How to detect immune cells

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to in vitro methods for detecting immune cells, and compositions and molecules for carrying them out.

[0002] Related Applications This application claims priority to Australian Provisional Application No. 2022902657, the entire contents of which are incorporated herein by reference. [Background technology]

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

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

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

[0006] Another important consideration during cellular immunotherapy protocols is the need to assess whether CAR T cells continue to expand in vivo after administration, which involves determining whether cells remain present in the patient's circulation at various time points after the initial infusion of cells.

[0007] Therefore, a need exists for methods and reagents for determining the presence of cellular immunotherapeutics, such as CAR T cells, in patient samples.

[0008] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, considered relevant, and / or combined with other pieces of prior art by a person skilled in the art. Summary of the Invention

[0009] The present invention finds particular use in the detection of genetically modified immune cells that are engineered to bind to dysfunctional P2X7 receptors on cancer cells. Thus, a fusion protein comprising: (i) a dysfunctional P2X7 receptor epitope portion; (ii) an Fc region of an antibody, preferably an Fc region of an antibody that has reduced affinity for an Fc receptor compared to a wild-type or naturally occurring Fc region.

[0010] The present invention provides (i) a peptide; (ii) an antibody Fc region, preferably an antibody Fc region having reduced affinity for an Fc receptor compared to a wild-type or naturally occurring Fc region; Fusion proteins are provided in which the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7 (preferably the amino acid sequence of SEQ ID NO: 14). Optionally, the peptide comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 7-69 or 122, or a sequence at least 80%, at least 81%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, provided that the sequence comprises at least the sequence set forth in SEQ ID NO: 14, 7, or 9.

[0011] The Fc region of the antibody can be an IgG, IgA, IgD, IgE, or IgM Fc region. Preferably, the Fc region is from an IgG antibody, such as an IgG1, IgG2, IgG2b, IgG3, or IgG4 antibody.

[0012] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chains.

[0013] The Fc region preferably contains one or more amino acid substitutions to reduce affinity for Fc receptors (FcR, including any of FcγRI, FcγRII, and FcγRIII), thereby reducing the ability of the fusion protein to elicit antibody-dependent cell-mediated toxicity (ADCC). Such substitutions are well known in the art and include, but are not limited to, "DANA" and "LALA" amino acid substitutions and variations thereof, as further defined herein.

[0014] In further embodiments, the Fc region may also contain substitutions that inhibit recruitment of complement C1q. Such mutations are also well known in the art and are further described herein.

[0015] In preferred embodiments, the fusion protein has an affinity for an FcR that is less than about 250 nM, preferably less than 500 nM, less than 1000 nM, and most preferably less than 2000 nM.

[0016] In a preferred embodiment, the Fc region of the fusion protein comprises one or more amino acid substitutions, relative to a naturally occurring Fc sequence, that prevent or reduce the ability of the Fc region to homodimerize. Preferably, the amino acid substitutions comprise one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules. The cysteine ​​residues in the Fc region may be substituted with any other amino acid residue, optionally with glycine, serine, alanine, lysine, and glutamic acid, preferably with glycine or serine.

[0017] The cysteine ​​residues for substitution are preferably one or more of the cysteine ​​residues located in the region of the Fc region corresponding to the hinge region of an immunoglobulin. Examples of IgG1 hinge regions and variations thereof, including cysteine-to-serine substitutions, are provided in Table 2 herein. The hinge region of an immunoglobulin (e.g., IgG1) contains three cysteine ​​residues (numbered C220, C226, and C229 according to EU numbering). Thus, in any embodiment, at least one, at least two, or all three of the cysteine ​​residues in the immunoglobulin hinge region are substituted. Preferably, at least two or all three of the cysteine ​​residues are substituted. More preferably, all cysteine ​​residues in the Fc region, such as the hinge region, are substituted. In a particularly preferred embodiment, at least one of C226 and C229 is substituted, and preferably both C226 and C229 are substituted.

[0018] Therefore, in a preferred embodiment, the fusion protein comprises a hinge region for linking a peptide (e.g., a dysfunctional P2X7 receptor epitope portion) and an Fc region of an antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences set forth in SEQ ID NOs: 76 to 113, or 136 to 137, or 141, or 142.

[0019] The present invention also provides heterodimeric asymmetric molecules comprising a fusion protein described herein (e.g., comprising a peptide of SEQ ID NO: 7 or 14, or a variant thereof exemplified by any of SEQ ID NOs: 2-69) and an antibody Fc region, and further comprising an antibody Fc region that does not contain the peptide. Such asymmetric heterodimeric molecules can be obtained using knobs-in-hole technology, as further described herein, to promote dimerization of non-identical Fc regions.

[0020] Preferably, the fusion protein or heterodimeric asymmetric molecule consists of or essentially consists of the peptide and the Fc region of an antibody, such that the fusion protein or heterodimeric asymmetric molecule does not contain the antigen-binding domain of an antibody (i.e., the fusion protein does not contain a VH, VL, Fab, Fv, or scFv derived from an antibody).

[0021] In any embodiment, the peptide (e.g., dysfunctional P2X7 receptor epitope portion) may comprise any amino acid sequence derived from a dysfunctional P2X7 receptor, but preferably comprises an epitope sequence that is found in a dysfunctional P2X7 receptor but not in a functional P2X7 receptor.

[0022] In a preferred embodiment, the amino acid sequence of the dysfunctional P2X7 receptor epitope portion comprises or at least consists of the amino acid sequence set forth in SEQ ID NO: 14. In a particularly preferred embodiment, the portion comprises at least the sequence set forth in SEQ ID NO: 7 or 9.

[0023] In any embodiment, the dysfunctional P2X7 receptor epitope portion comprises an amino acid sequence set forth in any of SEQ ID NOs: 7-69 or 122, or a sequence at least 80%, at least 81%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, provided that the sequence comprises at least the sequence set forth in SEQ ID NO: 14 or 7 or 9.

[0024] In any embodiment, the fusion protein comprises an amino acid sequence set forth in any of SEQ ID NOs: 145-158, 160, or 161, or a sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0025] In a further embodiment, the fusion protein of the present invention may comprise one or more modifications to allow detection of the fusion protein, including when the protein binds to an immune cell expressing an exogenous cell surface receptor comprising an antigen binding domain and an intracellular signaling domain (chimeric antigen receptor (CAR) or modified TCR). Preferably, the antigen binding domain of the receptor is for binding to the peptide described herein and / or for binding to a dysfunctional P2X7 receptor.

[0026] It will be understood that typically, the antigen-binding domain of the receptor will be capable of binding or recognizing the same peptide (e.g., dysfunctional P2X7 receptor epitope portion) contained in the fusion protein. Furthermore, it is within the skill of the art, with knowledge of the specific epitope and binding target of a given anti-dysfunctional P2X7 receptor CAR, to be able to design a suitable fusion protein according to the present invention for use in binding to a CAR.

[0027] The one or more modifications to the fusion protein may be selected from fluorescent moieties, metal particles (for use in cytometric time-of-flight, CyTOF, techniques), magnetic particles, chromophoric moieties, phosphorescent moieties, luminescent moieties, light-absorbing moieties, radioactive moieties, and chemically detectable moieties such as haptens, e.g., biotin, avidin, streptavidin, and derivatives thereof.

[0028] In the case of biotin or a magnetic moiety, the moiety may be conjugated to the fusion protein using any method known to one of skill in the art. In one example, the moiety is conjugated to the fusion protein via one or more lysine residues of the protein and / or at the amino terminus of the protein.

[0029] In a second aspect, the present invention also provides the use of the fusion protein of the first aspect or the polypeptide further described herein for detecting one or more genetically modified immune cells that express a receptor comprising an antigen-binding domain for binding to dysfunctional P2X7 receptor and / or peptide described herein.Preferably, the receptor expressed by immune cells is a chimeric antigen receptor (CAR) or optionally a modified T cell receptor (TCR).Preferably, the detection is an in vitro method to enable the determination of the presence of immune cells in a complex mixture, such as a biological sample obtained from a patient who has previously been treated with immune cells.

[0030] In a preferred embodiment of the second aspect, there is provided an in vitro method for detecting immune cells expressing a receptor comprising an antigen binding domain for binding to a dysfunctional P2X7 receptor, comprising: (i) providing a biological sample from a patient who has been treated 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; (ii) contacting 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 the polypeptide comprises a detection moiety to allow detection of the polypeptide; contacting, thereby allowing the formation of a cell-bound polypeptide complex; (iii) detecting the complex; Thereby, provide an in vitro method for detecting immune cells that express receptors that have antigen binding domains that bind to dysfunctional P2X7 receptors.Optionally, the method comprises first isolating the complex before detecting.

