234A and L235A (LALA) (see, e.g., WO 2012 / 130831). In addition, alterations can be made in the Fc region that result in altered (i.e., reduced) C1q binding and / or altered complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000) (e.g., G236R).

[0257] Further examples of modified Fc regions include those comprising "LALALS" (amino acid substitutions L234A / L235A / M428L / N434S, as described in Zalevsky et al., (2010) Nat. Biotechnol. 28:157-159) and LALAPG (L234A / L235A / P329G amino acid substitutions, as described in Gunn et al., (2021), Immunity 54:815).

[0258] In any embodiment, the Fc region of the Fc fusion protein of the present invention can comprise at least the "LALA" mutation (L234A and L235A) for reducing binding to FcR. The fusion protein can additionally or alternatively comprise the mutation G346R for eliminating the recruitment of complement C1q.

[0259] Other Fc modifications for use in the present invention include variants that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Such variants are also referred to herein as "knockout variants" or "KO variants". Variants that reduce binding to FcγR and complement can be used to reduce unwanted interactions mediated by the Fc region. Preferred knockout variants are described in US2008-0242845 A1, entitled "Fc Variants with Optimized Properties", published October 2, 2008, which is hereby expressly incorporated 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, numbered according to the EU index. Preferred substitutions include, but are not limited to, 234G, 235E, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, numbered according to the EU index. Preferred variants include 236R / 328R. The variants can 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 the IgG2 and IgG4 Fc regions. Hybrid isotypes can also be useful, such as the hybrid IgG1 / IgG2 isotype described in US Patent Serial No. 11 / 256,060. Other modifications for reducing FcγR and complement interactions include, but are not limited to, substituting 297A, 297D, 234A, 235A, 237A, 318A, 228P, 236E, ΔG236, 265G, 268Q, 297Q, 309L, 330S, 331S, 327Q, 220S, 226S, 229S, 238S, 233P, 234A, and 234V and removing glycosylation at position 297 by mutagenesis 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 the following document, which is hereby incorporated by reference in its entirety: Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0260] In some aspects, the Fc region of the fusion protein includes mutations in the complement (C1q) and / or Fcγ receptor (FcγR) binding sites. In some aspects, such mutations can render the fusion protein incapable of generating antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC).

[0261] The Fc region used in the context of the present invention preferably does not elicit cytotoxicity, such as antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cell cytotoxicity (CDC).

[0262] In some aspects, the Fc region can comprise one or more substitutions for reducing the affinity for FcRn and thereby reducing the serum or circulatory half-life of the fusion protein. Substitutions for reducing the affinity for FcRn are known in the art and are described, for example, in the following references: Ward et al., (2015), Mol. Immunol., 67:131-141; and Grevys et al., (2015), 194:5497-5508. Examples of substitutions include substitutions at one or more positions among Ile253, His310, and His435, such as I253A, H310A, and H435A.

[0263] The term "Fc region" also includes native sequence Fc regions and variant Fc regions. The Fc region can include the carboxyl terminus of the heavy chain. Antibodies produced by a host cell can undergo post-translational cleavage of one or more, particularly one or two amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain, or it can include a cleavage variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, which is also known as the EU index, as described in the following reference: 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 can be considered. Amino acid sequence variants of the Fc region of an antibody can be prepared by appropriate modification of the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues within the amino acid sequence of the Fc region of the antibody, and / or insertions and / or substitutions. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as inducing or supporting an anti-inflammatory response.

[0264] The Fc region of the antibody can be the Fc region of any antibody class, such as IgA, IgD, IgE, IgG, and IgM. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, as used in the context of the present invention, the antibody can be the Fc region of IgG. For example, the Fc region of the antibody can be the Fc region of IgG1, IgG2, IgG2b, IgG3, or IgG4. In some aspects, the fusion protein of the present invention comprises IgG of the Fc region of an antibody. In the context of the present invention, the Fc region of the antibody is the Fc region of IgG, preferably IgG1.

[0265] Linker region between the dysfunctional P2X7 receptor epitope and the Fc region

[0266] The dysfunctional P2X7 receptor epitope and the Fc region of an antibody (or serum albumin, transferrin, carboxy-terminal peptide of chorionic gonadotropin (CG) β-chain, non-exact 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) can be directly linked or linked through a linker sequence. The linker sequence can be the spacer sequences defined herein or exemplified in Table 1 or Table 3. Alternatively, the linker sequence can be any amino acid-based linker sequence commonly used in the art.

[0267] The linker is usually a peptide having a length up to 20 amino acids, but can also be up to 50 amino acids. The terms "linked to" or "fused to" refer to a covalent bond formed between two moieties, such as a peptide bond. Thus, in the context of the present invention, the linker can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the fusion proteins provided herein can comprise a linker between the epitope of the dysfunctional P2X7 receptor and the Fc region of an antibody, such as between the N-terminus of the Fc region and the C-terminus of the dysfunctional P2X7 receptor epitope. As another example, the fusion proteins provided herein can comprise a linker between the dysfunctional P2X7 receptor epitope and the Fc region of an antibody, such as between the C-terminus of the Fc region and the N-terminus of the dysfunctional P2X7 receptor epitope portion. In particular, the dysfunctional P2X7 receptor epitope portion can be fused to the N-terminus of the Fc region via a linker at the C-terminus. The advantage of such linkers is that they can make the different polypeptides of the fusion protein more likely to fold independently and function as expected. Thus, in the context of the present invention, the dysfunctional P2X7 receptor epitope portion and the Fc region of an antibody can be included in a single-chain multifunctional polypeptide.

[0268] In some aspects, the fusion proteins of the invention or the polypeptides used according to the invention comprise a peptide linker. In some aspects, the peptide linker connects the dysfunctional P2X7 receptor epitope moiety to the Fc region of an antibody. In some aspects, the peptide linker can comprise the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS). In some aspects, the peptide linker can comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer. The linker can be a series of repeating glycine and serine residues of different lengths (GS), i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker can be (GS)3 (i.e., GSGSGS) or longer such as (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 greater. Fusion proteins having linkers of such lengths are included within the scope of the invention. Preferably, n does not exceed 3 (i.e., when n equals 3, the linker is GSGSGS).

[0269] In a further embodiment, the linker can contain added amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linker region can also contain the sequence GSGK.

