Method for detecting immune cells

Abstract

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

Description

Technical Field

[0001] The present invention relates to in vitro methods for detecting immune cells and to compositions and molecules for performing such methods.

[0002] Cross - reference to related applications

[0003] This application claims priority to Australian Provisional Application AU 2022902657, the entire content of which is incorporated herein by reference. Background Art

[0004] Cell therapy for treating cancer and other disease states is a rapidly evolving field. The development of genetically modified 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 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 successes of CAR T therapy have 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 CARs are expanded in in vitro culture and then infused into patients, where they exert potent cytotoxicity following tumor antigen recognition and subsequent activation. Various factors in the manufacture and administration of these cells affect the in vivo persistence and long-term anti-tumor effects of CAR T cells.

[0007] Another important consideration in cell immunotherapy regimens is the need to assess whether CAR T cells continue to proliferate in vivo following administration. This includes determining whether the cells continue to be present in the patient's circulation at different time points after the initial infusion of the cells.

[0008] Accordingly, there is a need for methods and reagents for determining the presence of cell immunotherapeutic agents such as CAR T cells in patient samples.

[0009] Any reference in this specification to any prior art is not an admission or implication that such prior art forms part of the common general knowledge in any jurisdiction or that such prior art could 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

[0010] The present invention is particularly applicable to the detection of genetically modified immune cells that are engineered to bind to dysfunctional P2X7 receptors on cancer cells. Accordingly, there is provided a fusion protein comprising:

[0011] (i) an epitope portion of a dysfunctional P2X7 receptor; and

[0012] (ii) the Fc region of an antibody, preferably, wherein the Fc region has a reduced affinity for Fc receptors compared to a wild-type or naturally occurring Fc region.

[0013] The present invention provides a fusion protein comprising:

[0014] (i) a peptide; and

[0015] (ii) the Fc region of an antibody, preferably, wherein the Fc region has a reduced affinity for Fc receptors compared to a wild-type or naturally occurring Fc region,

[0016] wherein the peptide comprises the amino acid sequence of SEQ ID NO:7 (preferably the amino acid sequence of SEQ ID NO:14) 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 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 thereto, provided that the sequence comprises at least the sequence set forth in SEQ ID NO:14 or 7 or 9.

[0017] The Fc region of the antibody can be the Fc region of IgG, IgA, IgD, IgE or IgM. Preferably, the Fc region is from an IgG antibody, such as an IgG1 antibody, an IgG2 antibody, an IgG2b antibody, an IgG3 antibody or an IgG4 antibody.

[0018] 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.

[0019] The Fc region preferably comprises one or more amino acid substitutions that are used to reduce the affinity for Fc receptors (FcRs, including any one of FcγRI, FcγRII, and FcγRIII) and thereby reduce the ability of the fusion protein to elicit antibody-dependent cell-mediated cytotoxicity (ADCC). Such substitutions are well known in the art and include, but are not limited to, "DANA" and "LALA" amino acid substitutions and their variants, as further defined herein.

[0020] In additional embodiments, the Fc region may further comprise substitutions that eliminate the recruitment of complement C1q. Such mutations are also well known in the art and will be further described herein.

[0021] 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.

[0022] In a preferred embodiment, 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. Preferably, the amino acid substitutions include substitutions of one or more 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 glutamic acid, preferably glycine or serine.

[0023] 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 2 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 in 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.

[0024] Thus, in a preferred embodiment, the fusion protein comprises a hinge region for linking a peptide (e.g., a dysfunctional P2X7 receptor epitope moiety) 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.

[0025] The present invention also provides a heterodimeric asymmetric molecule comprising a fusion protein described herein (e.g., a peptide comprising SEQ ID NO: 7 or 14, or a variant thereof, as exemplified by any one of SEQ ID NOs: 2 to 69) and an Fc region of an antibody, and further comprising an Fc region of an antibody that does not comprise the peptide. Such asymmetric heterodimeric molecules can be obtained using, for example, the knob-in-hole technology further described herein to promote dimerization of non-identical Fc regions.

[0026] Preferably, the fusion protein or heterodimeric asymmetric molecule consists of or consists essentially of a peptide and an Fc region of an antibody such that the fusion protein or heterodimeric asymmetric molecule does not comprise the antigen-binding domain of the antibody (i.e., such that the fusion protein does not comprise VH, VL, Fab, Fv or scFv derived from an antibody).

[0027] In any embodiment, the peptide (e.g., a dysfunctional P2X7 receptor epitope moiety) can comprise any amino acid sequence derived from a dysfunctional P2X7 receptor, although preferably it comprises a sequence of an epitope found on the dysfunctional P2X7 receptor but not on the functional P2X7 receptor.

[0028] In a preferred embodiment, the amino acid sequence of the dysfunctional P2X7 receptor epitope moiety comprises at least the amino acid sequence listed in SEQ ID NO: 14 or consists at least of the amino acid sequence listed in SEQ ID NO: 14. In a particularly preferred embodiment, the moiety comprises at least the sequence listed in SEQ ID NO: 7 or 9.

[0029] In any embodiment, the dysfunctional P2X7 receptor epitope moiety comprises the amino acid sequence listed in any one of SEQ ID NOs: 7 to 69 or SEQ ID NO: 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 97%, at least 98% or at least 99% identical thereto, provided that the sequence comprises at least the sequence listed in SEQ ID NO: 14 or 7 or 9.

[0030] In any embodiment, the fusion protein comprises the amino acid sequence listed in any one of SEQ ID NOs: 145 to 158, 160, or 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 thereto.

[0031] In a further embodiment, the fusion protein of the invention may comprise one or more modifications that allow for the detection of the fusion protein, including when the protein binds to an immune cell expressing an exogenous cell surface receptor, wherein the exogenous cell surface receptor comprises an antigen-binding domain and an intracellular signaling domain (e.g., a chimeric antigen receptor (CAR) or a modified TCR). Preferably, the antigen-binding domain of the receptor is for binding to a peptide as described herein and / or for binding to a dysfunctional P2X7 receptor.

[0032] It will be understood that typically, the antigen-binding domain of the receptor will be capable of binding or recognizing the same peptide contained within the fusion protein (e.g., the epitope portion of the dysfunctional P2X7 receptor). Additionally, one skilled in the art of a given anti-dysfunctional P2X7 receptor CAR's specific epitope and binding target can design a suitable fusion protein for binding to the CAR according to the present invention.

[0033] The one or more modifications of the fusion protein may be selected from the following: a fluorescent moiety, metal particles (for Cytometric time offlight (CyTOF) methods), magnetic particles, chromophore moieties, phosphorescent moieties, luminescent moieties, light-absorbing moieties, radioactive moieties, and chemically detectable moieties such as haptens (e.g., biotin, avidin, streptavidin, and their derivatives).

[0034] In the case of a biotin or magnetic moiety, any method known to the skilled person may be used to conjugate the moiety to the fusion protein. In one example, the moiety is conjugated to the fusion protein via one or more lysine residues of the protein and / or at the amino terminus of the protein.

[0035] In a second aspect, the present invention also provides the use of the fusion protein of the first aspect or the polypeptide as further described herein for detecting one or more genetically modified immune cells, wherein the one or more genetically modified immune cells express a receptor comprising an antigen-binding domain for binding to a dysfunctional P2X7 receptor and / or a peptide as described herein. Preferably, the receptor expressed by the immune cell is a chimeric antigen receptor (CAR), or optionally, the receptor expressed by the immune cell is a modified T cell receptor (TCR). Preferably, the detection is an in vitro detection method for enabling the determination of the presence of immune cells in a complex mixture, such as a biological sample obtained from a patient who has previously received immune cell therapy.