[0031] Preferably, the cell is an immune cell that expresses a chimeric antigen receptor (CAR) for binding to dysfunctional P2X7 receptor.Therefore, an in vitro method for detecting an immune cell that expresses a chimeric antigen receptor (CAR) for binding to dysfunctional P2X7 receptor, comprising: (i) providing a biological sample from a patient who has been treated with immune cells, preferably immune effector cells, comprising a chimeric antigen receptor (CAR) for binding to a dysfunctional P2X7 receptor; (ii) contacting the sample with a polypeptide, wherein the polypeptide comprises an epitope of the dysfunctional P2X7 receptor recognized by the CAR, and the polypeptide comprises a detection moiety to enable detection of the polypeptide; contacting, thereby allowing the formation of a cell-bound polypeptide complex; (iii) detecting the complex; Thereby, also provided is an in vitro method for detecting the immune cell that expresses chimeric antigen receptor (CAR) for binding to dysfunctional P2X7 receptor.Optionally, the method comprises first isolating the complex before detecting.

[0032] 1. An in vitro method for detecting immune cells expressing an exogenous cell surface receptor, the method comprising: detecting an exogenous cell surface receptor comprising an antigen-binding domain for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or, optionally, the amino acid sequence of any of SEQ ID NOs: 2 to 69 and 122), the method comprising: (i) providing a biological sample from a patient treated with immune cells, preferably immune effector cells, wherein the cells express an exogenous cell surface receptor comprising an antigen-binding domain for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or, optionally, the amino acid sequence of any of SEQ ID NOs: 2-69 and 122); (ii) contacting the sample with a polypeptide, wherein the polypeptide comprises a peptide recognized by the antigen-binding domain of the receptor, and the polypeptide comprises a detection moiety to allow detection of the polypeptide; contacting, thereby allowing the formation of a cell-bound polypeptide complex; (iii) detecting the complex; Thereby, an in vitro method is provided for detecting immune cells expressing an exogenous cell surface receptor having an antigen-binding domain for binding to an exogenous cell surface receptor comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2-69 and 122). Optionally, the method comprises first isolating the complex prior to the detecting step.

[0033] Preferably, the cell is an immune cell expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2 to 69 and 122). Accordingly, there is provided an in vitro method for detecting immune cells expressing a chimeric antigen receptor (CAR) for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or 14 (or optionally the amino acid sequence of any of SEQ ID NOs: 2 to 69 and 122), comprising: (i) providing a biological sample from a patient who has been treated with immune cells, preferably immune effector cells, comprising a chimeric antigen receptor (CAR) for binding to a peptide; (ii) contacting the sample with a polypeptide, wherein the polypeptide comprises a peptide recognized by the CAR, and the polypeptide comprises a detection moiety to enable detection of the polypeptide; contacting, thereby allowing the formation of a cell-bound polypeptide complex; (iii) detecting the complex; and detecting an immune cell expressing a chimeric antigen receptor (CAR). Optionally, the method includes first isolating the complex prior to the detecting step.

[0034] In any embodiment of the second aspect of the invention, the polypeptide comprises the amino acid sequence of the fusion protein of any embodiment of the first aspect of the invention.

[0035] In any embodiment of the second aspect of the present invention, the polypeptide comprises a first portion comprising a peptide (e.g., an epitope of a dysfunctional P2X7 receptor) linked to a further amino acid sequence to promote the solubility and stability of the first portion. The further amino acid sequence linked to the dysfunctional P2X7 receptor epitope may comprise any suitable linker or hinge region, such as those exemplified in Table 1 and Table 3. Such a linker or hinge region may comprise an amino acid sequence consisting of glycine and serine repeats (the so-called "GS" linker sequence, and its variants as further defined herein). The hinge region may also comprise a sequence derived from an immunoglobulin hinge region, such as those defined in Table 3.

[0036] In a further embodiment, the polypeptide may be in the form of a fusion protein comprising an epitope of a dysfunctional P2X7 receptor linked to a further amino acid sequence, which may comprise serum albumin, transferrin, the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta chain, an imprecise repeat peptide sequence, a polypeptide sequence composed of a proline-alanine-serine polymer, an elastin-like peptide (ELP) repeat sequence, a homopolymer of glycine residues, or a gelatin-like protein.

[0037] Furthermore, the polypeptide may be in the form of a conjugate comprising 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 may be via an activated carboxylic acid of the amino acid sequence comprising an epitope of a dysfunctional P2X7 receptor.

[0038] According to the second aspect of the invention, the moiety for enabling detection of the polypeptide may 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.

[0039] Optionally, the polypeptide is labeled with a biotin moiety, and the method may further comprise contacting the cell (after step ii) with an anti-biotin antigen-binding protein, preferably the anti-biotin antigen-binding protein comprising one or more moieties to allow detection of the complex. Optionally, the one or more moieties to allow detection of the complex comprise a fluorophore (i.e., such that the anti-biotin antibody is fluorescently labeled).

[0040] Optionally, the polypeptide comprises a magnetic label, and the detecting step comprises: i) applying a magnetic field to the population of cells; ii) removing or discarding cells that are not attracted to the magnetic field; and iii) removing the magnetic field, thereby providing the population of immune cells that express a chimeric antigen receptor (CAR) for binding to the dysfunctional P2X7 receptor.

[0041] In particularly preferred embodiments of the second aspect of the invention, the exogenous cell surface receptor comprising an antigen binding domain (e.g., a chimeric antigen receptor CAR) comprises an antigen binding domain comprising the CDR amino acid sequence of PEP2-2-1 as described in PCT / AU2010 / 001070 (or in WO2011020155, or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508). More preferably, the antigen-binding domain of the receptor (e.g., CAR) comprises or consists of the amino acid sequence of the PEP2-2-1 antigen-binding protein described in PCT / AU2010 / 001070 (WO2011020155, or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), which are incorporated herein by reference.

[0042] In any embodiment, the biological sample from the patient may be a sample of whole peripheral blood or a derivative thereof, such as a peripheral mononuclear monocyte (buffy coat) preparation.

[0043] In a further embodiment, there is provided a kit for use in the methods described herein, comprising: a fusion protein or polypeptide capable of being bound by a receptor (e.g., CAR) for binding to a dysfunctional P2X7 receptor; Optionally, a kit is provided that includes the fusion protein or polypeptide and one or more reagents to allow detection of the complex.

[0044] Optionally, the kit comprises written instructions for use in the method of the second aspect of the invention.

[0045] As used herein, unless the context requires otherwise, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, ingredients, elements, or steps.

[0046] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and referring to the accompanying drawings, in which:

[0047] Sequence information [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Brief explanation of the drawings]

[0048] [Figure 1] Detection of untransduced T cells (UTD) (lower panel), donor T cells expressing anti-nfP2X7 CAR (middle panel), and T cells expressing anti-CD33 CAR (upper panel). [Figure 2] Flow cytometry analysis of untransduced T cells (UTD) (bottom panel), donor T cells expressing an anti-nfP2X7 CAR (middle panel), and Jurkat cells expressing an 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). [Figure 3]Percentage of CD25+ / CD69+ and PD-L1+ cells at 24 hours (A), 48 hours (B), and 72 hours (C) after detection with monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NOs: 158 and 149, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0049] An important consideration during cellular immunotherapy protocols is the need to assess whether CAR T cells continue to expand in vivo after administration. This involves determining whether the cells remain present in the patient's circulation at various time points after the initial infusion of cells. Methods for detecting CAR T cells exist, but these typically rely on modifications to the CAR or immune cells expressing the CAR (such as the inclusion of a fluorescent label or tag). This approach is less desirable because it requires further genetic modification of the cells and / or the infusion of a foreign substance into the subject.

[0050] The approach of the present invention is non-invasive, does not require modification of the CAR or the immune cells expressing the CAR, and allows for the rapid determination of the presence of genetically modified immune cells expressing a receptor for binding to a dysfunctional P2X7 receptor in a patient sample.

[0051] The present invention provides fusion proteins (monomeric, homodimeric or heterodimeric molecules derived therefrom) comprising a linear epitope derived from the P2X7 receptor (e.g., as exemplified by any of SEQ ID NOs: 14 or 7) and an Fc region of an antibody. Such fusion proteins have particular use in the in vitro detection of CAR T cells when linked to a moiety that allows for detection of the protein.

[0052] In a particularly preferred embodiment, the Fc fusion protein is designed to contain only a single copy of a linear epitope derived from the P2X7 receptor. This can be achieved by introducing amino acid substitutions into the Fc region to prevent homodimerization, as described further herein, or alternatively, by using well-known knob-into-hole technology to ensure the formation of an asymmetric heterodimeric molecule (e.g., comprising an E200 peptide-Fc fusion protein and an Fc region that does not contain the E200 peptide). Such a monomeric or asymmetric heterodimeric molecule has the advantage of reducing activation of target immune cells and preventing unnecessary exhaustion of target immune cells (as further described herein in the Examples), particularly when there is a need or intention to determine the function or activation status of CAR T cells after their detection. Without wishing to be bound by theory, the inventors believe this is due to a reduced ability of the molecule to cross-link either two different CAR receptors on one cell or two different CAR receptors on two separate CAR-expressing cells.

[0053] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0054] For purposes of interpreting this specification, the following definitions shall generally apply and, whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0055] As used herein, the term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings, or for either meaning.