[0270] The peptide linker can consist of a series of repeats of Thr-Pro (TP) that contain one or more additional amino acids at the N-terminus and C-terminus of the repeat sequence. For example, the linker can comprise or consist of the sequence GTPTPTPTPTGEF (also referred to as the TP5 linker). In a further aspect, the linker can be a shorter and / or α-helical rigid linker (e.g., A(EAAAK)3A, PAPAP, or a dipeptide such as LE or CC).

[0271] In a further embodiment, as an alternative or supplement to the glycine-serine based linker region described above, the fusion protein can comprise a dysfunctional P2X7 receptor epitope moiety that is linked to the Fc region of an antibody via a hinge region. The connection between the dysfunctional P2X7 receptor epitope moiety and the Fc region can include a combination of a hinge region and a linker region.

[0272] Examples of suitable hinge regions include hinge regions derived from immunoglobulins. The hinge region can be derived from IgG1, IgG2, IgG3, or IgG4 and can contain one or more amino acid substitutions (e.g., to prevent or reduce the likelihood of disulfide bond formation). Alternative hinge sequences can be derived from alternative immunoglobulin domains, CD8A, CD8B, CD4, or CD28, TRAC, TRBC, TRGC, TRDC.

[0273] Additional linker sequences may also include any sequence of the CL / CH1 domain of any length, but not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. The linker may be derived from the immunoglobulin heavy chain of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. The linker may be derived from the immunoglobulin light chain, such as Cκ or Cλ. The linker sequence may also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins.

[0274] Table 3 below provides non-limiting examples of suitable hinge regions for linking the dysfunctional P2X7 receptor epitope moiety to the Fc region in the molecules of the present invention.

[0275] It will be understood that the dysfunctional P2X7 receptor epitope moiety may be linked to the Fc region (or other protein sequences as defined herein) by more than one linker and / or more than one hinge region. For example, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety conjugated directly (N-terminus to C-terminus) to the Fc region. Alternatively, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety, followed by a linker region, and then the Fc region. In addition, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety, followed by a linker region, then a hinge region, and then the Fc region. In yet another embodiment, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety, followed by a linker region, then a hinge region, another linker region, and then the Fc region. Of course, those skilled in the art will understand that alternative configurations are possible (i.e., where the dysfunctional P2X7 receptor epitope moiety is linked to the C-terminus of the Fc region by one or more linkers and / or hinge regions).

[0276] Table 3: Further exemplary linker / hinge region sequences

[0277]

[0278]

[0279]

[0280] In certain embodiments, the dysfunctional P2X7 receptor epitope moiety is directly fused to the Fc region of an antibody (or the carboxy-terminal peptide of serum albumin, transferrin, chorionic gonadotropin (CG) β-chain, non-exact repeat peptide sequences, polypeptide sequences composed of proline-alanine-serine polymers, elastin-like peptide (ELP) repeats, homopolymers of glycine residues, or gelatin-like proteins) such that there is no linker between the two regions of the fusion protein or polypeptide.

[0281] In certain embodiments, the linker that couples the dysfunctional P2X7 receptor epitope moiety to the Fc region of an antibody (or serum albumin, transferrin, the carboxy-terminal peptide of chorionic gonadotropin (CG) β-chain, a non-exact repeat peptide sequence, a polypeptide sequence composed of a proline-alanine-serine polymer, an elastin-like peptide (ELP) repeat, a homopolymer of glycine residues, or a gelatin-like protein) is a cleavable linker.

[0282] Cleavable linkers are well known in the art and include, for example, the sequence defined in SEQ ID NO:144, which defines the cleavage site of human rhinovirus 3C protease. Proteases for cleaving such cleavage sites are also readily available from commercial suppliers (e.g., Pierce HRV 3C Protease).

[0283] 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 content of which is incorporated herein by reference.

[0284] The receptor and immune cells expressing the receptor

[0285] The present invention finds use in methods for enriching subpopulations of immune cells expressing a receptor comprising an antigen-binding domain that binds to a tumor-associated and tumor-specific antigen, such as a dysfunctional P2X7 receptor. The receptor is preferably a chimeric antigen receptor (CAR) or a variant thereof. The receptor can also be a modified TCR.

[0286] The antigen recognition domain of the receptor preferably recognizes a target antigen expressed on cancer cells. It will be understood that any number of different immune cells expressing different antigen recognition domains to bind different target antigens can be used in accordance with the present invention, but it will be necessary to use molecules that comprise epitopes that compete for binding to the cellular immunotherapeutic agent.

[0287] For example, immune cells can include receptors having antigen recognition domains for binding to any 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 (Connexin 2), CD117 (c-Kit), CD146 (MCAM), CD155 (PVR), CD171 (LI CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, especially EGFR, mesothelin, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, CD276, and PCSA.

[0288] In certain embodiments, the immune cells express a CAR (or a variant thereof) for binding to a dysfunctional P2X7 receptor. The extracellular portion of the CAR or its variant can comprise an nfP2X7 binding domain that recognizes the E200 (or E300 or E200-300 complex) epitope disclosed herein.

[0289] Generally, a CAR, its variant, or a TCR can comprise an extracellular domain (extracellular portion) containing 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 the CAR, its variant, or the TCR comprises an nfP2X7 binding domain that recognizes the E200 (or E300 or E200-300 complex) epitope disclosed herein.

[0290] Generally, the antigen recognition domain of a CAR or TCR comprises a binding polypeptide that includes 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 includes amino acid sequence homology to the CDR1, 2, and 3 domains of the V H and / or V L chains of an antibody that binds to a dysfunctional P2X7 receptor. As will be appreciated, the CAR will preferably be able to recognize the same dysfunctional P2X7 receptor epitope portion present on the fusion protein of the present invention.