[0036] In a preferred embodiment of the second aspect, there is provided an in vitro method for detecting an immune cell that expresses a receptor comprising an antigen-binding domain for binding to a dysfunctional P2X7 receptor, the method comprising:

[0037] (i) providing a biological sample from a patient who has received treatment with immune cells, preferably immune effector cells, wherein the cells express a receptor comprising an antigen-binding domain for binding to a dysfunctional P2X7 receptor;

[0038] (ii) contacting the sample with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a detection moiety for allowing detection of the polypeptide,

[0039] thereby allowing the formation of a complex of the polypeptide that binds to the cells;

[0040] (iii) detecting the complex,

[0041] thereby detecting an immune cell that expresses a receptor having an antigen-binding domain for binding to a dysfunctional P2X7 receptor. Optionally, the method includes a step of first separating the complex before the detecting step.

[0042] Preferably, the cell is an immune cell that expresses a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor. Accordingly, there is also provided an in vitro method for detecting an immune cell that expresses a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor, the method comprising:

[0043] (i) Provide a biological sample from a patient who has been treated with immune cells, preferably immune effector cells, wherein the immune cells comprise a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor;

[0044] (ii) Contact the sample with a polypeptide, wherein the polypeptide comprises an epitope of a dysfunctional P2X7 receptor recognized by the CAR, and wherein the polypeptide comprises a detection moiety for allowing detection of the polypeptide,

[0045] thereby allowing formation of a complex of the polypeptide that binds to the cells;

[0046] (iii) Detect the complex,

[0047] thereby detecting immune cells that express a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor. Optionally, the method includes a step of first isolating the complex prior to the detection step.

[0048] An in vitro method for detecting immune cells that express an exogenous cell surface receptor, the exogenous cell surface receptor comprising an antigen-binding domain for binding to a peptide comprising 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 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:

[0049] (i) Provide a biological sample from a patient who has been treated with immune cells, preferably immune effector cells, wherein the cells express an exogenous cell surface receptor, the exogenous cell surface receptor comprising an antigen-binding domain for binding to a peptide comprising 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 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);

[0050] (ii) Contact the sample with a polypeptide, wherein the polypeptide comprises a peptide recognized by the antigen-binding domain of the receptor, and wherein the polypeptide comprises a detection moiety for allowing detection of the polypeptide,

[0051] thereby allowing formation of a complex of the polypeptide that binds to the cells; and

[0052] (iii) Detecting said complex,

[0053] thereby detecting immune cells expressing an exogenous cell surface receptor having an antigen-binding domain for binding to a peptide comprising 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 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). Optionally, the method includes a step of first separating the complex before the detecting step.

[0054] Preferably, the cell is an immune cell expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising 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 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). Accordingly, also provided is an in vitro method for detecting an immune cell expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising 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 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:

[0055] (i) Providing a biological sample from a patient who has received treatment with immune cells, preferably immune effector cells, wherein the cells comprise a chimeric antigen receptor (CAR) for binding to the peptide;

[0056] (ii) Contacting the sample with a polypeptide, wherein the polypeptide comprises a peptide recognized by the CAR and wherein the polypeptide comprises a detection moiety for allowing detection of the polypeptide,

[0057] thereby allowing formation of a complex of the polypeptide that binds to the cells;

[0058] (iii) Detecting said complex,

[0059] thereby detecting immune cells expressing a chimeric antigen receptor (CAR). Optionally, the method includes a step of first separating the complex before the detecting step.

[0060] 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.

[0061] In any embodiment of the second aspect of the present invention, the polypeptide comprises a first portion that comprises a peptide (such as an epitope of a dysfunctional P2X7 receptor) linked to an additional amino acid sequence for promoting the solubility and stability of the first portion. The additional amino acid sequence linked to the epitope of the dysfunctional P2X7 receptor can comprise any suitable linker or hinge region, such as those exemplified in Tables 1 and 3. Such linker or hinge regions can comprise an amino acid sequence consisting of glycine and serine repeats (the so-called "GS" linker sequence, and variants thereof as further defined herein). The hinge region can also comprise a sequence derived from the hinge region of an immunoglobulin, such as those defined in Table 3.

[0062] In a further embodiment, the polypeptide can be in the form of a fusion protein that comprises an epitope of a dysfunctional P2X7 receptor linked to an additional amino acid sequence. The additional sequence can 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, an elastin-like peptide (ELP) repeat, a homopolymer of glycine residues, or a gelatin-like protein.

[0063] In addition, the polypeptide can be in the form of a conjugate that comprises a carbohydrate (such as polyethylene glycol (PEG)), a lipid, a liposome, a peptide, or an aptamer conjugated to an amino acid sequence comprising an epitope of a dysfunctional P2X7 receptor. In the case of a PEG conjugate, the conjugation can be carried out via an activated carboxylic acid of the amino acid sequence comprising the epitope of the dysfunctional P2X7 receptor.

[0064] According to the second aspect of the present invention, the moiety for allowing detection of the polypeptide can be any suitable detectable moiety, such as a fluorescent moiety, a magnetic particle, a chromophore moiety, a phosphorescent moiety, a luminescent moiety, a light-absorbing moiety, a radioactive moiety, and a chemically detectable moiety, such as a hapten (e.g., biotin, avidin, streptavidin, and derivatives thereof).

[0065] 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 for allowing detection of the complex. Optionally, one or more moieties for allowing detection of the complex comprise a fluorophore (i.e., such that the avidin antibody is fluorescently labeled).

[0066] Optionally, wherein the polypeptide comprises a magnetic label, the detecting step may comprise: 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 a dysfunctional P2X7 receptor.

[0067] In a particularly preferred embodiment of the second aspect of the present invention, an exogenous cell surface receptor (such as a chimeric antigen receptor, CAR) comprising an antigen-binding domain 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 patents 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 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 patents US 9,127,059, US 9,688,771 or US10,053,508) 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 patents US 9,127,059, US 9,688,771 or US10,053,508), and the above-mentioned documents are incorporated herein by reference.

[0068] In any embodiment, a biological sample from a patient may be a sample of whole peripheral blood or a derivative thereof, such as a preparation of peripheral mononuclear cells (buffy coat).

[0069] In a further embodiment, there is provided a kit for the methods described herein, the kit comprising:

[0070] - a fusion protein or polypeptide capable of binding to a receptor (such as a CAR) for binding to a dysfunctional P2X7 receptor;

[0071] - Optionally, one or more reagents for detecting the fusion protein or polypeptide and its complexes.

[0072] Optionally, the kit includes written instructions for the methods of the second aspect of the present invention.

[0073] As used herein, unless the context requires otherwise, the term "comprising" and variations thereof, such as "comprises", "including" and "containing", are not intended to exclude additional additives, components, wholes or steps.

[0074] Other aspects of the invention and further embodiments of the aspects described in the previous paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings.

[0075] Sequence information

[0076] Table 1: Exemplary sequences of dysfunctional P2X7 receptors and receptor epitope moieties

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[0086] Description of the drawings

[0087] Figure 1 : Detection of untransduced T cells (UTD) (bottom panel), donor T cells expressing anti-nfP2X7 CAR (middle panel), and T cells expressing anti-CD33 CAR (top panel).

[0088] Figure 2 : Flow cytometry analysis of untransduced T cells (UTD) (bottom panel), donor T cells expressing anti-nfP2X7 CAR (middle panel), and Jurkat cells expressing anti-nfP2X7 CAR (top panel) using three different biotinylated fusion proteins containing epitopes of the nfP2X7 receptor. The fusion proteins used were: DetR1 dimer (SEQ ID NO: 149); DetR1 monomer (SEQ ID NO: 158) and DetR2 monomer (SEQ ID NO: 146).

