In vivo detection of immune cells

Abstract

The present invention relates to radiolabeled molecules and related precursor molecules for the detection of immune cells expressing receptors comprising an antigen binding site and a signaling domain for binding to a P2X7 receptor of a dysfunction.

Description

Technical Field

[0001] The present invention relates to radiolabeled compounds for detecting immune cells, radiolabeled precursor compounds for preparing radiolabeled compounds, and their uses and methods.

[0002] Related Applications

[0003] This application claims priority to Australian Provisional Application AU 2022902654, the entire content of which is hereby incorporated by reference. Background Art

[0004] Cancer immunotherapy is a rapidly evolving field. The development of T cells expressing chimeric antigen receptors (CARs) has revolutionized adoptive cell therapy.

[0005] The potential of this approach has been demonstrated in clinical trials, in which CAR T cells have been infused into adult and pediatric patients with B cell malignancies, neuroblastoma, and sarcoma. To date, more than 500 clinical trials have emerged worldwide, which have been designed to test the efficacy of CAR T cells targeting 64 different tumor-associated antigens. Among them, 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, in the case of so-called "liquid" tumors, or when the CAR targets CD19, CD22, or B cell maturation antigen (BCMA), the most success of CAR T therapy has been observed.

[0006] There are still some challenges in the clinical application of CAR T cell therapy. Given the challenges associated with CAR T cell therapy, the development of methods for in vivo imaging and tracking CAR T cells has received increasing attention to gain a deeper understanding of their biological functions. These methods can also be used to monitor and adjust treatments involving CAR T cell therapy.

[0007] Current methods for monitoring infused CAR T cells include serum profiling of cytokines associated with T cell activation, direct counting of tumor-specific T cells in peripheral blood, and (repeated) tumor biopsies. However, these methods do not allow for real-time or quantitative monitoring of infused CAR T cells in vivo. Labeled antibodies for tracking T cells have also been developed. However, a disadvantage associated with this method is that the antigens expressed on target cells are usually also expressed to some extent in other tissues, which may lead to non-specific background uptake of the labeled antibodies.

[0008] Therefore, alternative methods for in vivo imaging and tracking immune cells such as CAR T cells are needed.

[0009] Any reference in the specification to any prior art is not an admission or suggestion that such prior art forms part of the common general knowledge in any jurisdiction, or that such prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0010] The present invention provides a radiolabeled molecule, the radiolabeled molecule comprising:

[0011] (i) a dysfunctional P2X7 receptor epitope portion that is recognized or capable of being bound by an antigen recognition domain of a receptor expressed on an immune cell, wherein the receptor is for binding to a dysfunctional P2X7 receptor and comprises a signal transduction domain; and

[0012] (ii) a radionuclide that is directly or indirectly linked to the epitope portion,

[0013] or a salt or solvate thereof.

[0014] The present invention provides a radiolabeled molecule, the radiolabeled molecule comprising:

[0015] (i) a peptide and

[0016] (ii) a radionuclide that is directly or indirectly linked to the peptide,

[0017] or a salt or solvate thereof,

[0018] wherein the peptide comprises or consists of: the amino acid sequence of a linear epitope derived from the P2X7 receptor, preferably the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:7. Optionally, the peptide comprises or consists of the following amino acid sequences: GHNYTTRNILPGLNITC (SEQ ID NO:2; also referred to herein as the "E200 epitope"), GHNYTTRNILPGLNIT (SEQ ID NO:3), KYYKENNVEKRTLIK (SEQ ID NO:4; also referred to herein as the "E300" epitope), or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6; also referred to herein as the "E200 / E300" or "composite" epitope), or any one of SEQ ID NO:2 to SEQ ID NO:69 and SEQ ID NO:122.

[0019] Preferably, the peptide is recognized or capable of being bound by the antigen recognition domain of an exogenous cell surface receptor (such as a chimeric antigen receptor, including those expressed on T cells) that contains an intracellular signaling domain.

[0020] Preferably, the radionuclide can be a β-emitting radioisotope (such as positron or β+ decay) or a γ-emitting radioisotope.

[0021] In any embodiment, the radionuclide is selected from: 11 C, 18 F, 44 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 90 Nb, 94m Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 177 Lu and 213 Bi.

[0022] The radionuclide can be directly linked to the epitope moiety, for example, directly to the amino acid side chain of the epitope moiety. Alternatively, the radionuclide can be indirectly linked to the epitope moiety, for example, the radionuclide can be included in a radiolabeled moiety conjugated to the epitope moiety. In some embodiments, the radiolabeled moiety contains a covalently bound radionuclide. In other embodiments, the radiolabeled moiety contains a chelator moiety capable of chelating the radionuclide, wherein the radionuclide is complexed with the chelator moiety. The chelator moiety can be selected from TMT, DOTA, TCMC, DO3A, CB-DO2A, NOTA, NETA, diamsar, DTPA, CHX-A”-DTPA, TETA, 1,4,7,10-tetraacetic acid, Te2A, HBED, 5HBED, HYBIC, DFO, DFOsq and HOPO.

[0023] In any embodiment, the radiolabeled moiety is conjugated to another peptide (such as an epitope moiety of a dysfunctional P2X7 receptor) that is recognized or capable of being bound by the antigen recognition domain of a receptor expressed on an immune cell, preferably wherein the receptor is for binding to a dysfunctional P2X7 receptor and contains a signaling domain.

[0024] On the other hand, the present invention provides a radiolabeled precursor molecule, which comprises:

[0025] (i) a dysfunctional P2X7 receptor epitope portion, which is recognized or capable of being bound by an antigen recognition domain of a receptor expressed on an immune cell, wherein the antigen recognition domain is for binding to the dysfunctional P2X7 receptor and the receptor comprises a signaling domain; and

[0026] (ii) one of the following:

[0027] (A) an atom or functional group capable of being converted into a radionuclide;

[0028] (B) a reactive functional group capable of conjugating with a radiolabeled cofactor; or

[0029] (C) a chelator moiety capable of chelating a radionuclide,

[0030] or a salt or solvate thereof.

[0031] In any embodiment, the atom or functional group can be any suitable atom or functional group capable of being converted into a radionuclide. The atom or functional group can be converted into a radionuclide, for example, by substitution, addition, or exchange with a compound containing a radionuclide. In some embodiments, the atom or functional group is capable of being 18 substituted with F. In some embodiments, the atom or functional group is capable of 125 undergoing isotope exchange with I.

[0032] In any embodiment, the reactive functional group can be any suitable reactive functional group capable of conjugating with a radiolabeled cofactor. It should be understood that the radiolabeled cofactor contains a radionuclide, which can be linked to the radiolabeled cofactor by a covalent bond or a non-covalent bond (e.g., by coordination). In certain embodiments, the reactive functional group is capable of reacting with the radiolabeled cofactor by click chemistry.

[0033] In any embodiment, the chelator moiety can be any suitable chelator moiety capable of chelating a radionuclide. In certain embodiments, the chelator moiety can be selected from TMT, DOTA, TCMC, DO3A, CB-DO2A, NOTA, NETA, diamsar, DTPA, CHX-A”-DTPA, TETA, 1,4,7,10-tetraacetic acid, Te2A, HBED, 5HBED, HYBIC, DFO, DFOsq, and HOPO.

[0034] In certain embodiments, a radiolabeled prosthetic group may be conjugated (or capable of further conjugation) with another dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by the antigen recognition domain of a receptor expressed on an immune cell, wherein the receptor is for binding to a dysfunctional P2X7 receptor and comprises a signaling domain.

[0035] In another aspect, the present invention provides a method for preparing a radiolabeled molecule, the method comprising:

[0036] - providing a radiolabeled precursor molecule as defined herein; and

[0037] - reacting the radiolabeled precursor molecule under conditions suitable for:

[0038] (i) converting an atom or functional group of (A) into a radionuclide;

[0039] (ii) conjugating a reactive functional group of (B) with a radiolabeled prosthetic group; or

[0040] (ii) chelating a chelator moiety of (C) with a radionuclide.

[0041] Thereby providing a radiolabeled molecule.

[0042] In another aspect, the present invention provides the use of the radiolabeled molecule described herein for detecting immune cells expressing a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor.

[0043] In another aspect, the present invention provides a method for detecting immune cells expressing a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor in a subject, the method comprising:

[0044] - administering to the subject the radiolabeled molecule described herein, the subject having been administered immune cells expressing a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor; and

[0045] - detecting the radiolabeled molecule in the subject.

[0046] In any embodiment of this aspect, the radiolabeled molecule is detected by performing a radionuclide scan. The radionuclide scan can be positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0047] In some embodiments in this regard, the method further comprises allowing the radiolabeled molecule to accumulate at a site in the subject where immune cells are found, prior to the step of detecting the radiolabeled molecule.

[0048] In some embodiments in this regard, the method further comprises administering to the subject, prior to the step of administering the radiolabeled molecule, immune cells that express a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor.

[0049] In another aspect, the invention provides a composition comprising the radiolabeled molecule described herein or a salt or solvate thereof, or the radiolabeled precursor molecule described herein or a salt or solvate thereof.