[0291] Although it will be understood that the methods of the present invention can be applied to enrich cells expressing any CAR that binds to a dysfunctional P2X7 receptor, in a preferred embodiment, the binding polypeptide of the CAR comprises the amino acid sequences of the CDRs of the VH and / or VL chains of an antibody as described in any one of the following, the entire contents of which are incorporated herein by reference: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any one of the corresponding US Patents 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 / AU2007 / 001540 (or the corresponding US Patent US 8,067,550), PCT / AU2007 / 001541 (or the corresponding US Publication US2010-0036101), PCT / AU2008 / 001364 (or any one of the corresponding US Patents US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding US Patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding US Patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or the corresponding Publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US 10,053,508) and PCT / AU2010 / 001741 (or the corresponding Publications WO 2011 / 075789 or US 8,835,609). Preferably, the antibody comprises the CDR amino acid sequence of PEP2-2-1 as described in PCT / AU2010 / 001070 (or any one of the corresponding US Patents US 9,127,059, US 9,688,771 or US 10,053,508) or the CDR amino acid sequence of BPM09 as described in PCT / AU2007 / 001541 (or the corresponding US Publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under the accession number 06080101. H and / or VL L chain of: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any one of the corresponding US Patents 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 / AU2007 / 001540 (or the corresponding US Patent US 8,067,550), PCT / AU2007 / 001541 (or the corresponding US Publication US2010-0036101), PCT / AU2008 / 001364 (or any one of the corresponding US Patents US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding US Patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding US Patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or the corresponding Publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US 10,053,508) and PCT / AU2010 / 001741 (or the corresponding Publications WO 2011 / 075789 or US 8,835,609). Preferably, the antibody comprises the CDR amino acid sequence of PEP2-2-1 as described in PCT / AU2010 / 001070 (or any one of the corresponding US Patents US 9,127,059, US 9,688,771 or US 10,053,508) or the CDR amino acid sequence of BPM09 as described in PCT / AU2007 / 001541 (or the corresponding US Publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under the accession number 06080101.

[0292] In a further embodiment, the binding polypeptide of the CAR comprises the amino acid sequence of the V H and / or V L chain of any of the antibodies described in any of the following, the entire contents of which are incorporated herein by reference: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any one of the corresponding US patents 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 US10,450,380), PCT / AU2007 / 001540 (or the corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or the corresponding US publication US2010-0036101), PCT / AU2008 / 001364 (or any one of the corresponding US patents US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding US patents US 8,597,643, US 9,328,155 or US10,238,716), PCT / AU2010 / 001070 (or the corresponding publication WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US10,053,508) and PCT / AU2010 / 001741 (or the corresponding publication WO 2011 / 075789 or US 8,835,609). Preferably, the antibody comprises the CDR amino acid sequence of PEP2-2-1 described in PCT / AU2010 / 001070 (or any one of the corresponding US patents US 9,127,059, US 9,688,771 or US10,053,508) or the CDR amino acid sequence of BPM09 described in PCT / AU2007 / 001541 (or the corresponding US publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under the accession number 06080101.

[0293] In a further embodiment, the binding polypeptide of the CAR comprises the amino acid sequence of an antibody or a fragment thereof as described in any one of the following, the entire contents of which are incorporated herein by reference: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any one of the corresponding US patents 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 / AU2007 / 001540 (or the corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or the corresponding US publication US2010-0036101), PCT / AU2008 / 001364 (or any one of the corresponding US patents US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or the corresponding publication WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US 10,053,508) and PCT / AU2010 / 001741 (or the corresponding publication WO 2011 / 075789 or US 8,835,609). Preferably, the antibody comprises the CDR amino acid sequence of PEP2-2-1 as described in PCT / AU2010 / 001070 (or any one of the corresponding US patents US 9,127,059, US 9,688,771 or US 10,053,508) or the CDR amino acid sequence of BPM09 as described in PCT / AU2007 / 001541 (or the corresponding US publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under accession number 06080101.

[0294] The CAR generally further comprises a signal peptide. A "signal peptide" refers to a peptide sequence that directs the intracellular trafficking and localization of a protein, such as trafficking and localization to a specific organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0295] Generally, an "antigen-binding domain" (or antigen-recognition domain) refers to the region of a CAR that specifically binds to an antigen (and thus is capable of targeting cells containing that antigen). A CAR can comprise one or more antigen-binding domains. Generally, the targeting region on a CAR is extracellular. The antigen-binding domain can comprise an antibody or an antibody-binding fragment thereof. The antigen-binding domain can comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a diabody, or a bispecific antibody. Any molecule that specifically binds to a given antigen, such as an affibody or a ligand-binding domain from a naturally occurring receptor, can be used as an antigen-binding domain. Generally, the antigen-binding domain is an scFv. Generally, in an scFv, the variable regions of the immunoglobulin heavy and light chains are fused via a flexible linker. Such a linker can be, for example, a "(G4 / S1)3-linker" and its variants, but those skilled in the art will understand that various linker sequences and forms can be used.

[0296] A CAR can also comprise a "hinge" region (sometimes referred to as a spacer or linker region) that connects the antigen-binding domain to the transmembrane domain. This is typically a hydrophilic region between the antigen-binding domain and the transmembrane domain. A CAR can comprise an extracellular hinge domain, but such a hinge can also be omitted. The hinge region can include, for example, the Fc fragment of an antibody or a fragment thereof, the hinge region of an antibody or a fragment thereof, the CH2 or CH3 region of an antibody, an accessory protein, an artificial hinge sequence, or a combination thereof. An example of a hinge region is the CD8α hinge.

[0297] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source of any such domain. When 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. When the key signaling and antigen-recognition modules (domains) are located on two (or even more) polypeptides, a CAR can have two (or more) transmembrane domains. Due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR, the splitting of the key 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).

[0298] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one normal effector function of the immune cell expressing the CAR. "Effector function" refers to a specific function of a cell. For example, in T cells, effector functions can be cytolytic activity or helper cell activity, including the secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transduces the signal for an effector function and directs the cell expressing the CAR to perform a specific function. The intracellular signaling domain can include any intact, mutated, or truncated portion of the intracellular signaling domain of a given protein that is sufficient to transduce a signal that initiates or blocks an immune cell effector function.

[0299] The function of the intracellular domain can be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination of these.

[0300] Examples of intracellular signaling domains for CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction upon antigen receptor binding.

[0301] The major cytoplasmic signaling sequences that act in a stimulatory manner can contain an ITAM (immunoreceptor tyrosine-based activation motif) signaling motif.

[0302] Examples of ITAMs containing major cytoplasmic signaling sequences commonly used in CARs are those derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Most prominently, the sequence derived from CD3ζ.

[0303] The cytoplasmic domain of a CAR can be designed to contain the CD3-ζ signaling domain either alone or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of a CAR can contain a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the part of the CAR that contains the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for lymphocytes to respond effectively to an antigen. Examples of co-stimulatory 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.

[0304] In some embodiments, the activating receptor (from which a portion of the signaling domain is derived) is the CD3 co-receptor complex or an Fc receptor.