[0089] Figure 3: The ratios of CD25+ / CD69+ to PD-L1+ cells 24 hours (A), 48 hours (B), and 72 hours (C) after detection using monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NO: 158 and 149, respectively). Detailed implementation

[0090] An important consideration during a cellular immunotherapy regimen is the need to evaluate whether CAR T cells continue to proliferate in vivo after administration. This includes determining whether the cells continue to be present in the patient's circulation at different time points after the initial infusion of the cells. Although there are methods for detecting CAR T cells, these generally rely on the modification of the CAR or immune cells expressing the CAR (e.g., inclusion of a fluorescent label or tag). This method is less than ideal because it requires further genetic modification of the cells and / or the infusion of foreign substances into the subject.

[0091] The method of the present invention is non-invasive, does not require modification of the CAR or immune cells expressing the CAR, and allows for the rapid determination of the presence of genetically modified immune cells in a patient sample, where the genetically modified immune cells express a receptor for binding to a dysfunctional P2X7 receptor.

[0092] The present invention provides fusion proteins (including monomeric, homodimeric, or heterodimeric molecules derived therefrom), which fusion proteins comprise a linear epitope derived from a P2X7 receptor (such as exemplified in any one of SEQ ID NO: 14 or 7) and the Fc region of an antibody. When conjugated to a moiety that permits the detection of the protein, such fusion proteins have a particular use in the in vitro detection of CAR T cells.

[0093] In a particularly preferred embodiment, the Fc fusion protein is designed to contain only a single copy of the linear epitope derived from the P2X7 receptor. As further described herein, this can be achieved by introducing amino acid substitutions into the Fc region to prevent homodimerization, or alternatively, using well-known "stub and socket" techniques to ensure the formation of an asymmetric heterodimeric molecule (e.g., comprising an E200 peptide-Fc fusion protein and an Fc region that does not contain the E200 peptide). Such monomeric or asymmetric heterodimeric molecules have the advantage of reducing the activation of target immune cells and preventing the unwanted depletion of target immune cells (as further described in the examples herein), particularly in situations where it is desired or intended to determine the function or activation status of CAR T cells after detection. Without wishing to be bound by theory, the inventors believe this is due to the reduced ability of the molecule to crosslink two different CAR receptors on one cell or two different CAR receptors on two independent CAR-expressing cells.

[0094] Definitions

[0095] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0096] For the purposes of interpreting this specification, the following definitions are generally applied, and where appropriate, terms used in the singular will also include the plural and vice versa.

[0097] As used herein, the term "and / or", e.g., "X and / or Y", will be understood to mean "X and Y" or "X or Y", and should be understood to provide explicit support for both meanings or either meaning.

[0098] The articles "a" and "an" as used herein refer to one or more than one (i.e., at least one) grammatical object of the article. By way of example, "a dysfunctional P2X7 receptor epitope moiety" means a dysfunctional P2X7 receptor epitope moiety or more than one dysfunctional P2X7 receptor epitope moiety.

[0099] As used herein, unless the context otherwise requires, the term "comprising" and variations thereof such as "comprises", "including" and "includes" are not intended to exclude additional additives, components, wholes or steps.

[0100] "Purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0101] "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.

[0102] 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, e.g., where the P2X7 monomer is a splice variant, allelic variant, SNP and isoform, including naturally occurring truncated or secreted forms of the monomer forming the P2X7 receptor (e.g., forms consisting of an extracellular domain sequence or a truncated form thereof), naturally occurring variant forms (e.g., alternative splicing forms) and naturally occurring allelic variants. In certain embodiments of the 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, e.g., various amino acids in the sequence shown in SEQ ID NO:1 may be substituted, deleted or inserted with residues.

[0103] "Functional P2X7 receptor" generally refers to the 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, enabling calcium ions and molecules up to 900 Da to enter the cytoplasm, and one of the consequences may be the induction of programmed cell death. Under normal steady state, the expression of the functional P2X7 receptor 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 the functional P2X7 receptor on red blood cells and other cell types.

[0104] "Dysfunctional P2X7 receptor" (also referred to as "non-functional" P2X7 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. The dysfunctional P2X7 receptor or (nfP2X7 receptor) generally refers to the 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. An example is the 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, and these 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 prolong 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. The dysfunctional P2X7 receptor is 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".

[0105] "Cancer-associated P2X7 receptor" generally refers to the P2X7 receptor that is found on cancer cells (including pre-tumor, tumor, malignant, benign, or metastatic cells) but not on non-cancerous or normal cells.

[0106] "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.

[0107] "E300 epitope" generally refers to an epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO:4) or its variants, as defined in SEQ ID NO:5.

[0108] "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).

[0109] As used herein, the term "antigen" is intended to include substances that bind to one or more antibodies or induce the production of one or more antibodies, and can include, but are not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and can be recognized by the adaptive immune system, which includes, but is 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.

[0110] "Epitope" generally refers to the portion of an antigen that is bound by 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.

[0111] With respect to the terms "bind", "specifically bind", or "specific for" referring to a receptor that recognizes and binds to a dysfunctional P2X7 receptor antigen-binding domain, it means that the receptor substantially does not recognize or bind to other antigens in the sample.

[0112] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as 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 the binding pair (such as 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.

[0113] 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 cell, such as pluripotent stem cells and early progenitor cell subsets that can mature or differentiate into somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / 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.

[0114] As used herein, the term "autologous" refers to any material that is derived from a subject and subsequently reintroduced into the same subject.

[0115] 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.

[0116] As used herein, the terms "engineered cell" and "genetically modified cell" can be used interchangeably. This 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, this term refers to the fact that cells, preferably immune cells, can be manipulated by recombinant methods known in the art to stably or transiently express peptides or proteins that are not expressed in these cells in their natural state. For example, immune cells are engineered to express an artificial construct, such as a chimeric antigen receptor, on their cell surface. For example, a CAR sequence can be delivered into a cell 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, a transposon (such as sleeping-beauty) or a variant thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0117] The amino acid structures and 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.

[0118] Table 2

[0119]

[0120] As used herein, the term "non-proteinogenic amino acid" refers to an amino acid having a side chain not present in the naturally occurring L-α-amino acids set forth in Table 2. Examples of non-proteinogenic amino acids and derivatives include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, tert-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of natural amino acids.

[0121] As used herein, the term "α-amino acid" refers to an amino acid having a single carbon atom (α-carbon atom) separating 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 an α-amino acid.

[0122] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms. Where appropriate, the alkyl may have a specific number of carbon atoms, e.g., C 1-6 alkyl, which includes alkyls having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight-chain or branched-chain arrangement. Examples of suitable alkyls 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.

[0123] 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 may be a subject (patient) suffering from a disease such as cancer. As used herein, the terms "subject" and "individual" may be used interchangeably.

[0124] Dysfunctional P2X7 receptor epitope moiety

[0125] The present invention also provides a fusion protein comprising a dysfunctional P2X7 receptor epitope moiety.

[0126] The dysfunctional P2X7 receptor epitope portion can be provided in the form of a dysfunctional P2X7 receptor or a fragment of a dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP. Such a receptor cannot extend the opening of non-selective calcium channels to apoptotic pores.

[0127] According to the present invention, the dysfunctional P2X7 receptor epitope portion is typically in the form of a peptide fragment of a dysfunctional P2X7 receptor. Generally, the peptide contains an epitope not found or not available for binding on a functional P2X7 receptor.

[0128] 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.

[0129] A series of peptide fragments of the dysfunctional P2X7 receptor are known and are discussed 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 US10,450,380), PCT / AU2008 / 001364 (and corresponding publications WO 2009 / 033233 and US 8,440,186, US 9,181,320, US 9,944,701 or US10,597,45) and PCT / AU2009 / 000869 (and corresponding publications WO 2010 / 000041 and US 8,597,643, US 9,328,155 or US10,238,716), and the entire contents thereof are incorporated herein by reference in their entirety. Exemplary peptides containing epitopes intended for use in the present invention are described below.