[0056] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "a dysfunctional P2X7 receptor epitope portion" means one dysfunctional P2X7 receptor epitope portion or two or more dysfunctional P2X7 receptor epitope portions.

[0057] As used herein, unless the context requires otherwise, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, ingredients, elements, or steps.

[0058] "Purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0059] "P2X7 receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, at least one of which has the amino acid sequence substantially as set forth in SEQ ID NO: 1 of Table 1 herein.

[0060] Insofar as P2X7 receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X7 receptor" encompasses naturally occurring variants of P2X7 receptor, for example, P2X7 monomers are isoforms (e.g., forms consisting of extracellular domain sequence or truncated forms thereof), naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants, including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of the monomers that form P2X7 receptor. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, P2X7 receptor may have a modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO: 1 may be substituted, deleted, or residues may be inserted.

[0061] "Functional P2X7 receptor" generally refers to the form of P2X7 receptor that has three intact binding sites or clefts for binding to ATP.When bound to ATP, functional receptor forms a non-selective sodium / calcium channel that converts into a pore-like structure that allows calcium ions and molecules up to 900 Da to enter the cell matrix, one of the consequences of which may be the induction of programmed cell death.In normal homeostasis, the expression of functional P2X7 receptor is generally limited to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes.In addition, there may be some expression of functional P2X7 receptor on erythrocytes and other cell types.

[0062] "Dysfunctional P2X7 receptor" (also called "non-functional" or (nf)P2X7) is a P2X7 receptor that has a defective response to ATP, such that it cannot form an apoptotic pore under physiological conditions. Dysfunctional P2X7 receptor or (nfP2X7 receptor) generally refers to a form of P2X7 receptor that has a different conformation from functional P2X7, whereby the receptor cannot form an apoptotic pore, but can still act as a non-selective channel by maintaining a single functional ATP-binding site located between adjacent monomers. One example occurs when one or more monomers have cis-isomerization at Pro210 (according to SEQ ID NO: 1). Isomerization can result from any molecular event that leads to misfolding of the monomer, including, for example, mutation of the monomer primary sequence or abnormal post-translational processing. One consequence of this isomerization is that the receptor cannot bind ATP at one, or more specifically, two, of the ATP-binding sites on the trimer, and as a result, cannot extend the opening of the channel. In such a situation, receptor cannot form pore, which limits the degree that calcium ions can enter into the cytosol.Dysfunctional P2X7 receptor is expressed on a wide range of epithelial cancers and hematopoietic cancers.As used herein, term " dysfunctional P2X7 receptor " can be used interchangeably with term " non-functional P2X7 receptor " or " nfP2X7 " receptor.

[0063] A "cancer-associated P2X7 receptor" is a P2X7 receptor that is generally found on cancer cells (including pre-neoplastic cells, neoplastic cells, malignant cells, benign cells, or metastatic cells) but not on non-cancerous or normal cells.

[0064] "E200 epitope" generally refers to the epitope having the sequence GHNYTTRNILPGLNITC (SEQ ID NO: 2), variants of which are exemplified in Table 1, including any of SEQ ID NOs: 3, or 7-69, and 122.

[0065] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO: 4) or a variant thereof, as defined in SEQ ID NO: 5.

[0066] "Composite epitope" generally refers to an epitope formed from the juxtaposition of the E200 epitope and the E300 epitope, or portions of these epitopes. An example of a composite epitope comprising the E200 epitope and the E300 epitope is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 6).

[0067] As used herein, the term "antigen" is intended to include a substance that binds to or induces the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and recognized by the adaptive immune system, including, but not limited to, antibodies or TCRs, or engineered molecules, including, but not limited to, transgenic TCRs, CARs, scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single-chain antibodies or multimers thereof, or any other molecule that can achieve high affinity binding to a structure.

[0068] "Epitope" generally refers to the portion of an antigen bound by the antigen-binding site of an antibody. An epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to a sequence of amino acids as in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope," i.e., an epitope in which the hypervariable loops of the CDRs bind to residues as they appear in the tertiary or quaternary protein structure.

[0069] With respect to a receptor that refers to an antigen-binding domain that recognizes and binds to a dysfunctional P2X7 receptor, the terms "binds to," "specifically binds to," or "specific for" are intended to mean that the receptor does not substantially recognize or bind to other antigens in a sample.

[0070] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present disclosure.

[0071] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system and performs a specific effector function, e.g., α-β T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic progenitor cell, such as an early progenitor cell subset that can mature or differentiate into pluripotent stem cells and somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (iPSCs and other artificial cell types). Preferred immune cells are cells with cytotoxic effector function, e.g., α-β T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or γ-delta T cells. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0072] As used herein, the term "autologous" refers to any material originating from the same subject into which it is subsequently reintroduced.

[0073] As used herein, the term "allogeneic" refers to any material derived from a different subject of the same species as the subject into which the material is being reintroduced.

[0074] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. These terms refer to the presence and / or expression of exogenous genes or nucleic acid sequences that, in turn, modify the genotype or phenotype of the cells or their progeny. The terms refer to the fact that cells, preferentially immune cells, can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins not naturally expressed in these cells. For example, immune cells can be engineered to express artificial constructs such as chimeric antigen receptors on their cell surface. For example, CAR sequences can be delivered into cells using adenovirus, adeno-associated virus (AAV)-based, retrovirus, or lentivirus vectors, or any other pseudotyped variants thereof, or any other gene delivery mechanism such as electroporation or lipofection with CRISPR / Cas9, transposons (e.g., Sleeping Beauty), or variants thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0075] The amino acid structures and single and three letter abbreviations used throughout this specification are defined in Table 2, which lists the 20 naturally occurring proteinogenic amino acids that occur in proteins as the L-isomer. Table 2 [ka] [Table 2]

[0076] As used herein, the term "non-proteinogenic amino acid" refers to an amino acid having a side chain that does not occur in the naturally occurring L-α-amino acids listed in Table 2. Examples of non-proteinogenic amino acids and derivatives include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of the natural amino acids.

[0077] As used herein, the term "α-amino acid" refers to an amino acid having a single carbon atom (α-carbon atom) separating its carboxyl terminus (C-terminus) and amino terminus (N-terminus). α-Amino acids include naturally occurring and non-naturally occurring L-amino acids, including their D-isomers and derivatives thereof, such as salts or derivatives in which functional groups are protected by suitable protecting groups. Unless otherwise specified, the term "amino acid" as used herein refers to an α-amino acid.

[0078] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 6 carbon atoms. Where appropriate, alkyl groups include alkyl groups having the specified number of carbon atoms, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. 1-6 Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 5-methylpentyl.

[0079] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey, or human. Preferably, the subject is a human. The subject may be a subject (patient) suffering from a disorder such as cancer. As used herein, the terms "subject" and "individual" may be used interchangeably.

[0080] Dysfunctional P2X7 receptor epitope region The present invention provides a fusion protein comprising a dysfunctional P2X7 receptor epitope portion.

[0081] The dysfunctional P2X7 receptor epitope portion can be provided in the form of dysfunctional P2X7 receptor or a fragment of dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP. Such receptor cannot widen the opening of non-selective calcium channel to apoptotic pore.

[0082] According to the present invention, the dysfunctional P2X7 receptor epitope portion is typically in the form of a peptide fragment of the dysfunctional P2X7 receptor. Typically, the peptide comprises an epitope that is not found on or is not available for binding to the functional P2X7 receptor.

[0083] In some embodiments, the peptide comprises a proline at amino acid 210 of the dysfunctional P2X7 receptor. In some embodiments, the peptide comprises one or more amino acid residues ranging from glycine at amino acid 200 to cysteine ​​at amino acid 216 of the dysfunctional P2X7 receptor.

[0084] A range of peptide fragments of dysfunctional P2X7 receptors are disclosed in PCT / AU2002 / 000061 (and corresponding publications WO2002 / 057306, and US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2008 / 001364 (and corresponding publications and PCT / AU2009 / 000869 (and corresponding publications WO2010 / 000041, and US8,597,643, US9,328,155, or US10,238,716), the contents of all of which are incorporated in their entirety. Exemplary peptides within these specifications that contain epitopes contemplated for use in the present invention are described below. [Table 3]

[0085] Non-limiting examples of variations of the E200 peptide sequence (including N-terminal and / or C-terminal extensions, and various linker, hinge, or spacer regions) are provided in Table 1.

[0086] The amino acid sequence of any one of SEQ ID NOs: 2 to 7 may comprise a portion of an epitope portion that can be recognized or bound by a receptor expressed on an immune cell (also referred to herein as the "recognition sequence" of the epitope portion).

[0087] 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.

[0088] In some embodiments, the N-terminus of the epitope moiety is a free amine (-NH2).

[0089] In some embodiments, the C-terminus of the epitope moiety is a free acid (—COOH). In some embodiments, the C-terminus is a derivative or analog of the free acid group, such as an ester (—COOC alkyl) or a primary or secondary amide (—CONHR, where R is selected from H and C alkyl). Advantageously, having a C-terminus that is a derivative or analog of the free acid group may improve the biological stability of the peptide compared to the free acid. In some embodiments, the C-terminus is a derivative or analog of the free acid group that includes a functional moiety, such as biotin.