[0305] In some embodiments, the co-stimulatory receptor (a portion of the signaling domain being derived from the co-stimulatory receptor) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0306] In some embodiments, the co-stimulatory receptor (a portion of the signaling domain being derived from the co-stimulatory receptor) is selected from CD28, OX40, or 4-1BB.

[0307] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order, with or without a linker. Short oligopeptides or polypeptides having a length preferably between 2 and 10 amino acids can form the linkage. A prominent linker is the glycine-serine dyad.

[0308] As an example, the cytoplasmic domain can comprise the signaling domain of CD3-ζ and the signaling domain of CD28. In another example, the cytoplasmic domain can comprise the signaling domain of CD3-ζ and the signaling domain of CD27. In a further example, the cytoplasmic domain can comprise the signaling domain of CD3-ζ, the signaling domain of CD28, and the signaling domain of CD27.

[0309] As described above, the extracellular portion or transmembrane domain or cytoplasmic domain of the CAR can further comprise a heterodimerization domain, with the aim of splitting the key signaling and antigen recognition modules of the CAR.

[0310] A CAR that binds to the fusion protein of the present invention or a polypeptide used according to the present invention, such as a CAR comprising the nfP2X7 E200 binding domain, can be designed to comprise any part or portion of the above-described domains herein in any order and / or combination, so as to produce a functional CAR.

[0311] The affinity of the dysfunctional P2X7 receptor binding domain of the CAR for the nfP2X7 recognition site E200 of the fusion protein of the present invention or a polypeptide used according to the present invention can vary, but the binding affinity can generally be in 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 a preferred embodiment, the binding affinity is at least about 1 nM or at least about 10 nM.

[0312] Receptors (such as CARs, their variants, or TCRs or their variants) are typically expressed by immune cells.

[0313] The immune cells can be the "engineered cells", "genetically modified cells" or "immune effector cells" described herein. Additionally, the immune cells can be immune cell precursors capable of differentiating into immune cells. Cells capable of differentiating into immune cells (e.g., T cells expressing a dysfunctional P2X7 CAR) can be stem cells, multi-lineage progenitors or induced pluripotent stem cells.

[0314] The immune cells can be white blood cells, 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.

[0315] In any embodiment, the immune cells can be T cells, where optionally the T cells do not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic level or functional level).

[0316] In any embodiment, the immune cells optionally do not express co-stimulatory molecules, which can 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 level or functional level).

[0317] In some embodiments, the immune cells include two or more different receptors (e.g., two or more CARs or variants thereof). The CARs can bind to different epitopes on the same target molecule (e.g., different epitopes on the dysfunctional P2X7 receptor). Alternatively, the CARs can bind to different target molecules such that only one CAR binds to the dysfunctional P2X7 receptor.

[0318] As used herein, the terms "different CARs" or "different chimeric antigen receptors" refer to any two or more CARs having non-identical antigen recognition domains and / or non-identical signaling domains. In one example, "different CARs" include two CARs having the same antigen recognition domain (e.g., both CARs can recognize the dysfunctional P2X7 receptor), but having different signaling domains, such as one CAR having a signaling domain containing a portion of an activating receptor while the other CAR has a signaling domain containing a portion of a co-stimulatory receptor. As will be understood, at least one of the two or more CARs within this embodiment will have an antigen recognition domain that recognizes the dysfunctional P2X7 receptor, and the other CARs can take any suitable form and can be directed against any suitable antigen.

[0319] Methods for enriching immune cells

[0320] It is well within the ability of a person skilled in the art to confirm the ability of a given fusion protein or polypeptide as defined herein to bind to the relevant receptor. For example, in the case of an nfP2X7 receptor-binding CAR, a person skilled in the art will be able to use conventional techniques to confirm the binding of the CAR to a polypeptide (or series of polypeptides), thereby determining the suitability of the polypeptide for the methods of the present invention.

[0321] It will be understood that the methods of the present invention can be used to isolate or enrich any population of immune cells expressing a receptor (including a CAR) comprising an antigen-binding domain that recognizes an epitope of a dysfunctional P2X7 receptor comprised on the fusion protein.

[0322] Furthermore, upon contacting the fusion protein or polypeptide of the present invention with any population of cells comprising target cells (i.e., cells capable of binding to an epitope of a dysfunctional P2X7 receptor on the fusion protein), a person skilled in the art can utilize conventional laboratory techniques to release the fusion protein / cell complex from the capture agent, thereby obtaining an enriched cell sample.

[0323] The separation of cell and protein populations using magnetic microbeads or macrobeads 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 of, for example, a shell of dextran molecules or silica-coated magnetic iron oxide. The solid support can also be a polymer containing magnetic material. Commercially available forms of such beads are also readily available and known, including microbeads (Miltenyi Biotec) and Dynabeads magnetic beads (Applied Biosystems) for use in conjunction with chromatography columns.

[0324] The use of microbeads (50 nm in diameter) is preferred over the use of macrobeads (1 - 5 μm in diameter) as these microbeads can be directly injected into a patient in need of treatment utilizing immune cells. Thus, in certain embodiments, it is not necessary to separate the immune cells from the polypeptide / cell complex of the second aspect of the present invention and the complex can be directly administered. In alternative embodiments, when the microbeads or macrobeads are linked to the fusion protein or polypeptide via a biotin moiety, the beads can be separated from the fusion protein or polypeptide by addition of an excess of free biotin.

[0325] Furthermore, a person skilled in the art will understand that multiple rounds of enrichment can be performed to increase the purity of the final cell composition.

[0326] In some embodiments, the present invention provides a method for enriching cells expressing a chimeric antigen receptor (CAR) in a composition rather than cells not expressing CAR, the method comprising contacting the cells with a fusion protein as described herein. In some embodiments, the cells expressing CAR are cells transduced with a nucleic acid molecule encoding CAR. In some embodiments, the cells expressing CAR are clones of cells expressing CAR transduced with a nucleic acid molecule encoding CAR.

[0327] Compositions and Their Uses

[0328] The present invention also provides a composition comprising, consisting essentially of, or consisting of one or more cells expressing one or more CARs for binding to a dysfunctional P2X7 receptor, wherein the one or more cells have been enriched according to one or more methods of the present invention.

[0329] As used herein, a composition comprising one or more cells expressing one or more CARs that have been enriched according to one or more methods of the present invention may contain 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 methods of the present invention" may contain other compounds and cells, provided that 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 methods of the present invention" means that the composition does not contain other functional cells except for the one or more cells expressing one or more CARs.