[0130] PCT Publication Peptide Sequence

[0131] WO 2002 / 057306 GHNYTTRNILPGLNIT(SEQ ID NO:3)

[0132] WO 2002 / 057306 GHNYTTRNILPGLNITC(SEQ ID NO:2) (also referred to herein as the "E200" epitope)

[0133] WO 2009 / 033233 KYYKENNVEKRTLIKVF (SEQ ID NO:4) (also referred to herein as the "E300" epitope)

[0134] WO 2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6) (also referred to herein as the "E200 / E300" or "composite" epitope)

[0135] 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).

[0136] The amino acid sequence of any one of SEQ ID NOs: 2 to 7 may comprise a part of an epitope portion recognized by a receptor expressed on an immune cell or capable of binding to a receptor expressed on an immune cell (also referred to herein as the "recognition sequence" of the epitope portion).

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

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

[0139] In some embodiments, the C-terminus of the epitope portion 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 to the free acid, having a C-terminus that is a derivative or analogue of the free acid group 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 comprises a functional moiety (such as biotin).

[0140] In any embodiment of the first or second aspect, the epitope that dysfunctions the P2X7 receptor comprises or consists of an epitope 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 a specific epitope of the dysfunctional P2X7 receptor.

[0141] 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 epitopes as defined herein. Obtaining the various polypeptides for use according to the present 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 comprising 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 additional amino acids from the native sequence of the P2X7 receptor may be included in the polypeptide, such as the N-terminal residue "DFP" of the epitope in the P2X7 receptor sequence and / or the C-terminal residue "TFHKT" of 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.

[0142] 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 to provide 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.

[0143] Including additional amino acid residues in the E200, E300 or composite epitope (or the extended epitope as discussed in the above paragraph) 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 epitopes as defined herein.

[0144] Fc region

[0145] In any embodiment, the amino acid sequence of the epitope of the dysfunctional P2X7 receptor may 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 may be fused to the C-terminal region of the Fc region of an antibody or a variant thereof via its N-terminal region.

[0146] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chain.

[0147] 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 dimerization of the molecule with the same molecule. It will be understood that any amino acid substitution that prevents dimerization of the Fc region may be used. Thus, in vivo, the Fc fusion proteins described herein may be monomeric proteins.

[0148] 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 substitutions of one or more cysteine residues to prevent the formation of disulfide bonds between Fc molecules. The cysteine residues in the Fc region may be substituted with any other amino acid residue, optionally glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

[0149] 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 the IgG1 hinge region and its variants (which include 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 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.

[0150] Thus, in a preferred embodiment, the fusion protein comprises a hinge region for linking the dysfunctional P2X7 receptor epitope moiety to the Fc region of the antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences described in SEQ ID NOs: 76 to 113, or 136 to 137, or 141, or 142.

[0151] 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 having a single copy of the epitope portion described herein and an Fc region that binds to another Fc region that does not comprise the epitope portion.

[0152] 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 method enables the expression and purification of heterodimeric fusion proteins in which each molecule has only one copy of the peptide epitope (e.g., the epitope portion derived from the E200 epitope described herein). Examples of "mortar and pestle" Fc pairings are provided herein as SEQ ID NO: 157 and 159 (mortar and pestle, respectively), 158 and 159, 160 and 162 (mortar and pestle, respectively), and 161 and 162. Accordingly, in any embodiment, the present invention provides a fusion protein comprising the 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 comprise the E200 peptide portion.

[0153] 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 comprise the E200 peptide fused thereto).

[0154] Preferably, the Fc region comprises one or more substitutions for eliminating or reducing effector functions (such as reduced binding and activation via FcR), as further described below.

[0155] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. In other words, the Fc region comprises two heavy chain fragments that comprise the C H 2 and C H 3 domains. In the context of the present invention, the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of the heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the C H 3 domain. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0156] 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 that can be attributed to the Fc region of an antibody and that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as the B cell receptor); and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, 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. Primary cells, NK cells, which mediate ADCC, 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). Non-limiting examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985)), 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, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA; and CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model, such as that 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 is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (e.g., see: 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 (e.g., see: Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929Al).

[0157] 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 positions among 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 an Fc region having one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, the substitutions are L234A and L235A (LALA) (see, e.g., WO 2012 / 130831). In addition, alterations can be made in the Fc region, resulting in altered (i.e., reduced) C1q binding and / or altered (i.e., reduced) 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).

[0158] 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).

[0159] 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.

[0160] 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 incorporated by reference in its entirety. 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, 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 the 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 Strohl, 2009, Current Opinion in Biotechnology 20:685-691, which is hereby incorporated by reference in its entirety.

[0161] 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).

[0162] 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 cytotoxicity (CDC).

[0163] 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 expression of a specific 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 referred to as the EU index, as described in the following reference: Kabat et al., Sequences of Proteins of Immunological Interest, Public Health Service, 5th Edition, 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 introducing appropriate modifications to 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, which has the desired characteristics, such as inducing or supporting an anti-inflammatory response.

[0164] The Fc region of an 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 that its heavy chain possesses. 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 an 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 an antibody is the Fc region of IgG, preferably IgG1.

[0165] Linker region between the dysfunctional P2X7 receptor epitope and the Fc region

[0166] The epitope of the dysfunctional P2X7 receptor and the Fc region of the antibody can be directly linked or linked via a linker sequence. The linker sequence can be a spacer sequence as 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.

[0167] Linkers are typically peptides that are up to 20 amino acids in length, but can 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, a 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 include a linker between the epitope of the dysfunctional P2X7 receptor and the Fc region of the 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 include a linker between the epitope of the dysfunctional P2X7 receptor and the Fc region of the antibody, such as between the C-terminus of the Fc region and the N-terminus of the dysfunctional P2X7 receptor moiety. In particular, the dysfunctional P2X7 receptor epitope moiety can be fused at the C-terminus to the N-terminus of the Fc region via a linker. The advantage of such linkers is that they can make it more likely that the different polypeptides of the fusion protein will fold independently and function as expected. Thus, in the context of the present invention, the dysfunctional P2X7 receptor epitope moiety and the Fc region of the antibody can be included in a single-chain multifunctional polypeptide.

[0168] In some aspects, the fusion proteins of the present invention include peptide linkers. In some aspects, the peptide linker links the dysfunctional P2X7 receptor epitope moiety to the Fc region of the antibody. In some aspects, the peptide linker can include the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Ser (GGGS) or Gly-Gly-Gly-Ser (GGGGS). In some aspects, the peptide linker can include 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 (GS) of different lengths, 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 present invention. Preferably, n does not exceed 3 (i.e., when n equals 3, the linker is GSGSGS).

[0169] In a further embodiment, the linker may comprise added amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linker region may also comprise the sequence GSGK.

[0170] The peptide linker may consist of a series of Thr-Pro (TP) repeats, which contain one or more additional amino acids at the N-terminus and C-terminus of the repeat sequence. For example, the linker may comprise the sequence GTPTPTPTPTGEF (also referred to as the TP5 linker) or consist of the sequence GTPTPTPTPTGEF. In a further aspect, the linker may be a shorter and / or α-helical rigid linker (e.g., A(EAAAK)3A, PAPAP or a dipeptide, such as LE or CC).

[0171] In a further embodiment, as an alternative or supplement to the glycine-serine-based linker region as described above, the fusion protein may comprise a dysfunctional P2X7 receptor epitope portion, which is linked to the Fc region of the antibody via a hinge region. The connection between the dysfunctional P2X7 receptor epitope portion and the Fc region may include a combination of a hinge region and a linker region.

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

[0173] 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.