[0050] The invention provides a radiolabeled precursor molecule comprising:

[0051] (i) a peptide; and

[0052] (ii) one of the following:

[0053] (A) an atom or functional group capable of being converted into a radionuclide;

[0054] (B) a reactive functional group capable of conjugating with a radiolabeled cofactor; or

[0055] (C) a chelator moiety capable of chelating a radionuclide,

[0056] or a salt or solvate thereof.

[0057] Wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 14 (preferably, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7). Optionally, the peptide comprises or consists of the following amino acid sequences: GHNYTTRNILPGLNITC (SEQ ID NO: 2; also referred to herein as the "E200 epitope"), GHNYTTRNILPGLNIT (SEQ ID NO: 3), KYYKENNVEKRTLIK (SEQ ID NO: 4; also referred to herein as the "E300" epitope), or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 6; also referred to herein as the "E200 / E300" or "composite" epitope), or any one of SEQ ID NOs: 2 to SEQ ID NO: 69 and SEQ ID NO: 122.

[0058] Preferably, the peptide is recognized or capable of being bound by an antigen recognition domain of an exogenous cell surface receptor comprising an intracellular signaling domain (such as a chimeric antigen receptor, including those expressed on T cells).

[0059] The present invention provides a method for detecting immune cells expressing an exogenous cell surface receptor comprising an intracellular signaling domain (such as a chimeric antigen receptor, including those expressed on T cells), wherein the receptor comprises an antigen recognition domain for binding to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 14 (preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 7);

[0060] - Administering to a subject a radiolabeled molecule as described herein, the subject having been administered immune cells expressing the receptor;

[0061] - Detecting the radiolabeled molecule in the subject.

[0062] Preferably, the radiolabeled molecule is detected by performing a radionuclide scan. The radionuclide scan can be positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0063] Optionally, the antigen recognition domain of the receptor is for binding to a peptide comprising or consisting of the following amino acid sequences: GHNYTTRNILPGLNITC (SEQ ID NO: 2; also referred to herein as the "E200 epitope"), GHNYTTRNILPGLNIT (SEQ ID NO: 3), KYYKENNVEKRTLIK (SEQ ID NO: 4; also referred to herein as the "E300" epitope), or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 6; also referred to herein as the "E200 / E300" or "composite" epitope), or any one of SEQ ID NO: 2 to SEQ ID NO: 69 and SEQ ID NO: 122.

[0064] In any aspect of the present invention, a dysfunctional P2X7 receptor epitope moiety can be provided in the form of a dysfunctional P2X7 receptor or a fragment of a dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly stacked monomers that cannot bind ATP. Such receptors are generally understood to not extend the opening of non-selective calcium channels to apoptotic pores.

[0065] In any aspect, the dysfunctional P2X7 receptor epitope moiety comprises or consists of a fragment of a dysfunctional P2X7 receptor. Exemplary fragments include peptides comprising the following amino acid sequences: GHNYTTRNILPGLNITC (SEQ ID NO:2; also referred to herein as the "E200 epitope"), GHNYTTRNILPGLNIT (SEQ ID NO:3), KYYKENNVEKRTLIK (SEQ ID NO:4; also referred to herein as the "E300" epitope), or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6; also referred to herein as the "E200 / E300" or "composite" epitope). Other exemplary peptide fragments are in Table 1 herein and comprise any one of SEQ ID NO:2 to SEQ ID NO:69 and SEQ ID NO:122. Preferably the dysfunctional P2X7 receptor epitope moiety comprises at least the sequence of SEQ ID NO:7.

[0066] The dysfunctional P2X7 receptor epitope moiety may additionally comprise a spacer that links the moiety to a radiolabel. Instances of such spacer sequences are further provided herein.

[0067] In a preferred embodiment of any aspect of the invention, the structure and spatial arrangement of the radiolabeled molecule or radiolabel precursor molecule of the invention is such that a minimum sequence of SEQ ID NO:14, more preferably SEQ ID NO:7, is available for binding to the antigen-binding domain of a receptor expressed on an immune cell, as described herein (e.g., a chimeric antigen receptor for binding to a dysfunctional P2X7 receptor). In other words, the structure is such that there is no steric hindrance that would prevent the antigen-binding domain from binding to the peptide.

[0068] In a preferred embodiment, the structure of the radiolabeled molecule or radiolabel precursor molecule of the invention provides a radionuclide that is directly or indirectly linked to the epitope moiety through the C-terminal region of the epitope moiety.

[0069] Alternatively, the structure of the radiolabeled molecule or radiolabel precursor molecule of the invention provides a radionuclide that is directly or indirectly linked to the epitope moiety through the N-terminal region of the epitope moiety.

[0070] In a preferred embodiment, the structure of the radiolabel precursor molecule of the invention provides an atom or functional group that is capable of being transformed into a radionuclide, the radionuclide being directly or indirectly linked to the epitope moiety through the C-terminal region of the epitope moiety.

[0071] In additional embodiments, the structure of the radiolabeled precursor molecule of the invention provides a reactive functional group capable of conjugating with a radiolabeled cofactor, which is directly or indirectly linked to the epitope moiety through the C-terminal region of the epitope moiety.

[0072] In additional embodiments, the structure of the radiolabeled precursor molecule of the invention provides a chelator moiety capable of chelating a radionuclide, which is directly or indirectly linked to the epitope moiety through the C-terminal region of the epitope moiety.

[0073] Alternatively, the structure of the radiolabeled precursor molecule of the invention provides: (A) an atom or functional group capable of being converted into a radionuclide; (B) a reactive functional group capable of conjugating with a radiolabeled cofactor; or (C) a chelator moiety capable of chelating a radionuclide, which is directly or indirectly linked to the epitope moiety through the N-terminal region of the epitope moiety.

[0074] According to any aspect of the invention, the radiolabeled molecule or radiolabeled precursor molecule can be in the form of a fusion protein. Optionally, the fusion protein comprises a peptide linked to the Fc region of an antibody (e.g., a linear epitope of the P2X7 receptor, such as those in SEQ ID NO:7 or SEQ ID NO:14, or as defined by any one of SEQ ID NO:2 to SEQ ID NO:69 and SEQ ID NO:122), as further described herein. Thus, the invention provides a fusion protein comprising a peptide derived from the P2X7 receptor (e.g., an epitope moiety of a dysfunctional P2X7 receptor) and the Fc region of an antibody, optionally wherein the fusion protein comprises a radiolabel or a moiety capable of being radiolabeled, or wherein the fusion protein comprises one of the following:

[0075] (A) an atom or functional group capable of being converted into a radionuclide;

[0076] (B) a reactive functional group capable of conjugating with a radiolabeled cofactor; or

[0077] (C) a chelator moiety capable of chelating a radionuclide,

[0078] or a salt or solvate thereof.

[0079] Examples of such fusion proteins are provided herein at SEQ ID NO:145 to SEQ ID NO:158, SEQ ID NO:160 and SEQ ID NO:161

[0080] In a preferred embodiment, a fusion protein comprising a dysfunctional P2X7 receptor epitope moiety and the Fc region of an antibody preferably comprises one or more modifications to the Fc region, such as to reduce effector function (by attenuating or reducing the ability to bind to Fc receptors and / or by reducing or eliminating the recruitment of complement C1q), shorten the serum half-life (by attenuating or reducing the ability to bind to the FcRN receptor), and / or reduce the tendency of the Fc region to aggregate and dimerize. Relevant amino acid substitutions for altering effector function, serum half-life, and aggregation are known to those of skill in the art and are further described herein, including as exemplified in Table 1.

[0081] The invention also provides a heterodimeric asymmetric molecule comprising a fusion protein as described herein (such as a peptide comprising SEQ ID NO:7 or 14, or a variant thereof, as exemplified by any one of SEQ ID NO:2 to SEQ ID NO:69) and the Fc region of an antibody, and further comprising the Fc region of an antibody that does not comprise the peptide. Such asymmetric heterodimeric molecules can be obtained using the knob-into-hole technology for promoting the dimerization of non-identical Fc regions, as further described herein.

[0082] Preferably, the fusion protein or heterodimeric asymmetric molecule consists of or consists essentially of a peptide and the Fc region of an antibody such that the fusion protein or heterodimeric asymmetric molecule does not comprise the antigen-binding domain of an antibody (such that the fusion protein does not comprise VH, VL, Fab, Fv, or scFv derived from an antibody).

[0083] It should be understood that a radiolabel, an atom or functional group capable of being converted into a radionuclide, a reactive functional group or chelator moiety capable of conjugating to a radiolabeled cofactor can be linked to or be part of the dysfunctional P2X7 receptor epitope moiety of the fusion protein, or can be linked to or be part of the Fc region of the fusion protein.

[0084] In any aspect, the dysfunctional P2X7 receptor epitope moiety is bound by or capable of being bound by an antigen-binding protein or an antigen-binding fragment thereof that binds to the dysfunctional P2X7 receptor, but is not bound by or capable of being bound by an antigen-binding protein or an antigen-binding fragment thereof that binds to the functional P2X7 receptor. Examples of suitable antigen-binding proteins or fragments thereof are further described herein.