[0330] A composition consisting of one or more cells expressing one or more CARs that have been enriched according to one or more methods of the present invention may contain components other than cells, such as compounds, proteins, pharmaceutically acceptable carriers, surfactants, preservatives, etc. In some embodiments, a composition consisting of one or more cells expressing one or more CARs that have been enriched according to one or more methods of the present invention may contain a non-significant amount of impurities.

[0331] In some embodiments, the amount of one or more cells expressing one or more CARs that bind to a dysfunctional P2X7 receptor 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 invention comprises a therapeutically effective amount of one or more cells expressing one or more CARs.

[0332] The composition or cell population obtained by the method of the invention can be used to treat a disease or disorder characterized by the expression of a dysfunctional P2X7 receptor.

[0333] In one embodiment, the subject in need of treatment includes a subject who has already suffered from a benign, pre-cancerous, non-metastatic tumor. In one embodiment, the cancer is pre-cancerous or pre-tumorous.

[0334] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer can be located in any organ or tissue, especially an organ or tissue with relatively high hemodynamic pressure, such as the lung, liver, kidney, pancreas, intestine, and brain. The secondary cancer can be detected in ascites and / or lymph nodes.

[0335] In one embodiment, the cancer can be substantially undetectable.

[0336] "Pre-cancerous" or "pre-tumorous" generally refers to a condition or growth that typically precedes or develops into cancer. A "pre-cancerous" growth may have cells characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of the cell cycle.

[0337] The cancer can be a solid or "liquid" tumor. In other words, the cancer can grow in tissues (malignant tumors, sarcomas, adenomas, etc.), or it can be a cancer present in body fluids such as blood or bone marrow (e.g., lymphoma and leukemia).

[0338] In certain preferred embodiments, the cancer to be treated can be a cancer characterized by a low expression level of a dysfunctional P2X7 receptor. Examples of such cancers include Burkitt lymphoma. However, immunohistochemical analysis of the surface expression of the dysfunctional P2X7 (nfP2X7) receptor in a patient's tumor biopsy revealed an IHC score range of 1+ to 3+. Thus, samples with low expression are present in a wide range of tumor types. Examples are found in various types of solid tumors, including but not limited to neuroblastoma, colorectal cancer, lung cancer, kidney cancer, skin cancer, breast cancer, brain cancer, and prostate cancer. This difference in expression levels in different tissues can be attributed to the formation of the tumor by cells in an early transformation state (the tissues with the highest receptor expression are likely the ones experiencing the highest proliferation rate).

[0339] Other examples of cancers that can be treated according to the methods of the present invention include blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, leukemias or lymphatic system malignancies, lung cancers (including small cell lung cancer (SCKC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or 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, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver tumors, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancers.

[0340] Examples

[0341] Example 1: Enrichment of nfP2X7 receptor-binding CAR T cells using biotinylated fusion proteins

[0342] Jurkat cells and / or primary CD4+ T cells 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 (3rd generation LV system) to express an anti-nfP2X7 chimeric antigen receptor (CAR), where the CAR contains an antigen-binding domain for binding to the E200 epitope of the P2X7 receptor.

[0343] By FACS sorting, the Jurkat nfP2X7 receptor cell line was cultured into single cell clones expressing nfP2X7-CAR. The CAR construct also contains a truncated EGFR (tEGFR) downstream of the ribosome skipping site (T2A) as a marker gene to detect successfully transduced cells when the CAR receptor is still occupied by the enrichment reagent (DetR1 or DetR2 as exemplary dimeric or monomeric Fc attenuated fusion proteins, as shown in Table 1) after enrichment.

[0344] To simulate the enrichment process, Jurkat wild-type cells (CAR-negative cells, tEGFR-negative cells) were mixed with Jurkat nfP2X7-CAR-tEGFR expressing cells at a ratio of 1:1, or primary generated nfP2X7 CAR T cell products were used. One round of positive selection was performed as described below.

[0345] Biotinylation was used to label the fusion protein containing the SEQ ID NO:149 sequence. The Fc fusion protein was conjugated with biotin by EZ-linkTM NHS-LC-LC-biotin (ThermoFisher, catalog number 21343) according to the manufacturer's instructions. The labeled Fc fusion protein was added to the mixture of uninduced and transduced cells at the designated concentration of 1 μg / ml or 2 μg / ml, and the cell / fusion protein mixture was incubated at room temperature for 10 minutes and washed in magnetic-activated cell sorting (MACS) cell separation buffer (Miltenyi Biotec) according to the manufacturer's instructions.

[0346] Method 1: Then anti-biotin microbeads (Miltenyi Biotec, 130-090-485) were added to the mixture and incubated for an additional 15 minutes at 4°C. Anti-biotin microbeads are magnetic particles coated with anti-biotin antibodies to allow separation of cells that bind to the anti-biotin antibody-coated microbeads. Then separation was performed according to the manufacturer's instructions.

[0347] The cell suspension was loaded onto an MS column (Miltenyi Biotec, 130-042-201) placed in the magnetic field of a MACS separator. The magnetically labeled material (i.e., the avidin containing the fusion protein and CAR T cells bound) remained within the column. The flow-through (including cells that did not bind to the fusion protein) was discarded.

[0348] Method 2: Cell enrichment was performed using EasySep technology from STEMCELL Technologies according to the manufacturer's instructions after the initial incubation with the designated Fc fusion protein as a comparison to the MACS technology from Miltenyi Biotec.

[0349] 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. The eluted material includes Jurkat cells or primary T cells that have been successfully transduced and express nfP2X7-CAR using a lentiviral vector and are thus able to bind to a biotinylated fusion protein containing the E200 epitope of the P2X7 receptor.

[0350] Figure 1 Shown are the pre-enrichment and post-enrichment enrichment protocol results at 0 hours and 48 hours of Jurkat nfP2X7 CAR T cell enrichment, where tEGFR was used as a marker gene to identify CAR-expressing cells. nfP2X7 CAR-positive cells were directly detected by indirect staining using the same LC-LC-biotinylated Fc attenuated fusion protein DetR1 or 2 (as defined in Table 1) and a second avidin antibody. After a single round of positive selection, the purity of CAR-expressing cells was >90%. At 48 hours post-enrichment ( Figure 1 B) shows that the viability and purity remained >90%.