[0174] Table 3 below provides non-limiting examples of suitable hinge regions for linking the dysfunctional P2X7 receptor epitope portion to the Fc region in the molecules of the present invention.

[0175] It will be understood that the dysfunctional P2X7 receptor epitope portion can be linked to the Fc region by more than one linker and / or more than one hinge region. For example, the fusion protein can comprise (N-terminus to C-terminus) the dysfunctional P2X7 receptor epitope portion directly conjugated to the Fc region. Alternatively, the fusion protein can comprise the dysfunctional P2X7 receptor epitope portion, followed by a linker region, and then the Fc region. In addition, the fusion protein can comprise the dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, and then the Fc region. In yet another embodiment, the fusion protein can comprise the dysfunctional P2X7 receptor epitope portion, 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 portion is linked to the C-terminus of the Fc region by one or more linkers and / or hinge regions).

[0176] Table 3: Further exemplary linker / hinge region sequences

[0177]

[0178]

[0179] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is directly fused to the Fc region of an antibody such that there is no linker between the two regions of the fusion protein.

[0180] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is linked to the Fc region of an antibody by a cleavable linker.

[0181] 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 for human rhinovirus 3C protease. Proteases for cleaving such cleavage sites are also readily available from commercial suppliers (e.g., Pierce HRV 3C protease).

[0182] Other known cleavable linkers and other linkers that can be used according to the present invention are disclosed in the following: Chen et al., (2013) Adv. Drug. Deliv. Rev. 65:1357-1369; the content of which is incorporated herein by reference.

[0183] In additional embodiments of the second aspect of the present invention, the dysfunctional P2X7 receptor epitope portion is linked to an additional amino acid sequence of a polypeptide by a spacer comprising a polysaccharide having at least 15 carbon atoms, the polysaccharide selected from the group consisting of dextran, amylopectin, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan, and chitin.

[0184] In yet a further alternative embodiment of the second aspect of the present invention, the polypeptide comprises a dysfunctional P2X7 receptor epitope moiety, which is linked via a spacer to a modification for allowing detection of the polypeptide, the spacer comprising a polysaccharide having at least 15 carbon atoms, the polysaccharide being selected from the group consisting of dextran, amylopectin, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan and chitin.

[0185] The spacer unit of the polypeptide for use according to the second aspect of the present invention preferably comprises at least 15 carbon atoms and is preferably selected from the group comprising oligopeptides, polyethylene glycol, enzymatically degradable units or affinity units, which provide a cleavable non-covalent linkage between the detection moiety and the dysfunctional P2X7 receptor epitope moiety.

[0186] Suitable oligopeptides contain at least 2 amino acids, preferably at most 10 amino acids. Most preferably, the oligopeptide has the amino acid sequence GGGSK. Preferred polyethylene glycol (PEG) contains from 10 to 200 ethylene glycol units.

[0187] As an enzymatically degradable spacer, any molecule that can be cleaved by a specific enzyme can be used. Suitable enzymatically degradable spacers S are, for example, polysaccharides, proteins, peptides, peptoids, polyesters, nucleic acids and derivatives thereof that can be cleaved by hydrolases. The enzymatically degradable spacer can consist of more than one different enzymatically degradable unit, which can be cleaved by the same or different enzymes.

[0188] Preferred polysaccharides are, for example, dextran, amylopectin, inulin, amylose, cellulose, hemicellulose, such as xylan or glucomannan, pectin, chitosan, or chitin.

[0189] The detection moiety and the dysfunctional P2X7 receptor epitope moiety can be coupled to the spacer covalently or non-covalently. Covalent or non-covalent conjugation methods are known to those skilled in the art. In the case of covalent binding between the detection moiety and / or the dysfunctional P2X7 receptor epitope moiety and the spacer, activated groups on the detection moiety and / or the dysfunctional P2X7 receptor epitope moiety or on the spacer react directly with functional groups on the spacer or on the detection moiety and / or the dysfunctional P2X7 receptor epitope moiety, or via a heterobifunctional linker molecule that first reacts with one binding partner and then with the other binding partner.

[0190] For the non-covalent or quasi-covalent coupling of the detection part D to the dysfunctional P2X7 receptor epitope part via a spacer, the spacer can be equipped with an affinity unit that is cleaved by a releasing agent when needed. The affinity unit includes, for example, biotin, avidin, and / or streptavidin, resulting in a quasi-covalent binding with a dissociation constant of less than 10M-9M.

[0191] The term "releasing agent" refers to any compound capable of binding to a part of the affinity unit. For example, the biotin-avidin affinity unit used as a spacer can be cleaved by streptavidin or an excess of free biotin can be added for cleavage. Through a competitive reaction, the affinity unit is cleaved, thereby releasing the detection part from the dysfunctional P2X7 receptor epitope part (or vice versa). Suitable affinity units and releasing agents were disclosed by James Hirsch et al. in Analytical Biochemistry 308(2002)343-357.

[0192] The affinity unit can be equipped with a detection means, that is, having a label that can be used for detection. The detection means can be the same as or different from those described for the detection part. Using an affinity unit equipped with a detection means as a spacer allows for further quantification of CAR cells.

[0193] Receptors and immune cells expressing the receptors

[0194] The present invention discovers the application in a method for detecting a subset of immune cells expressing a receptor that binds to the dysfunctional P2X7 receptor. The receptor is preferably a chimeric antigen receptor (CAR) or a variant thereof. The receptor can also be a modified TCR.

[0195] Generally, a CAR, its variant, or a TCR can include an extracellular domain (extracellular part) containing an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain can be connected to the transmembrane domain through a linker. The extracellular domain can also include a signal peptide. Preferably, the extracellular part of a CAR, its variant, or a TCR contains an nfP2X7 binding domain that recognizes the E200 (or E300 or E200-300 complex) epitope disclosed herein.

[0196] Generally, the antigen recognition domain of a CAR or a TCR contains a binding polypeptide that includes an amino acid sequence homologous to one or more complementarity-determining regions (CDRs) of an antibody that binds to the dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide includes the V H and / or V LAmino acid sequences homologous to the CDR1, 2, and 3 domains of the chain. As will be appreciated, the CAR will preferably be able to recognize the same dysfunctional P2X7 receptor epitope moiety present on the fusion proteins of the present invention.

[0197] Although it will be understood that any CAR that binds to the dysfunctional P2X7 receptor can be used in the methods of the present invention, in a preferred embodiment, the binding polypeptide of the CAR comprises the V of the antibody described in the following documents H and / or V LAmino acid sequences of the CDRs of the chain: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any one of the corresponding US patents US7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US9,663,584 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 publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US10,053,508) and PCT / AU2010 / 001741 (or the corresponding publications WO 2011 / 075789 or US 8,835,609), the entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 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 with the European Collection of Cell Cultures (ECACC) under the accession number 06080101.

[0198] In a further embodiment, the binding polypeptide of the CAR comprises the V of an antibody described in the following documents H and / or V LAmino acid sequences of the chains: 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), the entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequences of 2-2-1 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 sequences 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.

[0199] In a further embodiment, the binding polypeptide of the CAR comprises the amino acid sequence of an antibody or fragment thereof described in 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), the entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 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 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.

[0200] 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 (e.g., trafficking and localization to a specific organelle such as the endoplasmic reticulum and / or the cell surface).

[0201] 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 to form the scFv. 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.

[0202] 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.

[0203] 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 domain 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).

[0204] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one normal effector function of an 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 part of a protein that transduces signals for effector functions and directs the cell expressing the CAR to perform specific functions. The intracellular signaling domain can include any intact, mutated, or truncated part of the intracellular signaling domain of a given protein that is sufficient to transduce signals that initiate or block immune cell effector functions.