[0090] In an embodiment of either the first or second aspect, the epitope of the dysfunctional P2X7 receptor comprises or consists of an epitope that is found in the dysfunctional P2X7 receptor but not in the functional form of the P2X7 receptor.In other words, preferably, the polypeptide comprises or consists of an epitope that is specific for the dysfunctional P2X7 receptor.

[0091] In a further embodiment of the first or second aspect, the fusion protein comprises an epitope corresponding to E200, E300, or a combined E200 / E300 epitope as defined herein. It is within the skill of the art to obtain various polypeptides for use according to the present invention. For example, one skilled in the art will understand that it is possible to include additional amino acids N-terminal or C-terminal to the region comprising the epitope bound by the anti-nfP2X7 receptor CAR. As a non-limiting example, and typically in the context of E200 defined as having an amino acid sequence substantially as defined in SEQ ID NO: 2 or 7 (and having a minimal sequence as defined in SEQ ID NO: 14), additional amino acids derived from the native sequence of P2X7 can be included within the polypeptide, for example, residues "DFP" N-terminal to the epitope in the P2X7 receptor sequence, and / or residues "TFHKT" C-terminal to the epitope in the P2X7 receptor sequence. In any embodiment, the polypeptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids from the P2X7 receptor sequence in addition to the sequence of E200 or E300, or composite epitope.

[0092] In a preferred embodiment, the sequence of the E200 epitope is further modified to replace the cysteine ​​residue (residue 17 of SEQ ID NO: 2) with a serine residue (e.g., to provide the sequence of SEQ ID NO: 7). One of skill in the art will understand that this can be done to reduce the possibility of any disulfide bonds between the polypeptide and another molecule.

[0093] It is also within the skill of the art to include additional amino acid residues in an E200, E300, or composite epitope (or an extended epitope as discussed in the preceding paragraph), 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- and C-terminal regions of a peptide consisting of the amino acid sequence of the relevant epitope. Typically, such additional amino acids can be derived from a linker sequence (such as a peptide comprising glycine and serine residues) or from the hinge region of an immunoglobulin. Typically, no more than 30 amino acid residues, no more than 25 amino acid residues, or no more than 20 amino acid residues are added to the N- and / or C-terminal residues of an E200, E300, or composite epitope as defined herein.

[0094] Fc area In any embodiment, the amino acid sequence of the epitope of dysfunctional P2X7 receptor can be fused to the N-terminal region of the Fc region of antibody or its variant via its C-terminal region.In any embodiment, the amino acid sequence of the epitope of dysfunctional P2X7 receptor can be fused to the C-terminal region of the Fc region of antibody or its variant via its N-terminal region.

[0095] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chains.

[0096] The Fc region may contain one or more amino acid sequence modifications compared to the naturally occurring Fc sequence.The Fc region may contain one or more amino acid substitutions, such as substitution of one or more cysteine ​​residues, to prevent dimerization of the molecule into the same molecule.It will be understood that any amino acid substitution that prevents dimerization of the Fc region may be used.Therefore, in vivo, the Fc fusion protein described herein may be a monomeric protein.

[0097] Thus, the Fc region of the fusion protein contains one or more amino acid substitutions, relative to a naturally occurring Fc sequence, that prevent or reduce the ability of the Fc region to homodimerize. Preferably, the amino acid substitutions include one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules. The cysteine ​​residues in the Fc region may optionally be substituted with any other amino acid residue, including glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

[0098] The cysteine ​​residues for substitution are preferably one or more of the cysteine ​​residues located in the region of the Fc region corresponding to the hinge region of an immunoglobulin. Examples of IgG1 hinge regions and variations thereof, including cysteine-to-serine substitutions, are provided in Table 3 herein. The hinge region of an immunoglobulin (e.g., IgG1) contains three cysteine ​​residues (numbered C220, C226, and C229 according to EU numbering). Thus, in any embodiment, at least one, at least two, or all three of the cysteine ​​residues in the immunoglobulin hinge region are substituted. Preferably, at least two or all three of the cysteine ​​residues are substituted. More preferably, all cysteine ​​residues in the Fc region, such as the hinge region, are substituted. In a particularly preferred embodiment, at least one of C226 and C229 is substituted, and preferably both C226 and C229 are substituted.

[0099] Therefore, in a preferred embodiment, the fusion protein comprises a hinge region for linking the dysfunctional P2X7 receptor epitope portion and an Fc region of an antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences set forth in SEQ ID NOs: 76 to 113, or 136 to 137, or 141, or 142.

[0100] In further embodiments, the fusion protein region may comprise an Fc region corresponding to the Fc "hole" or "knob" for use in "knob-in-hole" heterodimers. The use of such Fc sequences is known in the art and provides asymmetric heterodimeric molecules that bind to an additional Fc region that does not contain the epitope moiety, including fusion proteins having a single copy of the epitope moiety and an Fc region described herein.

[0101] Those skilled in the art will be familiar with techniques and Fc sequences that allow for the formation of so-called monomeric fusion proteins, including, but not limited to, the use of the "knob-into-hole" IgG1 format (Ridgway et al., (1996), Protein Eng, 9:617-621). Such an approach in the context of the present invention allows for the expression and purification of heterodimeric fusion proteins with only one copy of a peptide epitope (e.g., an epitope portion derived from the E200 epitope described herein) per molecule. Examples of "knob-into-hole" Fc pairings are provided herein in SEQ ID NOS: 157 and 159 (knob and hole, respectively), 158 and 159, respectively, 160 and 162 (hole and knob, respectively), and 161 and 162, respectively. Thus, in any embodiment, the present invention provides a fusion protein comprising the amino acid sequence of any of SEQ ID NOs: 2-69 and 122 linked to an Fc region defined in SEQ ID NO: 160 or 162, wherein the fusion protein can form a heterodimer with an Fc region that does not include an E200 peptide portion.

[0102] Therefore, the fusion proteins of the present invention are preferably capable of forming heterodimeric molecules containing a single E200-containing amino acid sequence (in other words, the Fc portion of the fusion protein can heterodimerize with the Fc region of an antibody that does not contain an E200 peptide fused thereto).

[0103] Preferably, the Fc region comprises one or more substitutions to eliminate or reduce effector function, such as reducing binding and activation via FcR, as further described below.

[0104] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region is the C-terminal region of an antibody. H 2 domain and C H In the context of the present invention, the Fc region comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and by the C H The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively, and are held together by hydrophobic interactions of the three domains.

[0105] In some embodiments, the fusion protein does not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that an antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed 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). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO2013 / 120929A1).

[0106] In a preferred embodiment, the Fc fusion proteins of the present invention comprise an Fc region with reduced effector function. Fc regions with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutation in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). For example, an antibody variant may comprise an Fc region with one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, substitutions are L234A and L235A (LALA) (see, e.g., WO2012 / 130831). Additionally, changes can be made in the Fc region that result in altered (i.e., reduced) C1q binding and / or complement dependent cytotoxicity (CDC), as described, for example, in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000) (e.g., G236R).

[0107] Further examples of modified Fc regions include those containing "LALALS" (amino acid substitutions L234A / L235A / M428L / N434S, as described in Zalevsky et al., (2010) Nat. Biotechnol. 28:157-159), LALAPG (amino acid substitutions L234A / L235A / P329G, as described in Gunn et al., (2021, Immunity 54:815)).

[0108] In some embodiments, the Fc region of the Fc fusion proteins of the invention may contain at least the "LALA" mutation (L234A and L235A) to reduce binding to FcR. The fusion protein may additionally or alternatively contain the mutation G346R to inhibit recruitment of complement C1q.

[0109] Other Fc modifications for use in the present invention include variants that reduce or eliminate binding to FcγRs and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Such variants are also referred to herein as "knockout variants" or "KO variants." Variants that reduce binding to FcγRs and complement are useful for reducing undesired interactions mediated by the Fc region. Preferred knockout variants are those referred to as "Fc Variants with Optimized

[0033] Preferred modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, numbering according to the EU index. Preferred substitutions include 234G, 235G, 236R, 237K, 268G, 269K, 270K, 271K, 272K, 273K, 274K, 275K, 276K, 277K, 278K, 279K, 280K, 281K, 282K, 283K, 284K, 285K, 286K, 287K, 288K, 289K, 290K, 291K, 292K, 293K, 294K, 295K, 296K, 297K, 298K, 299K, 300K, 301K, 302K, 303K, 304K, 305K, 306K, 307K, 308K, 309K, 310K, 311K, 312K, 313K, 314K, 315K, 316K, 317K, 318K, 319K, 320K, 321K, 322K, 323K, 324K, 325K, 326K, 327K, 328K, 330K, 331K, 332K, 333K, 334K, 335K, 336K Examples of variants include, but are not limited to, 7R, 269R, 325L, and 328R, where numbering is according to the EU index. A preferred variant includes 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 IgG2 and IgG4 Fc regions. Hybrid isotypes, such as the hybrid IgG1 / IgG2 isotype described in U.S. Patent Application No. 11 / 256,060, can be useful.Other modifications to reduce FcγR and complement interactions include, but are not limited to, substitutions 297A, 297D, 234A, 235A, 237A, 318A, 228P, 236E, ΔG236, 265G, 268Q, 297Q, 309L, 330S, 331S, 327Q, 220S, 226S, 229S, 238S, 233P, 234A, and 234V, as well as removal of glycosylation at position 297 by mutation or enzymatic means, or by production in an organism such as a bacterium that does not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691, which is incorporated by reference in its entirety.