[0351] 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. CD4+ / CD8+ T cells were enriched using CD4+ / CD8+ microbeads (MACS). After enrichment, the cells were mixed in a 1:1 ratio and then transduced with a lentivirus expressing an anti-nfP2X7 CAR construct.

[0352] Figure 2 Shown are the pre-enrichment and post-enrichment enrichment protocol results at 0 hours and 48 hours of "Donor 12" nfP2X7 CAR T cell enrichment, where tEGFR was used as a marker gene to identify CAR-expressing cells. nfP2X7CAR-positive cells were directly detected by indirect staining using the same LC-LC-biotinylated Fc attenuated fusion protein and a second avidin antibody. After a single round of positive selection, the purity of CAR-expressing cells was >90%. At 48 hours post-enrichment ( Figure 2 B) shows that the viability and purity remained >90%.

[0353] Figure 3Shows the pre - enrichment and post - enrichment results of the enrichment protocol at 0 hours and 48 hours for the enrichment of nfP2X7 CAR T cells in "Donor 12" (using a CAR with an alternative nfP2X7 receptor - antigen - binding domain in the previous paragraph, "CAR2"), where tEGFR is used as a marker gene to identify CAR - expressing cells. The nfP2X7 CAR - positive cells are directly detected by indirect staining using the same LC - LC - biotinylated Fc - attenuated fusion protein DetR1 or 2 and a second anti - biotin antibody. After a single round of positive selection, the purity of CAR - expressing cells > 90%. At 48 hours after enrichment ( Figure 3 B) shows that the viability and purity are still > 90%.

[0354] Figure 4 Shows that compared to Figure 3 the pre - enrichment and post - enrichment results of the enrichment protocol using the EasySep technology at 0 hours and 48 hours for the enrichment of nfP2X7 CAR T cells in "Donor 12" compared to the MACS technology in Figure 4 where tEGFR is used as a marker gene to identify CAR - expressing cells. The nfP2X7 CAR - positive cells are directly detected by indirect staining using the same LC - LC - biotinylated Fc - attenuated fusion protein DetR1 or 2 and a second anti - biotin antibody. After a single round of positive selection, the purity of CAR - expressing cells > 90%. At 48 hours after enrichment (

[0355] The following table shows Figures 1 to 4 a summary of the enrichment conducted in

[0356]

[0357]

[0358] Figure 5 Shows the pre - enrichment and post - enrichment results of the enrichment protocol at 0 hours and 48 hours for the enrichment of nfP2X7 CAR T cells in "Donor 57", where tEGFR is used as a marker gene to identify CAR - expressing cells. The nfP2X7 CAR - positive cells are directly detected by indirect staining using the same LC - LC - biotinylated Fc - attenuated fusion protein DetR1 or 2 and a second anti - biotin antibody.

[0359] The enrichment was completed by the MACS technology. The data shows the enrichment of the dimeric Fc - attenuated fusion protein DetR1 (SEQ ID NO: 149) compared to the monomeric Fc - attenuated fusion proteins DetR1 (SEQ ID NO: 145) and DetR2 (SEQ ID NO: 146).

[0360] Condition:

[0361] 1. LC-LC-biotinylated dimer DetR1 Fc attenuation

[0362] 2. LC-LC-biotinylated monomer DetR1 Fc attenuation

[0363] 3. LC-LC-biotinylated monomer DetR1 Fc attenuation

[0364] Before separation, CAR expression was 55% by (DetR1) and 60% by tEGFR. According to the manufacturer's instructions for MACS separation using MS columns (see above), a total of 1×10E 7 T cells were labeled for 10 min at a concentration of 2 μg / mL at room temperature and then separation was continued.

[0365] After a single round of positive selection, the purity of CAR-expressing cells was >90%. The viability and purity were still >90% 48 hours after enrichment.

[0366] Cell counts ( = yield) and EGFR expression before and after separation showed CAR-positive and CAR-negative fractions. The use of a marker gene facilitated the direct detection of CAR.

[0367] The following table summarizes the results:

[0368]

[0369] Although these data indicate a higher yield using the dimeric form of the fusion protein, in practice, monomeric proteins are preferably used to avoid activating the immune cells being enriched. In addition, the above results may reflect that the dimeric protein has a more excellent biotinylation than the monomeric protein, thus improving the yield.

[0370] Figure 6 The purity and viability of post-separation CAR T cells were measured by a viability dye (7AAD, measured in the PerCP channel) and indirect CAR detection using the marker gene tEGFR (anti-EGFR cetuximab initially labeled with AF647, measured in the APC channel). Direct detection of the CAR receptor showed that the separated CAR-positive cells were still partially occupied. T cells expressing anti-nfP2X7 CAR were detected by staining the CAR T cells with biotinylated DetR2 (SEQ ID NO:146) and then with a secondary anti-biotin antibody in Vioblue (130-113-857 biotin antibody, Miltenyi Biotech).

[0371] Example 2: Enrichment using monomeric fusion proteins results in less activation and depletion

[0372] A series of enrichment experiments similar to those described in Example 1 were conducted. Briefly, Jurkat cells and / or primary CD4+ T cells and CD8+ T cells from healthy volunteer donors (donor 24), which were mixed at a 1:1 ratio after enrichment, were stably transduced with lentivirus (3rd generation LV system) to express a chimeric antigen receptor (CAR) targeting E200, wherein the CAR comprises an antigen-binding domain for binding to the E200 epitope of the P2X7 receptor.

[0373] CAR T cells were enriched using monomeric fusion proteins or dimeric fusion proteins, both of which contain a peptide moiety capable of binding to the CAR or the protein. The fusion proteins used in this experiment contain the amino acid sequences of SEQ ID NO:158 (monomer) and 149 (able to form homodimers in vitro).

[0374] Figure 7 Shown are the CD25+ / CD69+ expression levels (each measurement of T cell activation) and PD-1 expression levels (measurement of T cell exhaustion) at 24 hours, 48 hours, and 72 hours after enrichment using different concentrations of the fusion protein (10 ng / ml to 400 ng / ml).

[0375] The results show that enrichment using monomeric fusion proteins led to a significantly reduced T cell activation and a significantly reduced T cell exhaustion in a concentration-dependent manner compared to using dimeric fusion proteins.