[0085] A radiolabeled molecule or a radiolabeled precursor molecule may comprise two or more peptides as described herein (e.g., two or more dysfunctional P2X7 receptor epitope moieties). The two or more peptides may comprise the same sequence or different sequences, or consist thereof. For example, in any aspect, the radiolabeled molecule or the radiolabeled precursor molecule may comprise a peptide in the form of the E200 epitope and another peptide in the form of the E300 epitope. Alternatively, in any aspect, the radiolabeled molecule may comprise a peptide in the form of the E200 epitope and another peptide in the form of a composite epitope. Additionally, in any aspect, the radiolabeled molecule or the radiolabeled precursor molecule may comprise a first peptide in the form of the E200 epitope and another peptide in the form of the E200 epitope.

[0086] In any aspect of the present invention, the receptor comprising an antigen recognition domain and a signal transduction domain may be a chimeric antigen receptor (CAR) or a variant thereof, including ligand-based CARs, or a modified T cell receptor (TCR), etc.

[0087] In any aspect of the present invention, the immune cell may be a white blood cell, a peripheral blood mononuclear cell (PBMC), a lymphocyte, a T cell, a CD4+ T cell, a CD8+ T cell, a natural killer cell, a natural killer T cell, or a γδ T cell. In any embodiment, the cell may be a T cell, wherein 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 level or functional level). In any embodiment, the cell may be an immune cell, wherein optionally the cell does not express co-stimulatory molecules, which may be checkpoint, exhaustion, or apoptosis-related signal transduction receptors and ligands, such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L, and FAS-R (e.g., by knocking out one of these genes at the genetic level or functional level).

[0088] In a preferred embodiment of any aspect of the present invention, the immune cell is a T cell or other effector cell expressing a CAR comprising an antigen-binding domain for binding to a dysfunctional P2X7 receptor.

[0089] In another aspect, the present invention provides a formulation comprising the radiolabeled molecule described herein or a salt or solvate thereof, or the radiolabeled precursor molecule described herein or a salt or solvate thereof.

[0090] In another aspect, the present invention provides a kit comprising one or more of the following:

[0091] (i) The radiolabeled molecule described herein or a salt or solvate thereof;

[0092] (ii) The radiolabeled precursor molecule or a salt or solvate thereof as described herein;

[0093] (iii) The composition as described herein; or

[0094] (iv) The formulation as described herein.

[0095] Optionally, the kit includes instructions for use, or one or more reagents for use with the radiolabeled molecule or precursor molecule.

[0096] From the following description given by way of example and with reference to the accompanying drawings, other aspects of the present invention and other embodiments of the various aspects described in the foregoing paragraphs will become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 : A schematic diagram depicting an exemplary use of the radiolabeled molecule of the present invention for detecting immune cells enriched at tumor sites expressing dysfunctional P2X7 receptors.

[0098] Figure 2: a) Flow cytometry for detecting CAR-expressing immune cells bound by the monomeric E200-Fc fusion protein using an anti-His antibody. X-axis: His. b) Flow cytometry staining with an anti-biotin antibody of cells from mouse blood and bone marrow after ex vivo incubation with biotinylated monomeric molecules is detectable by the anti-biotin antibody to identify subsets of CAR-expressing cells. X-axis: monomeric fusion protein (DetR1, SEQ ID NO: 158).

[0099] Figure 3 : The proportions of CD25+ / CD69+ and PD-L1+ cells 72 hours after contact with monomeric or dimeric fusion proteins (having amino acid sequences of SEQ ID NO: 158 and SEQ ID NO: 149, respectively).

[0100] Sequence Information

[0101] Table 1: Exemplary sequences of dysfunctional P2X7 receptors and receptor epitope portions

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[0117] Detailed implementation manners

[0118] Reference will now be made in detail to certain embodiments of the present invention. While the invention will be described in conjunction with the embodiments, it should be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims.

[0119] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, and these methods and materials can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0120] It should be understood that the invention disclosed and defined in this specification includes all optional combinations of two or more separate features mentioned in the text or drawings or clearly apparent from the text or drawings. All such different combinations constitute various alternative aspects of the present invention.

[0121] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0122] The present invention provides a radiolabeled molecule, a composition and a kit comprising the radiolabeled molecule, which can be used to detect immune cells expressing a receptor and a signaling domain, such as CAR T cells. Additionally, the present invention provides a radiolabeled precursor molecule that can be used to produce the radiolabeled molecule.

[0123] The radiolabeled molecule comprises (i) a dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by an antigen recognition domain of a receptor expressed on an immune cell, wherein the receptor is for binding to the dysfunctional P2X7 receptor and comprises a signaling domain; and (ii) a radionuclide that is linked to the epitope moiety. The epitope moiety allows the radiolabeled molecule to specifically bind to the receptor expressed by the immune cell, and the radionuclide allows detection of the radiolabeled molecule and thus detection of the location and / or distribution of the immune cell expressing the receptor.

[0124] Accordingly, the radiolabeled molecule can advantageously provide a new method for real-time monitoring of the distribution and / or quantification of immune cells such as CAR T cells that bind to the dysfunctional P2X7 receptor in vivo.

[0125] In a particularly preferred embodiment of the invention, there is provided a fusion protein comprising a linear epitope derived from the P2X7 receptor (such as exemplified in either of SEQ ID NO:14 or SEQ ID NO:7) and the Fc region of an antibody or a heterodimeric molecule derived therefrom. The presence of the Fc region in the fusion protein provides particular advantages, including those related to preventing loss of the reagent from the circulation due to renal filtration. Thus, the molecule of the invention preferably comprises the Fc region from an antibody to assist in the stability of the protein in the circulation of a subject in whom detection of CAR T cells will be determined for it.

[0126] In a particularly preferred embodiment, the Fc fusion protein is designed to contain only a single copy of the linear epitope derived from the P2X7 receptor. As further described herein, this can be achieved by introducing amino acid substitutions into the Fc region to prevent homodimerization, or alternatively, using well-known "stapling" techniques to ensure formation of an asymmetric heterodimeric molecule (e.g., comprising an E200 peptide-Fc fusion protein and an Fc region that does not contain the E200 peptide). Such monomeric or asymmetric heterodimeric molecules have the advantage of reducing activation of target immune cells and preventing unwanted depletion of target immune cells (as further described in the examples herein). Without wishing to be bound by theory, the inventors believe this is due to the reduced ability of the molecule to crosslink two different CAR receptors on one cell or two different CAR receptors on two separate CAR-expressing cells.

[0127] Definitions

[0128] Unless otherwise defined, the 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.

[0129] For the purposes of interpreting this specification, the following definitions will generally apply, and terms used in the singular will also include the plural where appropriate, and vice versa.

[0130] As used herein, the term "and / or", such as "X and / or Y", will be understood to mean "X and Y" or "X or Y", and will be considered to provide explicit support for both meanings or either meaning.

[0131] The articles "a" and "an" are used herein to refer to one or more than one of the grammatical objects of the article (i.e., to at least one). For example, "a dysfunctional P2X7 receptor epitope moiety" means one dysfunctional P2X7 receptor epitope moiety or more than one dysfunctional P2X7 receptor epitope moiety.

[0132] As used herein, unless the context requires otherwise, the term "comprising" or variations thereof, such as "including" and "containing", are not intended to exclude additional additives, components, integers or steps.

[0133] "Purine receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0134] "P2X7 receptor" generally refers to a purine receptor formed by three protein subunits or monomers, at least one of which monomers has an amino acid sequence substantially as shown in SEQ ID NO:1 in Table 1 herein.

[0135] Insofar as the P2X7 receptor is formed by three monomers, it is "trimeric" or "trimer". "P2X7 receptor" encompasses naturally occurring variants of the P2X7 receptor, e.g., where the P2X7 monomers are splice variants, allelic variants, SNPs and isotypes, including naturally occurring truncated or secreted forms of the monomers forming the P2X7 receptor (e.g., forms consisting of extracellular domain sequences or truncated forms thereof), naturally occurring variant forms (e.g., alternative splice forms) and naturally occurring allelic variants. In certain embodiments of the invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the P2X7 receptor may have a modified amino acid sequence, e.g., various amino acids in the sequence shown in SEQ ID NO:1 may be substituted, deleted or residues may be inserted.

[0136] "Functional P2X7 receptor" generally refers to the form of the P2X7 receptor that has three complete binding sites or grooves for binding to ATP. When bound to ATP, the functional receptor forms a non-selective sodium / calcium channel that transforms into a pore-like structure capable of allowing calcium ions and molecules up to 900 Da to enter the cytosol, and one consequence may be the induction of programmed cell death. In normal homeostasis, the expression of the functional P2X7 receptor is generally limited to cells where programmed cell death occurs, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. Some expression of the functional P2X7 receptor may also be present on red blood cells and other cell types.