[0205] The function of the intracellular domain can be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination of these.

[0206] 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.

[0207] Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain immunoreceptor tyrosine-based activation motif (ITAM) signaling motifs.

[0208] Examples of ITAMs containing primary 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 prominent is the sequence derived from CD3ζ.

[0209] The cytoplasmic domain of a CAR can be designed to contain the CD3-ζ signaling domain 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 a 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.

[0210] In some embodiments, the activating receptor (from which a portion of the signaling domain is derived) is a CD3 co-receptor complex or an Fc receptor.

[0211] In some embodiments, the co-stimulatory receptor (a portion of the signaling domain is 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.

[0212] In some embodiments, the co-stimulatory receptor (a portion of the signaling domain is derived from the co-stimulatory receptor) is selected from the group consisting of: CD28, OX40, or 4-1BB.

[0213] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other randomly or in a specific order, with or without a linker. Short oligopeptides or short polypeptides having a length preferably between 2 and 10 amino acids can form the linkage. A prominent linker is the glycine-serine doublet.

[0214] 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.

[0215] As described above, the extracellular portion or transmembrane domain or cytoplasmic domain of the CAR can also comprise a heterodimerization domain, for the purpose of splitting the key signaling and antigen recognition modules of the CAR.

[0216] A CAR that binds to a radiolabeled molecule of the present invention, such as a CAR comprising an 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, thereby producing a functional CAR.

[0217] The affinity of the dysfunctional P2X7 receptor binding domain of the CAR for the nfP2X7 recognition site E200 of the radiolabeled molecule of 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.

[0218] Receptors such as CARs, their variants, or TCRs or their variants are typically expressed by immune cells.

[0219] The immune cells can be the "engineered cells", "genetically modified cells" or "immune effector cells" described herein. In addition, 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.

[0220] 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.

[0221] 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).

[0222] 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).

[0223] 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.

[0224] As used herein, the terms "different CARs" or "different chimeric antigen receptors" refer to any two or more CARs having different antigen recognition domains and / or different 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.

[0225] Methods for detecting immune cells

[0226] 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 a relevant receptor. For example, in the case of a CAR that binds to the nfP2X7 receptor, 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.

[0227] In any embodiment, cells complexed with a polypeptide as described herein in a biological sample are not re-introduced into the individual.

[0228] It will be understood that the methods of the present invention can be used to detect immune cells expressing a receptor (including a CAR) that comprises an antigen-binding domain that recognizes an epitope of a dysfunctional P2X7 receptor contained on a fusion protein. The methods and fusion proteins / polypeptides of the present invention are capable of detecting target cells that bind to the fusion protein / polypeptide by modifying the fusion protein or polypeptide to be detectable (also referred to herein as a "detection moiety").

[0229] In any embodiment of the second aspect of the present invention, the biological sample can be any patient sample containing immune cells that are desired to be detected. Preferably, the sample represents the level of circulating immune cells expressing a receptor that binds to a dysfunctional P2X7 receptor. Preferably, the sample is a sample of peripheral blood, such as EDTA-anticoagulated peripheral blood or a derivative thereof, such as PBMC (buffy coat).

[0230] It should be understood that the biological sample can be derived from tissues or other fluids in the body that contain immune cells. Thus, the sample can also be derived from solid tissue that has been homogenized to produce a single cell suspension (e.g., using a gentleMACS dissociator).

[0231] Furthermore, when contacting the fusion protein or polypeptide of the present invention with any cell population containing target cells (i.e., cells that are 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 detect the fusion protein / cell complex and thereby confirm the presence or absence of immune cells in the sample.

[0232] Furthermore, the methods of the present invention allow for the quantification of immune cells expressing a receptor for binding to a dysfunctional P2X7 receptor.

[0233] It should be understood that the fusion proteins or polypeptides for use according to the present invention may include any detection moiety having properties or functions that can be used for direct and indirect detection purposes, such as those detection moieties selected from the group consisting of: chromophore moieties, fluorescent moieties, phosphorescent moieties, luminescent moieties, light-absorbing moieties, radioactive moieties, and chemically detectable moieties, such as haptens (e.g., biotin, avidin, streptavidin, and their derivatives), or magnetic particles.

[0234] In a preferred embodiment, the detection moiety is a fluorescent dye, a magnetic particle, or biotin.

[0235] Suitable fluorescent moieties are those known from the field of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In these embodiments of the present invention, target cells labeled with the reagent are detected by exciting the detection moiety D and detecting the resulting emission (photoluminescence). In this embodiment, the detection moiety D is preferably a fluorescent moiety.

[0236] Useful fluorescent moieties can be protein-based (such as phycobiliproteins), polymeric (such as polyfluorene), organic small molecule dyes (such as xanthenes, such as fluorescein or rhodamine, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethene), or metal-organic complexes such as Ru, Eu, Pt complexes.

[0237] In one embodiment, the modification for allowing detection, especially a fluorescent dye, can be destroyed by oxidation in a photo- or chemical bleaching method (U.S. Patent No. 7,741,045 B2, EP 0810428 B1, or DE 10143757), such that the fluorescence is quenched.

[0238] Magnetic particles useful for achieving detection are preferably magnetic particles in the nano- to micron-size range, also known as magnetic beads 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. Suitable particles are commercially available from Miltenyi Biotec GmbH, Germany, under the trade names "MicroBeads" and "MACSiBeads".

[0239] Cells bound to the fusion protein or polypeptide comprising the modification for allowing detection can be detected by fluorescence emission, by applying a magnetic field, or by a chemical reaction of the chemically detectable moiety.

[0240] In one embodiment of the present invention, the detection moiety is a fluorescent moiety. Target cells labeled with a fluorescent dye conjugate are detected by exciting the fluorescent moiety and analyzing the resulting fluorescent signal. The excitation wavelength is typically selected according to the maximum absorption of the fluorescent moiety and is provided by a LASER source or an LED source known in the art. If several different detection moieties are used for multi-color / parameter detection, care should be taken to select fluorescent moieties with non-overlapping absorption spectra, at least non-overlapping maximum absorptions. In the case where the fluorescent moiety is the detection moiety, the target can be detected, for example, under a fluorescence microscope, in a flow cytometer, a spectrofluorometer, or a fluorescence scanner. The light emitted by chemiluminescence can be detected by similar instruments that omit excitation.

[0241] In another embodiment of the present invention, the detection moiety is a light-absorbing moiety, which is detected by the difference between the intensity of the irradiated light and the intensity of the transmitted or reflected light. The light-absorbing moiety can also be detected by photoacoustic imaging, which uses the absorption of a pulsed laser beam to generate an acoustic signal similar to an ultrasonic signal.

[0242] The radioactive detection moiety is detected by the radiation emitted by a radioisotope. Suitable instruments for detecting radioactive radiation include, for example, scintillation counters. In the case of β emission, an electron microscope can also be used for detection.

[0243] The transition metal isotope mass tag moiety is detected by mass spectrometry (such as ICP-MS), which is integrated in a mass cytometry instrument. Metal labeling can also be used to allow the detection and quantification of cells using the CyTOF (by mass cytometry) method. CyTOF allows the simultaneous quantification of multiple cell components by using an ICP-MS detector. For such applications, the fusion protein or polypeptide can be labeled with a lanthanide element group and can be linked to the fusion protein or polypeptide via an isotope polymer, such as diethylenetriaminepentaacetic acid (DTPA) chelators. This enables thiols or maleimides to be linked to the Fc region of the fusion protein or antibody via a reduced disulfide bond. Four to five polymers bind to the antibody, resulting in approximately 100 isotope atoms per antibody. The labeled fusion protein can be in solution, conjugated to beads, or surface-fixed. Cell staining follows the same procedure as fluorescent staining used for flow cytometry.