[0110] In some embodiments, the Fc region of the fusion protein comprises mutations to the complement (C1q) and / or Fcγ receptor (FcγR) binding sites, and in some embodiments, such mutations can render the fusion protein incapable of antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC).

[0111] The Fc region used in the context of the present invention preferably does not induce cytotoxicity, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0112] The term "Fc region" also includes native-sequence Fc regions and variant Fc regions. The Fc region may include the carboxyl terminus of the heavy chain. Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain the full-length heavy chain or a truncated variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to EU numbering, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the Fc region of an antibody may be contemplated. Amino acid sequence variants of the Fc region of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues in the amino acid sequence of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., inducing or supporting an anti-inflammatory response.

[0113] The Fc region of an antibody can be the Fc region of any of the antibody classes, such as IgA, IgD, IgE, IgG, and IgM. The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, when used in the context of the present invention, an antibody can be the Fc region of an IgG. For example, the Fc region of an antibody can be the Fc region of an IgG1, IgG2, IgG2b, IgG3, or IgG4. In some embodiments, the fusion protein of the present invention comprises the Fc region of an IgG. In the context of the present invention, the Fc region of an antibody is the Fc region of an IgG, preferably an IgG1.

[0114] Linker region between dysfunctional P2X7 receptor epitope and Fc region The dysfunctional P2X7 receptor epitope and the Fc region of the antibody can be linked directly or via a linker sequence. The linker sequence can be a spacer sequence as defined herein or as exemplified in Table 1 or Table 3. Alternatively, the linker sequence can be any amino acid-based linker sequence commonly used in the art.

[0115] The linker is usually a peptide having a length of up to 20 amino acids, but may be up to 50 amino acids in length. The term "linked to" or "fused to" refers to a covalent bond, such as a peptide bond, formed between two moieties. Thus, in the context of the present invention, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the fusion protein provided herein may comprise a linker between the epitope of a dysfunctional P2X7 receptor and the Fc region of an antibody, for example, between the N-terminus of the Fc region and the C-terminus of the dysfunctional P2X7 receptor epitope. As another example, the fusion protein provided herein may comprise a linker between the epitope of a dysfunctional P2X7 receptor and the Fc region of an antibody, for example, between the C-terminus of the Fc region and the N-terminus of the dysfunctional P2X7 receptor epitope moiety. In particular, dysfunctional P2X7 receptor epitope portion can be fused to the N-terminus of Fc region via a C-terminal linker.This linker has the advantage that different polypeptides of fusion protein can be independently folded and behave as expected.Therefore, in the context of the present invention, dysfunctional P2X7 receptor epitope portion and antibody Fc region can be comprised in a single chain multifunctional polypeptide.

[0116] In some embodiments, the fusion protein of the present invention comprises a peptide linker. In some embodiments, the peptide linker connects the dysfunctional P2X7 receptor epitope portion to the Fc region of the antibody. In some embodiments, the peptide linker can comprise the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Ser (GGGGS). In some embodiments, the peptide linker can comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or more. The linker can be of different lengths, i.e., a (GS)n series of repeated glycine and serine residues (GS), where n is any number from 1 to 15 or more. For example, the linker can be (GS)3 (i.e., GSGSGS) or longer, or (GS)11 or longer. It will be understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or longer. Fusion proteins with linkers of such lengths are included within the scope of the present invention. Preferably, n is 3 or less (i.e., when n is equal to 3, the linker is GSGSGS).

[0117] In further embodiments, the linker may include the inclusion of amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linker region may also include the sequence GSGK.

[0118] The peptide linker may consist of a repeating series of Thr-Pro (TP) with one or more additional amino acids N- and C-terminal to the repeat sequence. For example, the linker may comprise or consist of the sequence GTPTPTPTPTGEF (also known as a TP5 linker). In further embodiments, the linker may be a short and / or alpha-helical rigid linker (e.g., A(EAAAK)3A, PAPAP, or a dipeptide such as LE or CC).

[0119] In a further embodiment, instead of or in addition to the above-mentioned glycine-serine-based linker region, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety linked to the Fc region of an antibody via a hinge region. The link between the dysfunctional P2X7 receptor epitope moiety and the Fc region may comprise a combination of a hinge region and a linker region.

[0120] Examples of suitable hinge regions include those derived from immunoglobulins. The hinge region may be derived from IgG1, IgG2, IgG3, or IgG4 and may contain one or more amino acid substitutions (e.g., to prevent or reduce the possibility of disulfide bridge formation). Alternative hinge sequences may be derived from alternative immunoglobulin domains: CD8A, CD8B, CD4, or CD28, TRAC, TRBC, TRGC, TRDC.

[0121] Additional linker sequences can include any sequence of any length of the CL / CH1 domain, but do not include all residues of the CL / CH1 domain, e.g., the first 5-12 amino acid residues of the CL / CH1 domain. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linkers can also be derived from immunoglobulin light chains, e.g., Cκ or Cλ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences derived from hinge regions, and other naturally occurring sequences from other proteins.

[0122] Table 3 below provides non-limiting examples of suitable hinge regions for use in linking dysfunctional P2X7 receptor epitope moieties and Fc regions in the molecules of the invention.

[0123] 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 a dysfunctional P2X7 receptor epitope portion directly conjugated (N-terminus to C-terminus) to the Fc region. Alternatively, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, and then an Fc region. Still further, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, and then an Fc region. In yet another embodiment, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, another linker region, and then an Fc region. Of course, one skilled in the art will understand that alternative configurations are possible (i.e., the dysfunctional P2X7 receptor epitope portion is linked to the C-terminus of the Fc region via one or more linkers and / or hinge regions). [Table 4-1] [Table 4-2]

[0124] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is fused directly to the Fc region of the antibody, such that there is no linker between the two regions of the fusion protein.

[0125] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is linked to the Fc region of the antibody via a cleavable linker.

[0126] Cleavable linkers are well known in the art and include, for example, the sequence defined in SEQ ID NO: 144, which defines a cleavage site for the human rhinovirus 3C protease. Proteases for cleaving such cleavage sites are also readily available from commercial sources (e.g., Pierce HRV 3C Protease).

[0127] Other known cleavable linkers and other linkers that can be used in accordance with the present invention are disclosed in Chen et al., (2013) Adv. Drug. Deliv. Rev. 65:1357-1369, the contents of which are incorporated herein by reference.

[0128] In a further embodiment of the second aspect of the present invention, the dysfunctional P2X7 receptor epitope portion is linked to the further amino acid sequence of the polypeptide via a spacer comprising a polysaccharide having at least 15 carbon atoms selected from the group consisting of dextran, pullulan, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan, and chitin.

[0129] In yet a further embodiment of the second aspect of the present invention, the polypeptide comprises a dysfunctional P2X7 receptor epitope portion linked to a modification to allow detection of the polypeptide via a spacer comprising a polysaccharide having at least 15 carbon atoms selected from the group consisting of dextran, pullulan, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan, and chitin.

[0130] 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 consisting of an oligopeptide, a polyethylene glycol, an enzymatically degradable unit or an affinity unit, which provides a cleavable non-covalent linkage of the detection moiety and the dysfunctional P2X7 receptor epitope moiety.

[0131] Suitable oligopeptides contain at least 2 amino acids, preferably up to 10 amino acids. Most preferred are oligopeptides having the amino acid sequence GGGSK. Preferred polyethylene glycols (PEGs) contain 10 to 200 ethylene glycol units.

[0132] The enzymatically degradable spacer S can be any molecule that can be cleaved by a specific enzyme. Suitable enzymatically degradable spacers S include, for example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids, and derivatives thereof that can be cleaved by hydrolases. The enzymatically degradable spacer S can be composed of two or more different enzymatically degradable units that can be degraded by the same or different enzymes.

[0133] Preferred polysaccharides are, for example, dextran, pullulan, inulin, amylose, cellulose, hemicelluloses such as xylan or glucomannan, pectin, chitosan, or chitin.

[0134] The detection moiety and dysfunctional P2X7 receptor epitope moiety can be covalently or non-covalently bound to the spacer.Methods for covalent or non-covalent conjugation are known by those skilled in the art.In the case of the covalent bond between the detection moiety and / or dysfunctional P2X7 receptor epitope moiety and the spacer, the activation group on either the detection moiety and / or dysfunctional P2X7 receptor epitope moiety or on the spacer can be directly reacted with any functional group on either the spacer or the detection moiety and / or dysfunctional P2X7 receptor epitope moiety, or via a heterobifunctional linker molecule that first reacts with one and then reacts with the other binding partner.