[0376] Example 3: Efficiency, viability and yield after CART enrichment using biotin beads and MS columns

[0377] Enrichment of CAR T cells was performed using MACS technology according to "Method 1" in Example 1. T cells were obtained from three healthy donors (donors 50, 53, and 71, D50, D53, and D71).

[0378] Figure 8 Shown is that the enrichment using monomeric or dimeric fusion proteins was approximately equal, indicating that there was no significant loss of the enrichment potential of using monomeric fusion proteins compared to dimeric fusion proteins.

[0379] The results shown indicate two replicate experiments using T cells from donors 50 and 71.

[0380] Figure 9Shows the cell counts on day 1 and day 2 after MACS sorting, which were normalized to the expected maximum cell count. The results indicate that significantly higher cell counts were obtained when using the monomeric fusion protein for the enrichment process compared to using the homodimeric fusion protein. The results shown are for T cells obtained from two healthy donors (donor 50 and donor 71).

[0381] Figure 10 Shows the viability of cells on day 2 after MACS sorting, as measured by the percentage of 7AAD-negative cells in the cell population. The results indicate that the total cell viability was reduced when using the dimeric fusion protein for enrichment compared to using the monomeric fusion protein. The results shown indicate two replicate experiments performed using T cells from donors 50 and 71.

[0382] Figure 11 Shows the CD25+ / CD69+ expression and PD-1 expression of enriched CAR T cells on day 2 after MACS sorting.

[0383] The results indicate that cells enriched using the dimeric fusion protein had significantly higher levels of the activation markers CD25 and CD69 compared to cells enriched using the monomeric fusion protein. Additionally, cells enriched using the dimeric fusion protein had significantly higher levels of the exhaustion marker PD-1 compared to cells enriched using the monomeric fusion protein.

[0384] Overall, the results indicate that enriching CAR T cells using a monomeric fusion protein (such as having the amino acid sequence of SEQ ID NO:158) provides:

[0385] - A comparable level of enrichment of CAR-positive T cells as that obtained when using the dimeric fusion protein;

[0386] - Higher cell counts and higher cell viability, with improved cell recovery, compared to when using a homodimeric fusion protein (such as containing two copies of the E200-derived peptide) for enrichment;

[0387] - Less activation and less exhaustion of the enriched cells compared to cells obtained using the dimeric fusion protein.

[0388] These results indicate that an improved cell product was obtained using an enrichment method that includes using the monomeric fusion protein described herein compared to using a homodimeric fusion protein that contains two moieties (peptide sequences) capable of binding to the CAR and potentially causing cross-activation of CAR T cells.

[0389] Example 4: Enrichment using asymmetric heterodimeric molecules

[0390] Perform similar experiments using the heterodimeric asymmetric molecules described herein (e.g., such that the molecules comprise a dimer between an E200 peptide-Fc fusion protein and a different Fc region of an antibody; use the KIH technique). The results similarly show that, compared to using a homodimeric fusion protein comprising two copies of the E200 peptide (e.g., where the dimer is a homodimer of an E200-Fc fusion protein), using a heterodimeric asymmetric molecule comprising a single copy of the E200 peptide sequence for enriching CAR T cells results in significantly less T cell activation and significantly less T cell exhaustion in a concentration-dependent manner. These results suggest that, for the purpose of enriching CAR T cells, it is preferred to use an asymmetric heterodimeric molecule or a monomeric fusion protein (e.g., comprising a single amino acid sequence recognizable by the antigen-binding domain of a CAR) to minimize unwanted CAR T cell activation and exhaustion in a patient.

[0391] It will be understood that the invention, as disclosed and defined in this specification, extends to all alternative combinations of two or more of the separate features mentioned or evident from the text or drawings. All such different combinations constitute various alternative aspects of the invention.

Claims

1. A fusion protein, comprising: (i) An epitope portion of a dysfunctional P2X7 receptor; and (ii) The Fc region of an antibody.

2. The fusion protein according to claim 1, wherein the epitope portion of the dysfunctional P2X7 receptor comprises the sequence of an epitope found on the dysfunctional P2X7 receptor but not found on the functional P2X7 receptor.

3. The fusion protein according to claim 1 or 2, wherein the amino acid sequence of the epitope portion of the dysfunctional P2X7 receptor comprises or consists of at least the amino acid sequence shown in SEQ ID NO:7, preferably, the amino acid sequence of the epitope portion of the dysfunctional P2X7 receptor comprises or consists of the amino acid sequence of SEQ ID NO:

14.

4. The fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the epitope portion of the dysfunctional P2X7 receptor comprises or consists of the sequence shown in SEQ ID NO:9 or 122.

5. The fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the epitope portion of the dysfunctional P2X7 receptor comprises the amino acid sequence shown in any one of SEQ ID NO:7 to 69 or 122, or a sequence that is 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 to the amino acid sequence shown in any one of SEQ ID NO:7 to 69 or 122, provided that the sequence comprises at least the sequence shown 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 the antibody is the Fc region of IgG, IgA, IgD, IgE or IgM.

7. The fusion protein according to claim 6, wherein the Fc region is from an IgG antibody, preferably from 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 domain and the CH3 domain of the antibody.

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

10. The fusion protein according to claim 9, wherein the amino acid substitution comprises one or more substitutions of cysteine residues to prevent the formation of disulfide bonds between Fc molecules, optionally wherein the cysteine residue in the Fc region is substituted with any one of glycine, serine, alanine, lysine, and glutamic acid, preferably substituted with glycine or serine.

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

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

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

14. The fusion protein according to any one of claims 1 to 13, wherein the protein comprises a linker region for linking the dysfunctional P2X7 receptor epitope moiety to the Fc region of the antibody, optionally a cleavable linker region.

15. The fusion protein according to any one of claims 1 to 14, wherein the fusion protein comprises one or more modifications capable of detecting the fusion protein, including detecting the fusion protein when the fusion protein binds to a cell or a binding partner.

16. The fusion protein according to claim 15, wherein when bound to an immune cell expressing a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor (such as a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor), the one or more modifications allow for the capture of the fusion protein.

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

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

19. Use of the fusion protein or molecule according to any one of claims 1 to 18 for obtaining a population of immune cells that is enriched for cells expressing a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor.

20. The use according to claim 19, wherein the immune cells express a chimeric antigen receptor that binds to a dysfunctional P2X7 receptor.