[0137] "Dysfunctional P2X7 receptor" (also referred to as "non-functional" or (nf)P2X7) is a P2X7 receptor with impaired responsiveness to ATP such that it cannot form an apoptotic pore under physiological conditions. A dysfunctional P2X7 receptor or (nfP2X7 receptor) generally refers to the form of the P2X7 receptor that has a conformation different from that of the functional P2X7, whereby the receptor cannot form an apoptotic pore but can still function as a non-selective channel by maintaining a single functional ATP binding site located between adjacent monomers. An example is when one or more of these monomers have a cis-isomerization at Pro210 (according to SEQ ID NO:1). The isomerization may be caused by any molecular event that leads to misfolding of the monomer, including, for example, mutations in the primary sequence of the monomer or abnormal post-translational processing. One consequence of the isomerization is that the receptor cannot bind ATP at one or more, and more particularly two, ATP binding sites on the trimer, and thus cannot extend the opening of the channel. In these cases, the receptor cannot form a pore, which limits the extent to which calcium ions can enter the cytosol. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial and hematopoietic cancers. As used herein, the term "dysfunctional P2X7 receptor" may be used interchangeably with the terms "non-functional P2X7 receptor" or "nfP2X7 receptor".

[0138] "Cancer-associated P2X7 receptor" generally refers to a P2X7 receptor that is found on cancer cells (including pre-neoplastic, neoplastic, malignant, benign, or metastatic cells) but not on non-cancerous or normal cells.

[0139] "E200 epitope" generally refers to an epitope having the sequence GHNYTTRNILPGLNITC (SEQ ID NO:2). Variant examples thereof are shown in Table 1 and include any one of SEQ ID NO:3 or SEQ ID NO:7 to SEQ ID NO:69 or SEQ ID NO:155.

[0140] "E300 epitope" generally refers to an epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO:4) or a variant thereof, as defined by SEQ ID NO:5.

[0141] "Composite epitope" generally refers to an epitope formed by juxtaposing E200 and E300 epitopes or portions of these epitopes. An example of a composite epitope containing E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6).

[0142] As used herein, the term "antigen" is intended to include a substance that binds to or elicits the production of one or more antibodies and can include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. The term "antigen" as used herein refers to a molecular entity that can be expressed on a target cell and can be recognized by the adaptive immune system, which includes, but is not limited to, antibodies or TCRs or engineered molecules (including, but not limited to, transgenic TCRs, CARs, scFvs, or their multimers; Fab fragments or their multimers; antibodies or their multimers; single-chain antibodies or their multimers) or any other molecule that can bind to a structure with high affinity.

[0143] "Epitope" generally refers to the portion of an antigen that is bound by the antigen-binding site of an antibody. An epitope can be "linear", meaning that the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to an amino acid sequence in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope", i.e., an epitope in which the hypervariable loops of the CDRs bind to residues present in the tertiary or quaternary protein structure.

[0144] With respect to a receptor that mentions an antigen-binding domain that recognizes and binds to a dysfunctional P2X7 receptor, the terms "bind", "specifically bind", or "specific for" are intended to mean that the receptor substantially does not recognize or bind to other antigens in a sample.

[0145] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system and perform specific effector functions, such as α-β T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-δ T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages, or any hematopoietic progenitor cell such as a pluripotent stem cell and early progenitor cell subsets that can mature or differentiate into somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (such as iPSCs and other artificial cell types). Preferred immune cells are cells with cytotoxic effector functions, such as α-β T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or γ-δ T cells. "Effector function" means the specialized function of a cell. For example, in T cells, the effector function can be cytolytic activity or helper cell activity, including the secretion of cytokines.

[0146] As used herein, the term "amino acid" refers to a compound having an amino group and a carboxylic acid group. The amino acid can be an L- or D-isomer or a mixture thereof. The amino acid can have a naturally occurring side chain (see Table 1) or a non-protein side chain. The amino acid can also be further substituted at the α-position with a group selected from: -C 1-6 alkyl, -(CH2) n COR a 、-(CH2) n R b and -PO3H, where n is an integer selected from 1 to 8, and R a is -OH, -NH2, -NHC 1-3 alkyl, -OC 1-3 alkyl or -C 1-3 alkyl, and R b is -OH, -SH, -SC 1-3 alkyl, -OC 1-3 alkyl, -NH2, -NHC 1-3 alkyl or -NHC(C=NH)NH2, and wherein each alkyl group can be substituted with one or more groups selected from -OH, -NH2, -NHC 1-3 alkyl, -OC 1-3 alkyl, -SH, -SC 1-3 alkyl, -CO2H, -CO2C 1-3 alkyl, -CONH2 and -CONHC 1-3 alkyl.

[0147] The amino acid structures, single-letter, and three-letter abbreviations used throughout the specification are defined in Table 2, which lists the twenty naturally occurring proteinogenic amino acids that exist as L-isomers in proteins.

[0148] Table 2

[0149]

[0150]

[0151]

[0152] As used herein, the term "non-proteinogenic amino acid" refers to an amino acid having a side chain that is not present in the naturally occurring L-α-amino acids 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, tert-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of natural amino acids

[0153] As used herein, the term "α-amino acid" refers to an amino acid having a single carbon atom (α-carbon atom) that separates the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). α-Amino acids include naturally occurring and non-naturally occurring L-amino acids, their D-isomers, and derivatives such as salts or derivatives in which the functional groups are protected by suitable protecting groups. Unless otherwise specified, the term "amino acid" as used herein refers to α-amino acids.

[0154] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms. Where appropriate, an alkyl group may have a specific number of carbon atoms, e.g., C 1-6 alkyl, which includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight-chain or branched-chain arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 5-methylpentyl.

[0155] Suitable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0156] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as: sodium salts, potassium salts, lithium salts, calcium salts, magnesium salts, zinc salts, ammonium salts, and alkylammonium salts; salts formed with triethylamine; alkoxyammonium salts such as those formed with ethanolamine; and salts formed with ethylenediamine, choline, or amino acids (such as arginine, lysine, or histidine).

[0157] Basic nitrogen-containing groups can be quaternized with reagents such as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates such as dimethyl and diethyl sulfates; and the like.

[0158] Salts or other derivatives of the compounds of the present invention can be provided in solvated forms. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and can be formed during the crystallization of the compound with pharmaceutically acceptable solvents such as water, alcohols (such as methanol, ethanol, or isopropanol), DMSO, acetonitrile, dimethylformamide (DMF), etc., where the solvate forms part of the crystal lattice either by non-covalent binding or by occupying pores in the lattice. Hydrates are formed when the solvent is water and alcoholates are formed when the solvent is an alcohol. Solvates of the compounds of the present invention can be conveniently prepared or formed during the processes described herein. Generally, for the purposes of the compounds and methods provided herein, the solvated forms are considered equivalent to the non-solvated forms.

[0159] 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 can be a subject (patient) suffering from a disorder such as cancer, but the subject can also be a healthy subject.

[0160] Radioactively labeled molecule

[0161] It should be understood that the radiolabeled molecule of the present invention can be in any form, provided that it comprises (i) a dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by an antigen recognition domain expressed on an immune cell that binds to the dysfunctional P2X7 receptor, wherein the receptor comprises a signaling domain; and (ii) a radionuclide that is directly or indirectly linked to the epitope moiety.

[0162] Generally, the dysfunctional P2X7 receptor epitope moiety is in the form of a peptide. The dysfunctional P2X7 receptor epitope moiety is further described herein.

[0163] It should be understood that the presence of a radionuclide can affect the local charge field of nearby atoms. Therefore, it is preferred to link the radionuclide at a position in the radiolabeled molecule such that the radionuclide does not affect the binding and recognition of the epitope moiety by the antigen-binding domain of the receptor.

[0164] Accordingly, in some embodiments, the epitope moiety comprises one or more spacers between the dysfunctional P2X7 receptor epitope moiety and the radionuclide. A spacer is an amino acid sequence in the epitope moiety that can be present at the N-terminus or C-terminus of the recognition sequence of the epitope moiety. In the case where the radionuclide is directly or indirectly linked to the spacer of the epitope moiety, the spacer can sufficiently space the radionuclide from the recognition sequence so as not to affect the binding of the epitope moiety by the antigen-binding domain of the immune cell receptor.

[0165] In some embodiments, the spacer comprises 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 amino acids. In some embodiments, each spacer independently comprises no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 4 amino acids. Any minimum and maximum amounts can be combined to form a range, provided that the range is within 1 to 20 amino acids, such as 3 to 15 amino acids or 5 to 11 amino acids or 2 to 6 amino acids.

[0166] In some instances, the spacer comprises amino acid residues derived from a dysfunctional P2X7 receptor sequence, whether located at the N-terminus or C-terminus of the epitope sequence. For example, in some embodiments, the spacer is selected from the amino acid sequence at the C-terminus of the core E200 epitope sequence of the receptor, such as the sequence TFHKT (SEQ ID NO:138) and as exemplified in the peptide defined by SEQ ID NO:9. Alternatively or additionally, the spacer may comprise the amino acid sequence at the N-terminus of the core E200 epitope sequence of the receptor, such as the amino acid sequence DFP (SEQ ID NO:139) and as exemplified in the peptide of SEQ ID NO:140.