[0135] For non-covalent or quasi-covalent binding of the detection moiety D to the dysfunctional P2X7 receptor epitope moiety via a spacer, the spacer may be provided with an affinity unit that can be cleaved by a release agent as needed. The affinity unit may include, for example, biotin, avidin, and / or streptavidin, resulting in a quasi-covalent bond with a dissociation constant of less than 10 M.

[0136] The term " release agent " refers to any compound that can be bound to a part of affinity unit.For example, the biotin-avidin affinity unit used as spacer can be cleaved by streptavidin or by adding the access of free biotin.Through competitive reaction, the affinity unit is cleaved, thereby releasing the detection part from the dysfunctional P2X7 receptor epitope part (or vice versa).Suitable affinity unit and release agent are disclosed by James Hirsch et al. in Analytical Biochemistry 308 (2002) 343-357.

[0137] The affinity unit may be provided with a detection means, i.e., may have a label that can be used for detection. The detection means may be the same as or different from those described for the detection moiety. The use of the affinity unit provided with a detection means as a spacer allows for further quantification of CAR cells.

[0138] Receptors and immune cells that express them The present invention finds use in the method for detecting the subpopulation of immune cells that express the receptor for binding to dysfunctional P2X7 receptor.Receptor is preferably chimeric antigen receptor (CAR) or its variant.Receptor can also be modified TCR.

[0139] Generally, CAR, its variant or TCR can comprise an extracellular domain (extracellular portion) comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain. The extracellular domain can be linked to the transmembrane domain by a linker. The extracellular domain can also comprise a signal peptide. Preferably, the extracellular portion of CAR, its variant or TCR comprises the nfP2X7 binding domain that recognizes the E200 (or E300, or E200-300 complex) epitope disclosed herein.

[0140] Typically, the antigen recognition domain of the CAR or TCR comprises a binding polypeptide comprising amino acid sequence homology to one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide comprises one or more V of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L The CAR preferably comprises amino acid sequence homology to the CDR1, CDR2, and CDR3 domains of the chain. As can be seen, the CAR will preferably be able to recognize the same dysfunctional P2X7 receptor epitope portion as present on the fusion protein of the present invention.

[0141] While it will be appreciated that any CAR for binding to a dysfunctional P2X7 receptor can be used in accordance with the methods of the present invention, in a preferred embodiment, the binding polypeptide of the CAR is a CAR described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399, Nos. 617, 8,709,425, 9,663,584, or 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent No. 8,440,186, Nos. 9,181,320, 9,944,701, or 10,597,451), PCT / AU2008 / 001365 (or corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Pat. Nos. 8,597,643, 9,328,155, or 10,238, 716), PCT / AU2010 / 001070 (or any one of the corresponding publications WO / 2011 / 020155, US9,127,059, US9,688,771, or US10,053,508), and PCT / AU2010 / 001741 (or any one of the corresponding publications WO2011 / 075789 or US8,835,609). H and / or V LThe antibody comprises the amino acid sequences of the CDRs of the 2-2-1 chains, the entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequences of 2-2-1, as described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010-0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0142] In further embodiments, the CAR binding polypeptide is a polypeptide of interest described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,450). ,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or No. 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Patents Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Patents Nos. 8,597,643, 9,328,155 or 10,238,716). and the V of antibodies described in PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US9,127,059, US9,688,771, or US10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO2011 / 075789 or US8,835,609). H and / or V LThe entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequences of 2-2-1, as described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010-0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0143] In further embodiments, the CAR binding polypeptide is a polypeptide of interest described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,450,380). any one of PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or 10,597,451) Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Patent Nos. 8,597,643, 9,328,155, or 10,238,716), PCT / AU2010 / 001070 (or corresponding publications and PCT / AU2010 / 001741 (or any one of the corresponding publications WO2011 / 075789 or US8,835,609), the entire contents of which are incorporated herein by reference.Preferably, the antibody comprises the CDR amino acid sequences of 2-2-1, as described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010-0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0144] CARs also typically include a signal peptide. "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a specific organelle (such as the endothelial endoplasmic reticulum) and / or to the cell surface.

[0145] Generally, the "antigen-binding domain" (or antigen-recognition domain) refers to the region of a CAR that specifically binds to an antigen (and thus can target cells containing the antigen). A CAR may contain one or more antigen-binding domains. Generally, the target region on a CAR is extracellular. The antigen-binding domain may comprise an antibody or an antibody-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a bispecific antibody. Any molecule that specifically binds to a given antigen, such as an affibody or ligand-binding domain from a naturally occurring receptor, may be used as the antigen-binding domain. Often, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of an immunoglobulin heavy chain and a light chain are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3-linker" and variations thereof, although one skilled in the art will understand that a variety of linker sequences and formats may be used.

[0146] CARs may also include a "hinge" region (sometimes called a spacer region or linker region) that links the antigen-binding domain to the transmembrane domain. This is typically a hydrophilic region located between the antigen-binding domain and the transmembrane domain. CARs may include an extracellular hinge domain, but it is also possible to exclude such a hinge. The hinge region may include, for example, an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial hinge sequence, or a combination thereof. One example of a hinge region is the CD8α hinge.

[0147] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source for such a domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8α or CD28. If the main signaling and antigen recognition modules (domains) are located on two (or more) polypeptides, the CAR can have two (or more) transmembrane domains. The division of the main signaling and antigen recognition modules allows for small molecule-dependent, titratable, and reversible control of CAR cell expression (Wu et al., 2015, Science 350:293-303), which is due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR.

[0148] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to perform a specific function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector function.

[0149] The function of the intracellular domain may be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0150] Examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequence of the T cell receptor (TCR) and co-receptors that initiate signal transduction after antigen receptor binding.

[0151] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0152] Examples of ITAMs containing primary cytoplasmic signaling sequences commonly used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most notably, the sequence derived from CD3 zeta.

[0153] The cytoplasmic domain of the CAR can be designed to contain a CD3-zeta signaling domain, either alone or in combination with any desired cytoplasmic domain. The cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for efficient and sufficient lymphocyte response to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0154] 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.

[0155] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0156] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0157] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order, with or without a linker. A short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A prominent linker is a glycine-serine duplex.

[0158] As another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0159] As mentioned above, either the extracellular portion of the CAR, or the transmembrane domain, or the cytoplasmic domain may also contain a heterodimerization domain for the purpose of separating the main signaling and antigen recognition modules of the CAR.

[0160] A CAR that binds a radiolabeled molecule of the present invention, for example a CAR that comprises an nfP2X7E200 binding domain, may be designed to include any portion or part of the above-mentioned domains described herein in any order and / or combination that results in a functional CAR.

[0161] The affinity that dysfunctional P2X7 receptor binding domain of CAR binds to nfP2X7 recognition site E200 of radiolabeled molecule of the present invention can vary, but generally, binding affinity can be within the range of about 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM or about 1 nM, preferably at least about 10 pM or 1 pM.In preferred embodiments, binding affinity is at least about 1 nM or at least about 10 nM.

[0162] The receptor (e.g., a CAR, a variant thereof, or a TCR, or a variant thereof) is typically expressed by an immune cell.

[0163] The immune cells may be "engineered cells," "genetically modified cells," or "immune effector cells" as described herein. Furthermore, the immune cells may be immune cell precursors that can differentiate into immune cells. Cells that can differentiate into immune cells (e.g., T cells expressing a dysfunctional P2X7CAR) may be stem cells, multilineage progenitor cells, or induced pluripotent stem cells.

[0164] The immune cells can be leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells (including CD4+ T cells or CD8+ T cells), natural killer cells, natural killer T cells, or γδ T cells.

[0165] In any embodiment, the immune cell may be a T cell, and optionally the T cell does not express TcRαβ, PD1, CD3, or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0166] In any embodiment, the immune cells optionally do not express accessory molecules, which may be checkpoint, exhaustion, or apoptosis-related signaling receptors, and ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L, and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0167] In some embodiments, immune cells comprise two or more different receptors (for example, two or more CARs, or variants thereof). CARs can bind to different epitopes on the same target molecule (for example, different epitopes on dysfunctional P2X7 receptor). Alternatively, CARs can bind to different target molecules, so that only one of CARs binds to dysfunctional P2X7 receptor.

[0168] As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs that have either non-identical antigen recognition and / or non-identical signal transduction domains.In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize dysfunctional P2X7 receptors), but have different signal transduction domains, such as one CAR with a signal transduction domain that has a part of an activating receptor and the other CAR with a signal transduction domain that has a part of a costimulatory receptor.As will be understood, at least one of the two or more CARs in this embodiment has an antigen recognition domain that recognizes dysfunctional P2X7 receptors, and the other CAR can take any suitable form and be directed against any suitable antigen.

[0169] Methods for detecting immune cells It is well within the scope of those skilled in the art to confirm the ability of a given fusion protein or polypeptide defined herein to be bound by relevant receptors.For example, in the context of nfP2X7 receptor-binding CAR, those skilled in the art can use conventional techniques to confirm the binding of a polypeptide (or a series of polypeptides) by CAR, thereby determining the suitability of the polypeptide for use in the method of the present invention.