[0244] In the case where the Fc fusion protein retains the ability to dimerize, the complex of the fusion protein and cells can be purified by using protein A beads and then the cells can be quantified by using conventional flow cytometry techniques.

[0245] Alternatively, a fluorophore-labeled anti-Fc antibody can be used to bind to the fusion protein complex and detect the presence of the fusion protein complex.

[0246] In the case where the Fc fusion protein is biotinylated, avidin antibodies, optionally fluorescently labeled avidin antibodies, can be used to isolate and detect the complex.

[0247] Of course, in the case where the Fc fusion protein contains a fluorescent label, the fluorescence intensity can be directly determined as a means for detecting the presence and amount of cells in a sample.

[0248] Examples

[0249] Example 1: Flow cytometry detection of CAR T cells for binding to dysfunctional P2X7 receptors

[0250] A whole blood sample was treated with 2 ml of an NH4Cl-based red blood cell lysis solution (Beckman Coulter, Krefeld, Germany) for 10 min and washed with PBS containing 0.5% HSA. After removing the supernatant, the cells were resuspended and 100 μl was transferred to a new flow cytometry tube. After incubating with the fusion protein containing the sequences of SEQ ID NO:158 or 146 (DetR1 and DetR2, respectively) for 15 minutes, the cells were washed and incubated with an avidin antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), 7-AAD, CD3-APC, and CD45-KrO (all purchased from Beckman Coulter Immunotech, Marseille, France) for 15 minutes.

[0251] After the final wash step, the cells were acquired on a NAVIOS flow cytometer (Beckman Coulter, Krefeld, Germany). Cell debris was excluded based on light scattering characteristics and CART cells were defined as 7-AAD- / CD45+ / monocytes / CD3+ / CD19 CAR+.

[0252] Example 2: Detection of CAR T cells using an exemplary biotinylated fusion protein of the present invention

[0253] PBMCs were obtained from donors and enriched for CD4+ T cells and CD8+ T cells. The cells were then either not transduced or transduced with a lentiviral construct encoding a CAR using standard techniques. The construct used also included a sequence encoding EGFR to allow for indirect detection of successfully transduced cells.

[0254] Untransduced T cells (UTD), and T cells expressing an anti-nfP2X7 CAR or an anti-CD33 CAR (based on lintuzumab) were stained with the first-stage labeled anti-EGFR antibody cetuximab to detect the truncated EGFR, which is co-expressed downstream of the CAR receptor after the ribosomal skip (T2A) site.

[0255] UTD cells are negative for both CAR and the marker gene. For the marker of cetuximab, use the AF647 (AlexaFluor TM 647 antibody labeling kit from ThermoFisher according to the manufacturer's instructions, catalog number: A20186). Stain at 1 μg / mL.

[0256] Use the DetR1-Fc attenuated fusion protein (SEQ ID NO: 145) to detect T cells expressing anti-nfP2X7 CAR. The DetR1-Fc attenuated fusion protein was biotinylated using the NHS-LC-LC-biotin biotinylation kit from ThermoFisher (EZ-Link TM NHS-LC-LC-biotin, catalog number: 21343) and biotinylated according to the manufacturer's instructions. According to the manufacturer's instructions, use the anti-biotin antibody from Miltenyi Biotec (product number 130-113-857, biotin antibody, ) as the secondary antibody.

[0257] As Figure 1 shown, both UTD and anti-CD33 CAR T cells are negative after staining with the DetR1 biotinylated molecule, while cells expressing anti-nfP2X7 CAR show double-positive staining in terms of CAR-positive cells: first, positive by the truncated EGFR of the marker gene; second, positive by detecting the CAR receptor itself using the Fc fusion protein. Therefore, the detection reagent DetR1 (SEQ ID NO: 158) can be used to specifically identify cells expressing anti-nfP2X7 CAR.

[0258] Perform similar experiments using PBMCs obtained from two other individuals. The results (not shown) are similar to the above results.

[0259] Example 3: Detection of CAR T cells using exemplary monomeric and dimeric biotinylated fusion proteins of the present invention

[0260] Perform experiments similar to those described in Example 2. T cells were obtained from donors, either untransduced T cells (UTD) (bottom panel), or cells transduced with a lentiviral construct encoding anti-nfP2X7 CAR (middle panel). For comparison, Jurkat cells were also transduced with the same lentiviral construct.

[0261] Three different biotinylated fusion proteins containing epitopes of the nfP2X7 receptor were used to detect CAR-expressing cells. The fusion proteins used were: the DetR1 dimer (SEQ ID NO:149); the DetR1 monomer (SEQ ID NO:158); and the DetR2 monomer (SEQ ID NO:146). Figure 2 The results shown in Figure 2 indicate that monomeric or dimeric fusion proteins can be used to detect CAR-expressing cells. However, monomeric fusion proteins are preferred because they do not cause cross-linking.

[0262] Figure 3 Differences in the expression of activation markers (CD25 and CD69) and exhaustion marker PD-1 in CAR T cells are shown, for example, where the CAR T cells have been contacted with a monomeric fusion protein or a homodimeric fusion protein (e.g., containing two copies of the E200 epitope sequence) as described herein. The results show that contacting CAR T cells with a monomeric detection reagent (e.g., having the amino acid sequence of SEQ ID NO:158) results in significantly less activation and exhaustion of CAR T cells over a 72-hour period compared to a homodimeric detection reagent (e.g., having the amino acid sequence of SEQ ID NO:149). Thus, using a monomeric detection reagent (e.g., having only one copy of the E200 peptide for binding to the CAR) provides a particular advantage in terms of functional assessment of CAR T cells after their detection. For example, if it is desired to determine the function of CAR T cells after detection, it is preferred that the detection reagent does not cause unwanted activation or exhaustion of the CAR T cells.

[0263] Similar experiments were performed using the heterodimeric asymmetric molecules described herein (e.g., such that the molecule comprises a dimer between an E200 peptide-Fc fusion protein and a non-identical Fc region of an antibody; using the KIH technique). The results similarly show that contacting CAR T cells with a heterodimeric asymmetric molecule containing a single copy of the E200 peptide sequence results in significantly less T cell activation and significantly less T cell depletion in a concentration-dependent manner compared to using a dimeric fusion protein containing two copies of the E200 peptide (e.g., where the dimer is a homodimer of an E200-Fc fusion protein). These results indicate that for the purpose of detecting CAR T cells (especially if there is an intention to subsequently determine function or perform other in vitro assays), it is preferred to use asymmetric heterodimeric molecules or monomeric fusion proteins (i.e., containing a single E200 peptide sequence) to minimize unwanted activation and depletion of CAR T cells in the patient.

[0264] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the 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, wherein, The Fc region has a reduced affinity for Fc receptors compared to a wild-type or naturally occurring Fc region.

2. The fusion protein according to claim 1, wherein, The Fc region is an Fc region of IgG, IgA, IgD, IgE or IgM.

3. The fusion protein according to claim 1 or 2, wherein, The Fc region is from an IgG antibody, and the IgG antibody is, for example, an IgG1 antibody, an IgG2 antibody, an IgG2b antibody, an IgG3 antibody, or an IgG4 antibody.

4. The fusion protein according to any one of claims 1 to 3, wherein, 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.

5. The fusion protein according to any one of claims 1 to 3, wherein, The Fc region comprises one or more amino acid substitutions that are used to reduce the affinity for one or more of FcγRI, FcγRII, and FcγRIII and thereby reduce the ability of the fusion protein to elicit antibody-dependent cell-mediated cytotoxicity (ADCC).

6. The fusion protein according to any one of claims 1 to 5, wherein, The fusion protein has an affinity for FcR of less than about 250 nM, preferably less than 500 nM, less than 1000 nM, and most preferably less than 2000 nM.