[0167] It should be understood that the radiolabeled molecule or the molecule to be radiolabeled according to the present invention may be in the form of a fusion protein in further embodiments, wherein the fusion protein comprises a first amino acid sequence and a second amino acid sequence, and the first amino acid sequence contains an epitope of a dysfunctional P2X7 receptor. In some instances, the second amino acid sequence may comprise the "spacer" sequence outlined above. Alternatively, the second amino acid sequence may comprise the amino acid sequence of a protein for increasing the solubility or stability of the molecule. Thus, the second amino acid sequence may comprise an amino acid sequence derived from: an immunoglobulin, serum albumin, or other proteins, such as transferrin, the carboxy-terminal peptide of the beta chain of chorionic gonadotropin (CG), a non-exact repeat peptide sequence, a polypeptide sequence composed of a proline-alanine-serine polymer, an elastin-like peptide (ELP) repeat sequence), a homopolymer of glycine residues, or a gelatin-like protein).

[0168] In some instances, the second amino acid sequence may comprise an amino acid sequence from an immunoglobulin, such as the Fc region (e.g., comprising the CH2 and / or CH3 regions) or a variant thereof. Thus, the radiolabeled molecule or the molecule to be radiolabeled may be an Fc fusion protein composed of the amino acid sequence of an epitope of a dysfunctional P2X7 receptor linked to the Fc region of an antibody.

[0169] In any embodiment, the amino acid sequence of the epitope of a dysfunctional P2X7 receptor may be fused to the N-terminal region of the Fc region of an antibody or a variant thereof through its C-terminal region. In any embodiment, the amino acid sequence of the epitope of a dysfunctional P2X7 receptor may be fused to the C-terminal region of the Fc region of an antibody or a variant thereof through its N-terminal region.

[0170] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably comprising the CH2 and CH3 domains of the heavy chain.

[0171] Compared with the naturally occurring Fc sequence, the Fc region can contain one or more amino acid sequence modifications. The Fc region can contain one or more amino acid substitutions, such as substitutions of one or more cysteine residues, in order to prevent dimerization of the molecule with the same molecule. It should be understood that any amino acid substitution that prevents dimerization of the Fc region can be employed. Thus, in vivo, the Fc fusion proteins described herein can be monomeric proteins.

[0172] Accordingly, compared with the naturally occurring Fc sequence, the Fc region of the fusion protein can contain one or more amino acid substitutions that prevent or reduce the ability of the Fc region to homodimerize. Preferably, the amino acid substitution includes one or more substitutions of cysteine residues in order to prevent the formation of disulfide bonds between Fc molecules. The cysteine residues in the Fc region can be substituted with any other amino acid residue, optionally substituted with glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

[0173] The substituted cysteine residues are preferably one or more of the cysteine residues located in the region of the Fc region corresponding to the hinge region of the immunoglobulin. Examples of the IgG1 hinge region and its variants in which cysteine is substituted with serine are provided in Table 3 herein. The hinge region of an immunoglobulin (such as 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 cysteine residues in the immunoglobulin hinge region are substituted. Preferably, at least two or all three cysteine residues are substituted. More preferably, all cysteine residues in the Fc region, such as the hinge region, are substituted. In a particularly preferred embodiment, at least one of C226 and C229 is substituted, preferably both C226 and C229 are substituted.

[0174] Accordingly, in a preferred embodiment, the fusion protein comprises a hinge region for linking the epitope portion of the dysfunctional P2X7 receptor and the Fc region of the antibody, wherein the hinge region contains an amino acid sequence corresponding to any one of the sequences shown in SEQ ID NOs: 76 to 113, or SEQ ID NOs: 136 to 137 or SEQ ID NO: 141 or SEQ ID NO: 142.

[0175] In a further embodiment, the fusion protein region can contain an Fc region corresponding to the Fc "mortar" or "pestle" in a "mortar and pestle" heterodimer. The use of such Fc sequences is known in the art and provides an asymmetric heterodimeric molecule comprising a fusion protein having a single copy of the epitope portion as described herein and an Fc region that binds to another Fc region that does not contain the epitope portion.

[0176] Those skilled in the art should be familiar with the techniques and Fc sequences capable of forming so-called monomeric fusion proteins, including but not limited to the use of the "stalk and socket" IgG1 form (Ridgway et al., 1996, Protein Eng, Vol. 9: pp. 617-621). In the context of the present invention, such methods enable the expression and purification of heterodimeric fusion proteins, each molecule of which has only one copy of the peptide epitope (e.g., the epitope portion derived from the E200 epitope as described herein). Examples of "stalk and socket" Fc pairings are provided herein as SEQ ID NO: 157 and SEQ ID NO: 159 (stalk and socket respectively), SEQ ID NO: 158 and SEQ ID NO: 159, SEQ ID NO: 160 and SEQ ID NO: 162 (socket and stalk respectively), and SEQ ID NO: 161 and SEQ ID NO: 162. Thus, in any embodiment, the present invention provides a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 2 to SEQ ID NO: 69 and SEQ ID NO: 122 linked to an Fc region as defined by SEQ ID NO: 160 or SEQ ID NO: 162, wherein the fusion protein is capable of forming a heterodimer with an Fc region that does not contain the E200 peptide moiety.

[0177] Thus, the fusion proteins of the present invention are preferably fusion proteins capable of forming heterodimeric molecules comprising a single E200-containing amino acid sequence. (In other words, the Fc portion of the fusion protein can form a heterodimer with the Fc region of an antibody that does not contain the E200 peptide fused thereto).

[0178] In additional embodiments, the Fc region can contain one or more substitutions for eliminating or reducing effector functions, such as reducing binding and activation via FcR, as further described below.

[0179] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. In other words, the Fc region contains two heavy chain fragments, which heavy chain fragments contain the C H 2 and C H 3 domains. In the context of the present invention, the Fc region contains two heavy chain fragments, preferably the CH2 and CH3 domains of the heavy chain. The two heavy chain fragments are joined together by two or more disulfide bonds and by hydrophobic interactions of the C H 3 domain. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0180] In some aspects, the fusion protein does not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to determine the reduction / consumption of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains FcRn binding ability. The major cell mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. Vol. 9: pp. 457-492, 1991, Table 3 on page 464.

[0181] Non-limiting examples of in vitro assays for assessing the ADCC activity of a test molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, Vol. 83: pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, Vol. 82: pp. 1499-1502, 1985, 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. Vol. 166: pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA) and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in an animal model, as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA, Vol. 95: pp. 652-656, 1998. C1q binding assays can also be performed to determine that the antibody does not bind C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, Vol. 202: p. 163, 1996; Cragg, M.S. et al., Blood, Vol. 101: pp. 1045-1052, 2003; and Cragg, M.S. and M.J. Glennie, Blood, Vol. 103: pp. 2738-2743, 2004). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example: Petkova, S.B. et al., Int'l. Immunol. Vol. 18 No. 12: pp. 1759-1769, 2006; WO 2013 / 120929 A1).

[0182] Fc regions with reduced effector functions include Fc regions in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). For example, an antibody variant can comprise an Fc region having one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. For example, the substitutions are L234A and L235A (LALA) (see, e.g., WO 2012 / 130831). Additionally, the Fc region can be altered to result in an alteration (i.e., reduction) in C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. Vol. 164: pp. 4178-4184, 2000 (e.g., G236R).

[0183] Additional examples of modified Fc regions include Fc regions comprising "LALALS" (amino acid substitutions L234A / L235A / M428L / N434S as described in Zalevsky et al., Nat. Biotechnol. Vol. 28: pp. 157-159, 2010), LALAPG (L234A / L235A / P329G amino acid substitution as described in Gunn et al. (Immunity, Vol. 54: p. 815, 2021)).

[0184] In any embodiment, the Fc region of the Fc fusion protein of the invention can comprise at least the "LALA" mutation (L234A and L235A) for reducing binding to FcR. The fusion protein can additionally or alternatively comprise the mutation G346R for eliminating recruitment of complement C1q.

[0185] Other Fc modifications for use in the present invention include variants that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Such variants are also referred to herein as "knockout variants" or "KO variants". Variants that reduce binding to FcγR and complement can be used to reduce unwanted interactions mediated by the Fc region. Preferred knockout variants are described in US 2008-0242845 A1, published Oct. 2, 2008, entitled "Fc Variants with Optimized Properties", which is hereby expressly incorporated by reference. Preferred modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index. Preferred substitutions include, but are not limited to, 234G, 235E, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index. Preferred variants include 236R / 328R. The variants can be used in any IgG isotype or in the context of the Fc region of an IgG isotype, including but not limited to human IgG1, IgG2, IgG3, and / or IgG4. Preferred IgG Fc regions for reducing FcγR and complement binding and reducing Fc-mediated effector functions are the IgG2 and IgG4 Fc regions. Hybrid isotypes can also be used, such as the hybrid IgG1 / IgG2 isotype described in US Serial No. 11 / 256,060. Other modifications for reducing FcγR and complement interactions include, but are not limited to, substituting 297A, 297D, 234A, 235A, 237A, 318A, 228P, 236E, ΔG236, 265G, 268Q, 297Q, 309L, 330S, 331S, 327Q, 220S, 226S, 229S, 238S, 233P, 234A, and 234V, and removing glycosylation at position 297 by mutagenesis or enzymatic means or by production in organisms that do not glycosylate proteins such as bacteria. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology, Vol. 20: pp. 685-691, which is hereby incorporated by reference in its entirety.