[0170] In any embodiment, the cells in the biological sample complexed with a polypeptide described herein are not reintroduced into the individual.

[0171] It will be understood that the method of the present invention can be used to detect immune cells expressing receptors (including CARs) that contain an antigen-binding domain that recognizes the epitope of the dysfunctional P2X7 receptor contained in the fusion protein.The method and fusion protein / polypeptide of the present invention allow for the detection of target cells that bind to the fusion protein / polypeptide through modifications to the fusion protein or polypeptide (also referred to herein as "detection moiety") that allow for its detection.

[0172] In any embodiment of the second aspect of the present invention, biological sample can be any patient sample that contains the immune cell that is desired to be detected.Preferably, sample represents the level of circulating immune cell that expresses the receptor that binds to dysfunctional P2X7 receptor.Preferably, sample is peripheral blood, for example, EDTA anticoagulated peripheral blood, or its derivative, for example, PBMC (buffy coat) sample.

[0173] It will be understood that a biological sample may be derived from tissues or other fluids within the body that contain immune cells. Thus, a sample may also be derived from solid tissue that has been homogenized to produce a single-cell suspension (e.g., using a gentleMACS Dissociator).

[0174] Furthermore, when the fusion protein or polypeptide of the present invention is contacted with any population of cells, including target cells (i.e., cells that can bind to the dysfunctional P2X7 receptor epitope on the fusion protein), one skilled in the art can use routine laboratory techniques to detect the fusion protein / cell complexes and thereby confirm the presence or absence of immune cells in the sample.

[0175] Still further, the method of the present invention allows for the quantification of immune cells expressing the receptor for binding to the dysfunctional P2X7 receptor.

[0176] It will be understood that fusion proteins or polypeptides for use according to the invention may comprise any detection moiety having properties or functions that can be used for direct and indirect detection purposes, such as those 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 derivatives thereof, or magnetic particles.

[0177] In preferred embodiments, the detection moiety is a fluorescent dye, a magnetic particle, or biotin.

[0178] Suitable fluorescent moieties are those known from the field of immunofluorescence techniques, e.g., flow cytometry or fluorescence microscopy. In these embodiments of the invention, target cells labeled with a reagent are detected by exciting detection moiety D and detecting the resulting emission (photoluminescence). In this embodiment, detection moiety D is preferably a fluorescent moiety.

[0179] Useful fluorescent moieties can be protein-based such as phycobiliproteins, polymer-based such as polyfluorenes, small organic molecule dyes such as xanthenes like fluorescein, or rhodamines, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethenes, or metal-organic complexes such as Ru, Eu, and Pt complexes.

[0180] In one embodiment, modifications to allow detection, particularly fluorescent dyes, can be destroyed by oxidation in a photo- or chemical bleaching procedure (U.S. Patent No. 7,741,045 B2, EP 0810 428 B1, or DE 10143757), such that fluorescence is quenched.

[0181] Magnetic particles useful for enabling detection are preferably nano- to micro-scale magnetic particles, also known in the art as magnetic beads. The average diameter of the beads can range from 10 nm to 10 μm. Biocompatible magnetic particles are commercially available and consist, for example, of dextran molecules or of magnetic iron oxide coated with a silica shell. 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."

[0182] Cells that bind to a fusion protein or polypeptide containing a modification to enable detection can be detected by fluorescence, by application of a magnetic field, or by chemical reaction of a chemically detectable moiety.

[0183] 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 wavelength of excitation is typically selected according to the absorption maximum of the fluorescent moiety and is provided by a laser or LED source, as is known in the art. When using multiple different detection moieties for multi-color / parameter detection, care must be taken to select the fluorescent moieties so that they do not have overlapping absorption spectra, or at least overlapping absorption maxima. In the case of a fluorescent moiety as the detection moiety, the target can be detected, for example, under a fluorescence microscope, in a flow cytometer, a spectrofluorometer, or a fluorescence scanner. Light emitted by chemiluminescence can be detected by similar instrumentation, omitting excitation.

[0184] In another embodiment of the invention, the detection moiety is a light-absorbing moiety that is detected by the difference between the irradiated light intensity and the transmitted or reflected light intensity. Light-absorbing moieties can also be detected by photoacoustic imaging, which uses the absorption of a pulsed laser beam to generate acoustic, such as ultrasound, signals.

[0185] Radioactive detection moieties are detected via radiation emitted by radioisotopes. Suitable instruments for detecting radioactive radiation include, for example, scintillation counters. In the case of beta radiation, electron microscopes can also be used for detection.

[0186] The transition metal isotope mass tag moiety is detected by mass spectrometry, such as ICP-MS, integrated into mass cytometry instrumentation. Metal labels can also be useful for enabling cell detection and quantification using CyTOF (time-of-flight cytometry) methodology. CyTOF allows for the simultaneous quantification of multiple cellular components using an ICP-MS detector. For such applications, fusion proteins or polypeptides can be labeled with elemental lanthanide groups and linked to the fusion protein or polypeptide via an isotopic polymer containing, for example, a diethylenetriaminepentaacetic acid (DTPA) chelator. This allows thiols or maleimides to be attached to the Fc region of the fusion protein or antibody via a reduced disulfide bond. Four to five polymers are attached to the antibody, resulting in approximately 100 isotopic atoms per antibody. The tagged fusion protein can be in solution, conjugated to beads, or surface-immobilized. Cell staining follows the same procedure as fluorescent staining for flow cytometry.

[0187] For Fc fusion proteins that retain dimerization ability, complexes of fusion protein and cells may be purified using protein A beads, and the cells may then be quantified using conventional flow cytometry techniques.

[0188] Alternatively, a fluorophore-labeled anti-Fc antibody may be used to bind to and detect the presence of the fusion protein complex.

[0189] If the Fc fusion protein is biotinylated, an anti-biotin antibody, optionally fluorescently labeled, may be used to isolate and detect the complex.

[0190] Of course, in situations where the Fc fusion protein contains a fluorescent label, the degree of fluorescence can be determined directly as a means to detect the presence and quantity of cells in a sample. [Example]

[0191]

[0192] Example 1: Flow cytometry detection of CAR T cells for binding to dysfunctional P2X7 receptors Whole blood samples were treated with 2 ml of NH4Cl-based red blood cell lysis solution (Beckman Coulter, Krefeld, Germany) for 10 minutes 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 15 minutes of incubation with fusion proteins containing SEQ ID NO: 158 or 146 (DetR1 and DetR2, respectively), the cells were washed and incubated for 15 minutes with anti-biotin antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), 7-AAD, CD3-APC, and CD45-KrO (all purchased from Beckman Coulter Immunotech, Marseille, France).

[0193] After a final washing step, cells are acquired on a NAVIOS flow cytometer (Beckman Coulter, Krefeld, Germany). Cell debris is excluded based on light scatter characteristics, and CAR T cells are defined as 7-AAD- / CD45+ / mononuclear cells / CD3+ / CD19 CAR+.

[0194] Example 2: Detection of CAR T cells using exemplary biotinylated fusion proteins of the invention PBMCs were obtained from donors and enriched for CD4+ and CD8+ T cells. Cells were then either left untransduced or transduced with a lentiviral construct encoding a CAR using standard techniques. The constructs used also contained sequences encoding EGFR to allow for indirect detection of successfully transduced cells.

[0195] Untransduced T cells (UTD) and anti-nfP2X7CAR or anti-CD33 CAR (based on lintuzumab) were stained with the primary labeled anti-EGFR antibody cetuximab to detect the co-expressed truncated EGFR downstream of the CAR receptor after the ribosomal skip (T2A) site.

[0196] UTD cells are negative for both CAR and marker genes. For cetuximab labeling, ThermoFisher's AF647 (Alexa Fluor™ 647 Antibody Labeling Kit, Catalog No.: A20186) was used according to the manufacturer's instructions. Staining was performed at 1 μg / mL.

[0197] Detection of anti-nfP2X7CAR-expressing T cells was performed using a biotinylated DetR1-Fc attenuated fusion protein (SEQ ID NO: 145) using ThermoFisher's NHS-LC-LC-Biotinylation Kit (EZ-Link™ NHS-LC-LC-Biotin Catalog No.: 21343) according to the manufacturer's instructions. As a secondary antibody, anti-biotin (130-113-857, Biotin Antibody, VioBlue®) from Miltenyi Biotec was used according to the manufacturer's instructions.

[0198] As shown in Figure 1, UTD and anti-CD33 CAR T cells were both negative after staining with the DetR1 biotinylated molecule, while cells expressing the anti-nfP2X7CAR showed double positive staining for CAR-positive cells: first, via the marker gene truncated EGFR, and second, via the CAR receptor itself using an Fc fusion protein. Thus, the detection reagent DetR1 (SEQ ID NO: 158) can be used to specifically identify anti-nfP2X7CAR-expressing cells.

[0199] Similar experiments were performed using PBMCs obtained from two other individuals, and the results (not shown) were similar to those described above.