21. A method for obtaining a population of immune cells or for enriching a population of cells that express a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen on a cancer cell, the method comprising: (i) Provide a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen on a cancer cell; (ii) Contact 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 that allows for capture of the polypeptide; Thereby forming a complex of the polypeptide and the cells; (iii) Separate the complex from the population of cells, Thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen on a cancer cell.

22. A method for obtaining a population of immune cells or for enriching a population of cells that express a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen on a cancer cell, the method comprising: (i) Provide a mixed population of immune cells, preferably a mixed population of immune effector cells, wherein a subpopulation of the cells expresses a receptor comprising an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen on a cancer cell; (ii) Contact the mixed 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 that allows for capture of the polypeptide; Thereby forming a complex of the polypeptide and the cells; (iii) Separate the complex from the population of cells, (iii) Separate the complex from the population of cells, 23. The method according to claim 21 or 22, wherein the cells are immune cells that express a chimeric antigen receptor (CAR) that binds to a tumor-associated antigen or a tumor-specific antigen on a cancer cell, and wherein the polypeptide comprises an epitope recognized by the CAR, thereby obtaining or enriching a population of immune cells that express the CAR.

24. The method according to any one of claims 21 to 23, wherein the immune cells express a receptor that binds to a tumor-associated antigen or a tumor-specific antigen, and the tumor-associated antigen or tumor-specific antigen is selected from: dysfunctional (nf) P2X7 receptor, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, PCSA, CD19, CD20, Clec9a, CD276, PD-L1, and PD-L2.

25. A method for obtaining a population of immune cells or for enriching a population of cells that express a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor, the method comprising: (i) Provide a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor; (ii) Contact the population of cells with a polypeptide, wherein the polypeptide comprises an epitope of the dysfunctional P2X7 receptor recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a moiety that allows for capture of the polypeptide; Thereby forming a complex of the polypeptide and the cells; (iii) Separate the complex from the population of cells, Thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to a dysfunctional P2X7 receptor.

26. A method for obtaining a population of immune cells or for enriching a population of cells expressing a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor, the method comprising: (i) Provide a mixed population of immune cells, preferably a mixed population of immune effector cells, wherein a subpopulation of the cells expresses a receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor; (ii) Contact the mixed population of cells with a polypeptide, wherein the polypeptide comprises an epitope of the dysfunctional P2X7 receptor recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a moiety that allows for capture of the polypeptide; Thereby forming a complex of the polypeptide and the cells; (iii) Separate the complex from the population of cells, Thereby obtaining a population of immune cells expressing a receptor having an antigen-binding domain that binds to a dysfunctional P2X7 receptor.

27. The method according to claim 25 or 26, wherein the cells to be obtained or enriched are immune cells expressing a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor.

28. A method for obtaining a population of immune cells expressing a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor, the method comprising: (i) Provide a mixed population of immune cells, preferably a mixed population of immune effector cells, wherein the population comprises cells that have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor; or wherein the cells express a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor; (ii) Contact the cell population with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the CAR, and wherein the polypeptide comprises a moiety that allows for capture of the polypeptide; Thereby forming a complex of the polypeptide and the cells; (iii) Separate the complex from the population, Thereby obtaining a population of immune cells that express a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor.

29. A method for enriching a population of immune cells expressing a chimeric antigen receptor comprising an antigen-binding domain that binds to a dysfunctional P2X7 receptor, the method comprising: (i) Provide a population of immune cells, preferably a population of immune effector cells, wherein the cells have been transduced with a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor; (ii) Contact the cell population with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the CAR, and wherein the polypeptide comprises a moiety that allows for capture of the polypeptide, Thereby forming a complex of the polypeptide and the cells; (iii) Separate the complex of cells expressing a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor and that is bound to a fusion protein from the mixed population, Thereby enriching a population of immune cells that express a chimeric antigen receptor (CAR) that binds to a dysfunctional P2X7 receptor.

30. The method according to any one of claims 21 to 29, wherein the polypeptide comprises a first portion comprising an epitope of a tumor-associated antigen or a tumor-specific antigen (such as a dysfunctional P2X7 receptor) linked to an additional amino acid sequence for promoting the solubility and stability of the first portion.

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

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

33. The method according to claim 31 or 32, wherein the polypeptide is in the form of a fusion protein, the fusion protein comprising an epitope of a dysfunctional P2X7 receptor linked to an additional amino acid sequence, optionally wherein the additional sequence is selected from serum albumin, transferrin, the carboxy-terminal peptide of the beta chain of chorionic gonadotropin (CG), a non-exact repeat peptide sequence, a polypeptide sequence composed of a proline-alanine-serine polymer, an elastin-like peptide (ELP) repeat, a homopolymer of glycine residues, or a gelatin-like protein.

34. The method according to any one of claims 21 to 29, wherein the polypeptide is in the form of a conjugate, which comprises a carbohydrate, a lipid, a liposome, a peptide, and an aptamer conjugated to an amino acid sequence comprising an epitope of a dysfunctional P2X7 receptor.

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

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

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

38. The method according to any one of claims 21 to 35, wherein the polypeptide comprises a magnetic label, and the separation step comprises i) applying a magnetic field to the cell population; ii) removing or discarding the cells not attracted by the magnetic field; iii) removing the magnetic field, thereby providing a population of immune cells expressing the receptor.

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

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

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

42. The method according to claim 41, wherein the immune cells are T cells.

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

45. The method according to claim 41 or 42, wherein the cells are obtained from or derived from an allogeneic donor who does not require treatment.

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

47. The composition according to claim 46, wherein the composition comprises greater 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) that binds to a tumor-associated antigen or a tumor-specific antigen.

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

49. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject the composition according to claim 46 or 47.

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

51. A kit for the method according to any one of claims 21 to 45, the kit comprising: - The fusion protein according to any one of claims 1 to 17, the heterodimer according to claim 18, or a polypeptide, the polypeptide comprising an epitope of a tumor-associated antigen or a tumor-specific antigen and a moiety that allows for capture of the polypeptide; - Optionally, one or more reagents that allow for separation of the fusion protein and its complex.

52. The fusion protein according to any one of claims 1 to 17 or the heterodimer according to claim 18, which comprises an amino acid sequence as shown in any one of SEQ ID NO: 145 to 158 or 160 and 161.

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

54. The method according to any one of claims 21 to 45, wherein the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 145 to 158 or 160 or 161.

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

56. The method according to claim 55, wherein the asymmetric heterodimer comprises a fusion protein, and the fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 157, 157, 160 or 161.

Images