[0186] In some aspects, the Fc region contains mutations in the complement (C1q) and / or Fcγ receptor (FcγR) binding sites. In some aspects, such mutations can render the fusion protein unable to mediate antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC).

[0187] The Fc region, as used in the context of the present invention, preferably does not trigger cytotoxicity, such as antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cell cytotoxicity (CDC).

[0188] In some aspects, the Fc region can comprise one or more substitutions for reducing the affinity for FcRn, thereby shortening the serum or circulatory half-life of the fusion protein. Substitutions for reducing the affinity for FcRn are known in the art and are described, for example, in Ward et al., 2015, Mol. Immunol., Vol. 67: pp. 131-141 and Grevys et al., 2015, Vol. 194: pp. 5497-5508. Examples of substitutions include substitutions at one or more of Ile253, His310, and His435, such as I253A, H310A, and H435A.

[0189] The term "Fc region" also includes native sequence Fc regions and variant Fc regions. The Fc region can comprise the carboxyl terminus of the heavy chain. Antibodies produced by a host cell can undergo post-translational cleavage of one or more, particularly one or two amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can comprise the full-length heavy chain, or it can comprise a cleavage variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991. Amino acid sequence variants of the Fc region of an antibody can be contemplated. Amino acid sequence variants of the Fc region of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the Fc region of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, e.g., inducing or supporting an anti-inflammatory response.

[0190] The Fc region of the antibody can be the Fc region of any class of antibody (such as IgA, IgD, IgE, IgG, and IgM). The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, as used in the context of the present invention, the antibody can be the Fc region of IgG. For example, the Fc region of the antibody can be the Fc region of IgG1, IgG2, IgG2b, IgG3, or IgG4. In some aspects, the fusion protein of the present invention comprises IgG of the Fc region of an antibody. In the context of the present invention, the Fc region of the antibody is the Fc region of IgG (preferably IgG1).

[0191] The epitope of the dysfunctional P2X7 receptor and the Fc region amino acid sequence can be linked or fused directly or via a linker sequence. The linker sequence can be a spacer sequence as defined herein or exemplified in Table 1 or 3. Alternatively, the linker sequence can be any amino acid-based linker sequence commonly used in the art.

[0192] The linker is typically a peptide up to 20 amino acids in length, but can also be up to 50 amino acids in length. The terms "linked" or "fused" refer to a covalent bond formed between two moieties, such as a peptide bond. Thus, in the context of the present invention, the linker can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the fusion proteins provided herein can comprise a linker between the epitope of the dysfunctional P2X7 receptor and the Fc region of the antibody (such as between the N-terminus of the Fc region and the C-terminus of the epitope of the dysfunctional P2X7 receptor). Again, for example, the fusion proteins provided herein can comprise a linker between the epitope of the dysfunctional P2X7 receptor and the Fc region of the antibody (such as between the C-terminus of the Fc region and the N-terminus of the portion of the epitope of the dysfunctional P2X7 receptor). In particular, the portion of the epitope of the dysfunctional P2X7 receptor can be fused at the C-terminus to the N-terminus of the Fc region via a linker. Such linkers have the advantage that they can allow the different polypeptides of the fusion protein to fold independently more likely and function as expected. Thus, in the context of the present invention, the portion of the epitope of the dysfunctional P2X7 receptor and the Fc region of the antibody can be comprised in a single-chain multifunctional polypeptide.

[0193] In some aspects, the fusion proteins of the invention comprise a peptide linker. In some aspects, the peptide linker connects the dysfunctional P2X7 receptor epitope moiety to the Fc region of an antibody. In some aspects, the peptide linker may comprise the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS). In some aspects, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer. The linker may be a series of repeating glycine and serine residues of varying lengths (GS), i.e., (GS)n, where n is any value from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS) or longer, (GS)11 or longer. It should be understood that n can be any value, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or greater. Fusion proteins having linkers of such lengths are included within the scope of the invention. Preferably n is not greater than 3 (i.e., when n equals 3, the linker is GSGSGS).

[0194] In additional embodiments, the linker may include amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linking region may also comprise the sequence GSGK.

[0195] The peptide linker may consist of a series of repeating Thr-Pro (TP), with one or more additional amino acids at the N and C termini of the repeating sequence. For example, the linker may comprise or consist of the sequence GTPTPTPTPTGEF (also referred to as the TP5 linker). In additional aspects, the linker may be a short and / or α-helical rigid linker (e.g., A(EAAAK)3A, PAPAP, or dipeptides such as LE or CC).

[0196] In additional embodiments, as an alternative to or in addition to the glycine-serine-based linking region described above, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety linked to the Fc region of an antibody via a hinge region. The linkage between the dysfunctional P2X7 receptor epitope moiety and the Fc region may include a combination of a hinge region and a linking region.

[0197] Examples of suitable hinge regions include hinge regions derived from immunoglobulins. The hinge region may be derived from IgG1, IgG2, IgG3, or IgG4, and may comprise one or more amino acid substitutions (e.g., to prevent or reduce the likelihood of disulfide bridge formation). Alternative hinge sequences may be derived from alternative immunoglobulin domains CD8A, CD8B, CD4, or CD28, TRAC, TRBC, TRGC, TRDC.

[0198] Table 3 below provides non-limiting examples of suitable hinge regions for linking the dysfunctional P2X7 receptor epitope portion and the Fc region in the molecules of the present invention.

[0199] It should 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 (N to C terminus) directly conjugated to the Fc region. Alternatively, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, and then the Fc region. Additionally, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, and then the Fc region. In 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 the Fc region. Of course, those skilled in the art should understand that alternative configurations are possible (i.e., where the dysfunctional P2X7 receptor epitope portion is linked to the C-terminus of the Fc region by one or more linkers and / or hinge regions).

[0200] Table 3: Additional exemplary linker / hinge region sequences

[0201]

[0202]

[0203]

[0204] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is directly fused to the Fc region of an antibody such that there is no linker between the two regions of the fusion protein.

[0205] The radionuclide can be any radionuclide suitable for nuclear medicine (such as nuclear medicine tomography imaging). The radionuclide can allow for detection of the radiolabeled compound of the present invention, for example, by radionuclide scanning. In some embodiments, the radionuclide is a positron-emitting radioisotope that can be detected by positron emission tomography (PET). In some embodiments, the radionuclide is a γ-emitting isotope that can be detected by single photon emission computed tomography (SPECT).

[0206] The radionuclide can be linked to the radiolabeled compound of the present invention by a covalent bond or a non-covalent bond (such as by coordination). In some embodiments, the radionuclide is selected from carbon-11 ( 11 C), fluorine-18 ( 18 F), scandium-44 ( 44 Sc), copper-62, copper-64, and copper-67 ( 62Cu, 64 Cu, 67 Cu), gallium-67 and gallium-68( 67 Ga, 68 Ga), yttrium-86 and yttrium-90( 86 Y, 90 Y), zirconium-89( 89 Zr), niobium-90( 90 Nb), technetium-94 and technetium-99( 94m Tc, 99m Tc), indium-111( 111 In), iodine-123, iodine-124, iodine-125 and iodine-131( 123 I, 124 I, 125 I, 131 I), lutetium-177( 177 Lu) and bismuth-123( 213 Bi). In some embodiments, the radionuclide is selected from radioisotopes of C, F, Sc, Cu, Ga, Y, Zr, Nb, Tc, In, I, Lu, and Bi.

[0207] The radionuclide can be directly linked to the epitope moiety, for example, linked to an amino acid side chain. In cases where the epitope moiety contains a spacer, the radionuclide can be directly linked to the amino acid side chain of the spacer, which can sufficiently space the radionuclide from the recognition sequence so as not to affect the binding of the epitope moiety. In embodiments where the radionuclide is directly linked to the epitope moiety, the radionuclide can be selected from 11 C, 18 F, and 99m Tc. Examples of amino acids directly linked to the radionuclide include fluorine-18 labeled tyrosine( 18 F-Tyr) and technetium-99 labeled histidine( 99m Tc-His).

[0208] The radionuclide can alternatively be indirectly linked to the epitope moiety, for example, the radionuclide can be included in a radiolabeled moiety conjugated to the epitope moiety. In some embodiments, the radiolabeled moiety is conjugated to the amino acid side chain of the epitope moiety. In some embodiments, the radiolabeled moiety is conjugated to the N-terminus or C-terminus of the epitope moiety.

[0209] The radionuclide can be linked to the radiolabeled moiety by a covalent bond. Thus, in some embodiments, the radiolabeled moiety contains a covalently bound radionuclide. In these embodiments, the radionuclide can be selected from 11 C, 18 F, 123 I,124 I、 125 I and 131 I.

[0210] The radionuclide can alternatively be linked to the radiolabeled moiety by non-covalent bonds (e.g., by coordination). Thus, in some embodiments, the radiolabeled moiety comprises a chelator moiety capable of chelating the radionuclide, wherein the radionuclide is complexed with the chelator moiety. In these embodiments, the radionuclide can be selected from 44 Sc, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 90 Nb, 94m Tc, 99m Tc, 111 In, 177 Lu and 213 Bi.