[0200] Example 3: Detection of CAR T cells using exemplary monomeric and dimeric biotinylated fusion proteins of the invention An experiment similar to that described in Example 2 was performed. T cells were obtained from donors and were either untransduced T cells (UTD) (lower panel) or transduced with a lentiviral construct encoding an anti-nfP2X7 CAR (middle panel). For comparison, Jurkat cells were transduced with the same lentiviral construct.

[0201] CAR-expressing cells were detected 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). The results shown in Figure 2 indicate that either monomeric or dimeric fusion proteins can be used to detect CAR-expressing cells. However, monomeric fusion proteins are preferred because they do not lead to cross-linking.

[0202] [

[0203] Figure 3 shows the differential expression of activation markers (CD25 and CD69) and the exhaustion marker PD-1 in CAR T cells contacted with either a monomeric fusion protein or a homodimeric fusion protein (e.g., comprising two copies of the E200 epitope sequence), e.g., as described herein. The results indicate that contacting CAR T cells with a monomeric detection reagent (e.g., having the amino acid sequence of SEQ ID NO: 158) leads to significantly less CAR T cell activation and exhaustion 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) offers certain advantages in relation to functional assessment of CAR T cells following their detection. For example, if it is desirable to determine the function of CAR T cells following detection, it is preferable that the detection reagent not result in undesired activation or exhaustion of CAR T cells.

[0204] Similar experiments are performed using the heterodimeric asymmetric molecules described herein (e.g., such that the molecules comprise dimerization between an E200 peptide-Fc fusion protein and a non-identical Fc region of an antibody using KIH technology). The results similarly show that contacting CAR T cells with heterodimeric asymmetric molecules comprising a single copy of the E200 peptide sequence leads to significantly less T cell activation and significantly less T cell exhaustion in a concentration-dependent manner compared to using a homodimeric fusion protein comprising two copies of the E200 peptide (e.g., the dimer is a homodimer of E200-Fc fusion protein). These results indicate that it is preferable to use asymmetric heterodimeric molecules or monomeric fusion proteins (i.e., comprising a single E200 peptide sequence) for the purposes of CAR T cell detection (especially if there is an intention to subsequently determine function or perform other in vitro analyses) to minimize undesired activation and exhaustion of CAR T cells in patients.

[0205] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

Claims

1. A fusion protein comprising: (i) Dysfunctional P2X ７ a receptor epitope portion; (ii) an Fc region of an antibody, wherein the Fc region has a reduced affinity for an Fc receptor compared to a wild-type or naturally occurring Fc region.

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

3. 3. The fusion protein of claim 1 or 2, wherein the Fc region is from an IgG antibody, such as an IgG1, IgG2, IgG2b, IgG3, or 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 and CH3 domains of the heavy chain.

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

6. The fusion protein of 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, most preferably less than 2000 nM.

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

8. 8. The fusion protein of claim 7, wherein the amino acid substitutions include one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules.

9. 9. The fusion protein of claim 8, wherein the one or more substitutions for the cysteine ​​residues are glycine, serine, alanine, lysine, or glutamic acid, preferably glycine or serine.

10. Dysfunctional P2X ７ The receptor epitope portion is a dysfunctional P2X ７ Receptors are found, but functional P2X ７ The fusion protein of any one of claims 1 to 9, comprising an amino acid sequence of an epitope not found in the receptor.

11. Dysfunctional P2X ７ The fusion protein according to any one of claims 1 to 10, wherein the amino acid sequence of the receptor epitope portion comprises or at least consists of the amino acid sequence shown in SEQ ID NO:

14.

12. Dysfunctional P2X ７ The fusion protein according to any one of claims 1 to 11, wherein the amino acid sequence of the receptor epitope portion comprises or at least consists of the amino acid sequence shown in SEQ ID NO: 7, 9 or 122.

13. Dysfunctional P2X ７ 12. The fusion protein of any one of claims 1 to 11, wherein the amino acid sequence of the receptor epitope portion comprises, or at least consists of, an amino acid sequence set forth in any of SEQ ID NOs: 7 to 69 or 122, or a sequence at least 80%, at least 81%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, with the proviso that the sequence comprises at least the sequence set forth in SEQ ID NO: 14 or 7.

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

15. The fusion protein of claim 14, wherein the one or more modifications to the fusion protein can be selected from fluorescent moieties, metal labels (such as lanthanide elements), 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 derivatives thereof.

16. Dysfunctional P2X ７ Use of a fusion protein according to any one of claims 1 to 15 in a method for detecting genetically modified immune cells expressing a receptor comprising an antigen-binding domain for binding to the receptor.

17. 17. The use according to claim 16, wherein the immune cells are chimeric antigen receptor (CAR) cells.

18. 18. The use of claim 17, wherein the use is for determining the presence of said immune cells in a complex mixture, such as a biological sample obtained from a patient previously treated with said immune cells.

19. Dysfunctional P2X ７ 1. An in vitro method for detecting immune cells expressing a receptor comprising an antigen-binding domain for binding to a receptor, comprising: (i) providing a biological sample from a patient who has been treated with immune cells, preferably immune effector cells, wherein said cells contain dysfunctional P2X ７ providing a biological sample expressing a receptor comprising an antigen binding domain for binding to the receptor; (ii) contacting the sample with a polypeptide, wherein the polypeptide is a dysfunctional P2X receptor that is recognized by the antigen-binding domain of the receptor. ７ a polypeptide comprising an epitope of a receptor, said polypeptide comprising a detection moiety to allow detection of said polypeptide; contacting, thereby allowing the formation of a complex of said polypeptide bound to said cell; (iii) detecting the complex; This leads to dysfunctional P2X ７ An in vitro method for detecting immune cells expressing a receptor having an antigen-binding domain for binding to the receptor.

20. Dysfunctional P2X ７ 1. An in vitro method for detecting immune cells expressing a chimeric antigen receptor (CAR) for binding to a receptor, comprising: (i) Dysfunctional P2X ７ providing a biological sample from a patient who has been treated with immune cells, preferably immune effector cells, comprising a chimeric antigen receptor (CAR) for binding to a receptor; (ii) contacting the sample with a polypeptide, wherein the polypeptide is a dysfunctional P2X recognized by the CAR. ７ a polypeptide comprising an epitope of a receptor, said polypeptide comprising a detection moiety to allow detection of said polypeptide; contacting, thereby allowing the formation of a complex of said polypeptide bound to said cell; (iii) detecting the complex; This leads to dysfunctional P2X ７ An in vitro method for detecting immune cells expressing a chimeric antigen receptor (CAR) for receptor binding.

21. 21. The method of claim 19 or 20, wherein the method comprises first isolating the complex prior to the detecting step.

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

23. The polypeptide comprises a first moiety, a dysfunctional P2X polypeptide, linked to a further moiety to enhance solubility and stability of the first moiety. ７ 22. The method of any one of claims 19 to 21, comprising a first portion comprising an epitope of a receptor.

24. The further portion is a linker or hinge region of the dysfunctional P2X, such as an amino acid sequence exemplified in Tables 1 and 3. ７ 24. The method of claim 23, wherein the spacer comprises an amino acid sequence linked to a receptor epitope or a polysaccharide having at least 15 carbon atoms selected from the group consisting of dextran, pullulan, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan, and chitin.

25. The polypeptide is a dysfunctional P2X polypeptide linked to an additional amino acid sequence selected from serum albumin, transferrin, the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta chain, an imprecise repeat peptide sequence, a polypeptide sequence consisting of a proline-alanine-serine polymer, an elastin-like peptide (ELP) repeat sequence), a homopolymer of glycine residues, or a gelatin-like protein. ７ 24. The method of claim 23 in the form of a fusion protein comprising an epitope of the receptor.

26. The polypeptide may be a carbohydrate, a lipid, a liposome, a peptide, or a dysfunctional P2X. ７ 24. The method of any one of claims 19 to 23, in the form of a conjugate comprising an aptamer conjugated to an amino acid sequence comprising said epitope of a receptor.

27. 27. The method of any one of claims 19 to 26, wherein the moiety for allowing detection of the polypeptide is 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.

28. 28. The method of claim 27, wherein when the polypeptide is labeled with a biotin moiety, the method comprises (after step ii) contacting the cell with an anti-biotin antigen-binding protein, preferably wherein the anti-biotin antigen-binding protein comprises one or more moieties to allow detection of the complex.

29. 29. The method of claim 28, wherein the anti-biotin antigen binding protein comprises a fluorophore.

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

31. A kit for use in the method of any one of claims 19 to 30, comprising: -Dysfunctional P2X ７ a fusion protein or polypeptide capable of being bound by a receptor (e.g., CAR) for binding to the receptor; and - optionally a kit comprising said fusion protein or polypeptide and one or more reagents to allow detection of the complex.

32. The fusion protein of any one of claims 1 to 15, wherein the protein comprises an amino acid sequence set forth in any of SEQ ID NOs: 145-158, 160, or 161.

33. 30. The method of any one of claims 19 to 29, wherein the fusion protein comprises an amino acid sequence set forth in any of SEQ ID NOs: 145-158, 160, or 161.

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