7. The fusion protein according to any one of claims 1 to 6, wherein, The Fc region of the fusion protein comprises one or more amino acid substitutions compared to a naturally occurring Fc sequence, and the one or more amino acid substitutions prevent or reduce the ability of the Fc region to homodimerize.

8. The fusion protein according to claim 7, wherein, The amino acid substitution comprises substitution of one or more cysteine residues to prevent the formation of disulfide bonds between Fc molecules.

9. The fusion protein according to claim 8, wherein, The substitution of the one or more cysteine residues is a substitution with glycine, serine, alanine, lysine, or glutamic acid, preferably glycine or serine.

10. The fusion protein according to any one of claims 1 to 9, wherein, The dysfunctional P2X7 receptor epitope moiety comprises the amino acid sequence of an epitope found on the dysfunctional P2X7 receptor and not found on the functional P2X7 receptor.

11. The fusion protein according to any one of claims 1 to 10, wherein, The amino acid sequence of the dysfunctional P2X7 receptor epitope moiety comprises at least the amino acid sequence listed in SEQ ID NO:14 or consists at least of the amino acid sequence listed in SEQ ID NO:

14.

12. The fusion protein according to any one of claims 1 to 11, wherein, The amino acid sequence of the dysfunctional P2X7 receptor epitope moiety comprises at least the amino acid sequence listed in SEQ ID NO:7 or 9 or 122 or consists at least of the amino acid sequence listed in SEQ ID NO:7 or 9 or 122.

13. The fusion protein according to any one of claims 1 to 11, wherein, The amino acid sequence of the dysfunctional P2X7 receptor epitope moiety comprises at least the following amino acid sequence or consists at least of the following amino acid sequence: the amino acid sequence listed in any one of SEQ ID NO:7 to 69 or SEQ ID NO: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 97%, at least 98% or at least 99% identical thereto, provided that the sequence comprises at least the sequence listed in SEQ ID NO:14 or 7.

14. The fusion protein according to any one of claims 1 to 12, wherein, The fusion protein comprises one or more modifications for enabling the detection of the fusion protein or a complex comprising the fusion protein.

15. The fusion protein according to claim 14, wherein, The one or more modifications of the fusion protein can be selected from the following: a fluorescent moiety, a metal label (such as a lanthanide element), a magnetic particle, a chromophore moiety, a phosphorescent moiety, a luminescent moiety, a light-absorbing moiety, a radioactive moiety, and a chemically detectable moiety, such as a hapten, for example biotin, avidin, streptavidin, and derivatives thereof.

16. Use of the fusion protein according to any one of claims 1 to 15 in a method for detecting genetically modified immune cells, wherein the genetically modified immune cells express a receptor comprising an antigen-binding domain, and the antigen-binding domain is for binding to a dysfunctional P2X7 receptor.

17. Use according to claim 16, wherein, The immune cell is a chimeric antigen receptor (CAR) cell.

18. Use according to claim 17, wherein,The use is for determining the presence of the immune cell in a complex mixture, such as a biological sample obtained from a patient who has previously received treatment with the immune cell.

19. An in vitro method for detecting immune cells, wherein the immune cells express a receptor comprising an antigen-binding domain for binding to a dysfunctional P2X7 receptor, the method comprising: (i) Provide a biological sample from a patient who has received treatment with an immune cell, preferably an immune effector cell, wherein the cell expresses a receptor comprising the antigen-binding domain for binding to the dysfunctional P2X7 receptor; (ii) Contact the sample 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 detection moiety for allowing the detection of the polypeptide, thereby allowing the formation of a complex of the polypeptide bound to the cell; and (iii) Detect the complex, thereby detecting the immune cell that expresses a receptor having an antigen-binding domain for binding to the dysfunctional P2X7 receptor.

20. An in vitro method for detecting immune cells, wherein the immune cells express a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor, the method comprising: (i) Provide a biological sample from a patient who has received treatment with an immune cell, preferably an immune effector cell, wherein the immune cell comprises a chimeric antigen receptor (CAR) for binding to the dysfunctional P2X7 receptor; (ii) Contact the sample with a polypeptide, wherein the polypeptide comprises an epitope of the dysfunctional P2X7 receptor recognized by the CAR, and wherein the polypeptide comprises a detection moiety for allowing the detection of the polypeptide, thereby allowing the formation of a complex of the polypeptide bound to the cell; (iii) Detect the complex, thereby detecting the immune cell that expresses a chimeric antigen receptor (CAR) for binding to the dysfunctional P2X7 receptor.

21. The method according to claim 19 or 20, wherein The method includes a step of first separating the complex before the detecting step.

22. The method according to any one of claims 19 to 21, wherein The polypeptide comprises the amino acid sequence of the fusion protein as defined in any one of claims 1 to 15.

23. The method according to any one of claims 19 to 21, wherein The polypeptide comprises a first portion that comprises an epitope of the dysfunctional P2X7 receptor linked to an additional portion for promoting the solubility and stability of the first portion.

24. The method according to claim 23, wherein The additional portion is an amino acid sequence that is linked to an epitope of the dysfunctional P2X7 receptor, such as a linker or hinge region, for example, the amino acid sequences exemplified in Tables 1 and 3; or the additional portion is a spacer comprising a polysaccharide having at least 15 carbon atoms, the polysaccharide being selected from the group consisting of dextran, amylopectin, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan, and chitin.

25. The method according to claim 23, wherein The polypeptide is in the form of a fusion protein that comprises an epitope of the dysfunctional P2X7 receptor linked to an additional amino acid sequence, the additional amino acid sequence being selected from serum albumin, transferrin, the carboxy-terminal peptide of the β-chain of chorionic gonadotropin (CG), a non-exact repeat peptide sequence, a polypeptide sequence consisting of a polymer of proline-alanine-serine, an elastin-like peptide (ELP) repeat sequence, a homopolymer of glycine residues, or a gelatin-like protein.

26. The method according to any one of claims 19 to 23, wherein The polypeptide is in the form of a conjugate that comprises a carbohydrate, lipid, liposome, peptide, or aptamer conjugated to the amino acid sequence comprising the epitope of the dysfunctional P2X7 receptor.

27. The method according to any one of claims 19 to 26, wherein The portion for allowing detection of the polypeptide is any suitable detectable portion, such as a fluorescent portion, magnetic particle, chromophore portion, phosphorescent portion, luminescent portion, light-absorbing portion, radioactive portion, and chemically detectable portion, such as a hapten, for example, biotin, avidin, streptavidin, and derivatives thereof.

28. The method according to claim 27, wherein When the polypeptide is labeled with a biotin moiety, the method comprises the step of contacting the cell (after step ii) with an avidin antigen-binding protein, preferably, wherein the avidin antigen-binding protein comprises one or more portions for allowing detection of the complex.

29. The method according to claim 28, wherein The avidin antigen-binding protein comprises a fluorophore.

30. The method according to any one of claims 19 to 29, wherein The biological sample from the patient is a sample of peripheral blood or a derivative thereof, such as a sample of serum, plasma, or a preparation of peripheral mononuclear cells (buffy coat).

31. A kit for use in the method according to any one of claims 19 to 30, the kit comprising: - A fusion protein or polypeptide capable of binding to a receptor (e.g., a CAR) for binding to a dysfunctional P2X7 receptor; - Optionally, one or more reagents for allowing detection of the fusion protein or polypeptide and its complex.

32. The fusion protein according to any one of claims 1 to 15, wherein, The protein comprises an amino acid sequence as listed in any one of SEQ ID NO: 145 to 158, 160, or 161.

33. The method according to any one of claims 19 to 29, wherein, The fusion protein comprises an amino acid sequence as listed in any one of SEQ ID NO: 145 to 158, 160, or 161.

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