[0211] The chelator moiety can be any suitable chelator capable of chelating a radionuclide. In some embodiments, the chelator moiety is selected from TMT (6,6”-bis[N,N”,N”'-tetrakis(carboxymethyl)aminomethyl]-4'-(3-amino-4-methoxyphenyl)-2,2':6',2”-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-NN',N”(N”'-tetraacetic acid, also known as cyclen tetraacetic acid), TCMC (tetra-bisamide of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tri(acetic acid)-10-(2-thioethyl)acetamide), CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), NOTA (1,4,7-triazacyclononane-triacetic acid), NETA ({4-[2-(bis-carboxymethyl-aminoethyl]-7-carboxymethyl-[1,4,7]triazacyclononane-1-yl}), diamsar (3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine), DTPA (pentetic acid or diethylenetriaminepentaacetic acid), CHX-A”-DTPA ([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), Te2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), HBED (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid), 5HBED (3,3'-((ethane-1,2-diylbis((carboxymethyl)aza-diyl))bis(methylene))bis(4-hydroxybenzenesulfonate)), HYBIC (6-hydrazinonicotinic acid), DFO (deferoxamine), DFOsq (DFO-squaramide) and HOPO (3,4,3-(LI-1,2-HOPO) or other chelators as described herein.

[0212] The chelator moiety can be conjugated directly to the epitope moiety or indirectly to the epitope moiety via a linker, which can comprise a peptide or a chemical group. The linker can be any suitable linker known in the art, provided that the presence of the linker does not substantially affect the ability of the chelator moiety to complex the radionuclide and / or does not affect the ability of the epitope moiety to bind to immune cells. The chelator moiety can be conjugated to the epitope moiety or the linker (if present) by any suitable means. As a non-limiting example, in the case where the chelator moiety is DOTA, DOTA can be conjugated to the epitope moiety or the linker, for example, by forming an amide or ester bond with a suitable functional group (such as an amine or a hydroxyl group) on the epitope moiety or the linker, through at least one of the carboxylic acid groups of DOTA. Alternatively, DOTA can be conjugated to the epitope moiety or the linker through at least one of the carbon atoms in the tetraazacyclododecane ring and / or through at least one of the methylene groups of at least one of the four carboxylic acid groups of DOTA.

[0213] In any embodiment, the radiolabeled moiety can be further conjugated to another dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by a receptor expressed on immune cells, wherein the receptor comprises an antigen recognition domain and a signal transduction domain for binding the dysfunctional P2X7 receptor. That is, as described elsewhere herein, the radiolabeled compounds of the present invention can comprise one or more dysfunctional P2X7 receptor epitope moieties. In the case where the radiolabeled compounds of the present invention comprise two or more dysfunctional P2X7 receptor epitope moieties, the epitope moieties can comprise the same sequence or different sequences or be composed thereof.

[0214] Radiolabeled precursor molecule

[0215] In a molecule labeled with a radionuclide, the radionuclide may be prone to decay and may have a relatively short half-life. For this reason, the radiolabeled molecule may need to be prepared shortly before its intended use (e.g., before being administered to a subject and detected in vivo by radionuclide scanning), such that the radiolabeled molecule can be used within the expected lifetime of the radionuclide. Ideally, the radiolabeled molecule is prepared from a precursor by a minimum number of reaction steps, which can allow for the efficient preparation of the radiolabeled molecule. As a non-limiting example, the radiolabeled molecule can be prepared from a radiolabeled precursor compound in about 30 minutes.

[0216] Accordingly, the present invention also provides a radiolabeled precursor molecule comprising:

[0217] (i) A dysfunctional P2X7 receptor epitope moiety, said dysfunctional P2X7 receptor epitope moiety being recognized or capable of being bound by the antigen-binding domain of a receptor expressed on an immune cell, wherein said receptor is for binding to a dysfunctional P2X7 receptor and comprises a signaling domain; and

[0218] (ii) A radionuclide precursor moiety selected from:

[0219] (A) An atom or functional group capable of being converted into a radionuclide;

[0220] (B) A reactive functional group capable of being conjugated to a radiolabeled cofactor; or

[0221] (C) A chelator moiety capable of chelating a radionuclide,

[0222] or a salt or solvate thereof.

[0223] The radiolabeled precursor molecules of the present invention can be used to prepare the radiolabeled molecules as described herein. Accordingly, the present invention also provides the use of the radiolabeled precursor molecules for the preparation of the radiolabeled molecules as described herein.

[0224] It will be apparent to those skilled in the art that the epitope moiety will generally be any suitable epitope moiety for the radiolabeled molecule as defined herein. However, the epitope moiety is not directly or indirectly linked to the radionuclide, but is directly or indirectly linked to a moiety that can be converted into a radionuclide or can be conjugated or chelated with a radionuclide (such as any one of (A), (B) or (C) as described above).

[0225] The radiolabeled precursor molecule can comprise a radionuclide precursor moiety conjugated to the epitope moiety, wherein the radiolabeled precursor moiety comprises one of (A), (B) or (C) above. Thus, any one of (A), (B) and (C) can be present in the epitope moiety or in the radiolabeled precursor moiety conjugated to the epitope moiety.

[0226] Any suitable atom or functional group known in the art that can be converted into a radionuclide can be used. The atom or functional group can be converted into a radionuclide, for example, by substitution, addition or exchange with a compound containing the radionuclide. As a non-limiting example, the atom or functional group can be any suitable leaving group that can be substituted by a radionuclide (e.g. 18 F). In the case where the atom or functional group is present in the epitope moiety, the atom or functional group can be, for example, the hydroxyl group of a tyrosine side chain, which can be substituted by 18 F to provide 18F-Tyr. In the case where an atom or functional group is present in the radiolabeled precursor moiety, the atom or functional group can be, for example, any suitable leaving group as described in Krishnan et al.( 18 F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry, November 7, 2017; Vol. 23, No. 62: pp. 15553-15577). As another non-limiting example, the atom or functional group can be an iodine atom capable of exchanging with 125 I or other iodine isotopes.

[0227] Any suitable reactive functional group capable of conjugating a radiolabeled cofactor known in the art can be used. It should be understood that the reactive functional group will be capable of forming a covalent bond with a complementary reactive functional group present on the radiolabeled cofactor. In the case where the reactive functional group is present in the epitope moiety, the reactive functional group can be, for example, a reactive functional group of an amino acid side chain (e.g., a cysteine thiol group). In the case where the reactive group is present in the radiolabeled precursor moiety, the reactive group can be conjugated to the epitope moiety through any suitable linker, which can comprise a peptide or a chemical group.

[0228] In some embodiments, the reactive functional group is an amino group, which can form an amide bond with a radiolabeled co - ligand containing a carboxylic acid group. In some embodiments, the reactive functional group is a carboxylic acid group, which can form an amide bond with a radiolabeled co - ligand containing an amino group. In some embodiments, the reactive functional group is a hydroxyl group, which can form an ester bond with a radiolabeled co - ligand containing a carboxylic acid group. In some embodiments, the reactive functional group is a carboxylic acid group, which can form an ester bond with a radiolabeled co - ligand containing a hydroxyl group. In some embodiments, the reactive functional group contains a leaving group (such as, but not limited to, halogen, tosylate, mesylate, triflate, etc.), which can couple with a radiolabeled co - ligand containing a nucleophilic group (such as, but not limited to, thiol, hydroxyl, amine, or carboxylic acid) by nucleophilic substitution. In some embodiments, the reactive functional group contains a nucleophilic group (such as, but not limited to, thiol, hydroxyl, amine, or carboxylic acid), which can couple with a radiolabeled co - ligand containing a leaving group (such as, but not limited to, halogen, tosylate, mesylate, triflate, etc.) by nucleophilic substitution. In some embodiments, the reactive functional group contains a group represented by an open valence (such as a general alkyl group of R - CH2 -), and this group can be connected to the radiolabeled co - ligand by a single covalent bond. This list is not exhaustive and is only intended for illustration. Any other reactions and reagents known to facilitate intermolecular coupling are incorporated herein. Any one of these compounds and the corresponding conjugates is contemplated within the present invention. As another non - limiting example, the reactive functional group (or complementary reactive functional group) may be able to form a covalent bond with a nitrogen - containing functional group (such as an amine) or a sulfur - containing functional group (such as a thiol), such as - C(=O)CH=CH2, - S(=O)CH=CH2, - S(=O)2CH=CH2, - C(=O)CH=CH - CH2NR2, - S(=O)CH=CH - CH2NR2, - S(=O)2CH=CH - CH2NR2, C(=O)C≡CH, S(=O)C≡CH, S(=O)2C≡CH, α,β - unsaturated ketone, α,β - unsaturated ester, α,β - unsaturated amide, α,β - unsaturated sulfone, α,β - unsaturated sulfoxide, α,β - unsaturated sulfonamide, propargyl ketone, propargyl ester, propargyl amide, propargyl sulfone, propargyl sulfoxide, propargyl sulfonamide, maleimide, α - chloroamide, disulfide, 5 - fluoro - 2,4 - dinitrobenzene, etc., or any other amine or thiol - modifying functional group known in the art, including Krishnan et al.( 18Those described in "F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry, November 7, 2017; Vol. 23, No. 62: pp. 15553-15577". It should be understood that the reactive functional groups present in the radiolabeling precursor compound and the complementary reactive functional groups present in the radiolabeling co - ligand can be reversed.

[0229] In some embodiments, the reactive functional group is capable of reacting with the radiolabeling co - ligand by click chemistry, such as, for example, as described by Krishnan et al.( 18 "F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry, November 7, 2017; Vol. 23, No. 62: pp. 15553-15577". In some embodiments, the reactive functional group is an alkyne, which can react with a radiolabeling co - ligand containing an azide group by click chemistry. In some embodiments, the reactive functional group is an azide, which can react with a radiolabeling co - ligand containing an alkyne group by click chemistry. Suitable alkynes include strained alkynes such as dibenzocyclooctyne (DBCO), bicyclononyne (BCN), monofluorooctyne (MOFO), and difluorocyclooctyne (DIFO). In some embodiments, the reactive functional group is a tetrazine, which can react with a radiolabeling co - ligand containing an alkene by click chemistry. In some embodiments, the reactive functional group is an alkene, which can react with a radiolabeling co - ligand containing a tetrazine by click chemistry. Suitable alkenes include strained alkenes such as trans - cyclooctene (TCO), cyclooctyne, and norbornene.

[0230] The radiolabeling co - ligand can be any suitable radiolabeling co - ligand capable of conjugating to the epitope moiety by reacting with the reactive functional group of the epitope moiety. Non - limiting examples of radiolabeling co - ligands containing a covalently - bound radionuclide include Krishnan et al.( 18Those described in "F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry, November 7, 2017; Vol. 23, No. 62: pp. 15553-15577). In the case where the radionuclide is linked to the radiolabeled cofactor by a non-covalent bond, the radiolabeled moiety can be a chelator moiety capable of chelating the radionuclide, wherein the radionuclide is complexed with the chelator moiety. In this case, the chelator moiety can be the same chelator moiety as defined for the radiolabeled compounds described herein.

[0231] The radiolabeled cofactor can be further conjugated (or capable of being further conjugated) to another dysfunctional P2X7 receptor epitope moiety recognized or capable of being bound by a receptor expressed on an immune cell, wherein the receptor comprises an antigen recognition domain and a signal transduction domain for binding to the dysfunctional P2X7 receptor.

[0232] Any suitable chelator moiety capable of chelating a radionuclide can be used. The chelator moiety can be present in the epitope moiety. In this case, the chelator moiety can be, for example, a histidine residue capable of chelating a radionuclide (e.g. 99m Tc, to provide 99m Tc-His). Alternatively, the chelator moiety can be present in a radiolabeled precursor conjugated to the epitope moiety. In this case, the chelator moiety can be the same chelator moiety as defined for the radiolabeled compounds described herein. Additionally, the chelator moiety can be conjugated to the epitope moiety via any suitable linker as described herein.

[0233] Dysfunctional P2X7 receptor epitope moiety

[0234] The dysfunctional P2X7 receptor epitope moiety can be provided in the form of a dysfunctional P2X7 receptor or a fragment of a dysfunctional P2X7 receptor, which receptor has at least one of three ATP binding sites formed at the interface between adjacent correctly stacked monomers that cannot bind ATP. Such receptors cannot extend the opening of non-selective calcium channels to apoptotic pores.

[0235] According to the present invention, the dysfunctional P2X7 receptor epitope moiety is generally in the form of a peptide fragment of a dysfunctional P2X7 receptor. Thus, in a particularly preferred embodiment, the radiolabeled molecule of the present invention comprises:

[0236] (i) A peptide that is recognized or capable of being bound by the antigen recognition domain of a receptor expressed on an immune cell, wherein the receptor is for binding to a dysfunctional P2X7 receptor and comprises a signaling domain; and

[0237] (ii) A radionuclide that is directly or indirectly linked to the epitope moiety,

[0238] or a salt or solvate thereof.

[0239] In addition, the present invention provides a radiolabeled precursor molecule, which comprises:

[0240] (i) A peptide that is recognized or capable of being bound by the antigen binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding to a dysfunctional P2X7 receptor and comprises a signaling domain; and

[0241] (ii) A radionuclide precursor moiety selected from:

[0242] (A) An atom or functional group capable of being converted into a radionuclide;

[0243] (B) A reactive functional group capable of conjugating with a radiolabeled cofactor; or

[0244] (C) A chelator moiety capable of chelating a radionuclide,

[0245] or a salt or solvate thereof.

[0246] Generally, the peptide comprises an epitope that is not present on a functional P2X7 receptor or is not available for binding on a functional P2X7 receptor.

[0247] In some embodiments, the peptide comprises a proline at amino acid position 210 of a dysfunctional P2X7 receptor. In some embodiments, the peptide comprises one or more amino acid residues from glycine at amino acid position 200 to cysteine at amino acid position 216 of a dysfunctional P2X7 receptor, including the terminal values.

[0248] A series of peptide fragments of the dysfunctional P2X7 receptor are known and discussed in PCT / AU2002 / 000061 (and in the corresponding publications WO 2002 / 057306 and US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584 or US 10,450,380), PCT / AU2008 / 001364 (and in the corresponding publications WO 2009 / 033233 and US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,45), and PCT / AU2009 / 000869 (and in the corresponding publications WO 2010 / 000041 and US 8,597,643, US 9,328,155 or US 10,238,716), the contents of all of these documents being incorporated herein by reference in their entirety. Exemplary peptides containing epitopes contemplated for use in the present invention are described below within these specifications.

[0249] PCT published peptide sequences

[0250] WO 2002 / 057306 GHNYTTRNILPGLNIT (SEQ ID NO:3)

[0251] WO 2002 / 057306 GHNYTTRNILPGLNITC (SEQ ID NO:2) (also referred to herein as the "E200" epitope)

[0253] WO 2009 / 033233 KYYKENNVEKRTLIKVF (SEQ ID NO:4) (also referred to herein as the "E300" epitope)

[0255] WO 2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO:6) (also referred to herein as the "E200 / E300" or "composite" epitope)

[0256] Non-limiting examples of variants of the E200 peptide sequence (including those having N- and / or C-terminal extensions, and various linkers, hinges or spacers) are provided in Table 1.

[0257] The amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:7 may comprise a part of an epitope portion that is recognized or capable of being bound by a receptor expressed on an immune cell (also referred to herein as the "recognition sequence" of the epitope portion).

[0258] In some embodiments, the epitope moiety comprises or consists of an amino acid sequence selected from any of the peptide sequences listed in Table 1 above.

[0259] In the case where the peptides in Table 1 have histidine residues, the radionuclide can be conjugated to the epitope moiety through two histidine residues. In the case where the sequence has cysteine residues, the conjugation can be carried out by using an F18 compound as described herein such as N-[N-(S)-1,3-dicarboxypropyl]carbamoyl]-4- 18 F]fluorobenzyl-L-cysteine( 18 F-DCFBC) to label the cysteine residue. In the case where the sequence has lysine residues, the coupling of the radionuclide can be through the lysine residue.

[0260] In some embodiments, the N-terminus of the epitope moiety is a free amine (-NH2).

[0261] In some embodiments, the C-terminus of the epitope moiety is a free acid (-COOH). In some embodiments, the C-terminus is a derivative or analogue of the free acid group, such as an ester (-COOC1-6alkyl) or a primary or secondary amide (-CONHR4, where R4 is selected from H and C1-6alkyl). Advantageously, compared to the free acid, having a C-terminus that is a derivative or analogue of the free acid group can improve the biological stability of the peptide. In some embodiments, the C-terminus is a derivative or analogue of the free acid group that contains a functional moiety such as biotin.

[0262] Receptors and immune cells expressing them

[0263] In any embodiment, the receptor comprising an antigen-binding domain and a signaling domain for binding to a dysfunctional P2X7 receptor is preferably a chimeric antigen receptor (CAR) or a variant thereof. The receptor can also be a modified TCR.

[0264] Generally, a CAR, its variant or a TCR can comprise an extracellular domain (extracellular portion) containing an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain can be linked to the transmembrane domain by a linker. The extracellular domain can also contain a signal peptide. Preferably, the extracellular portion of the CAR, its variant or the TCR comprises an nfP2X7 binding domain that recognizes an epitope such as E200 (or E300 or E200-300 complex) as disclosed herein.

[0265] Generally, the antigen recognition domain comprises a binding polypeptide that contains an amino acid sequence homologous to one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide contains an amino acid sequence homologous to the CDR1, CDR2, and CDR3 domains of the V H and / or V L chain of an antibody that binds to a dysfunctional P2X7 receptor.

[0266] In a preferred embodiment, the binding polypeptide comprises the V of the antibody described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584 or US10,450,380), PCT / AU2007 / 001540 (or in the corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in the corresponding US publication US2010-0036101), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181,320, US 9,944,701 or US10,597,451), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US10,053,508) and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609) H and / or V LThe amino acid sequences of the CDRs of the chain, the entire contents of these documents 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 US patents US 9,127,059, US 9,688,771 or US10,053,508) or the CDR amino acid sequences of BPM09 as described in PCT / AU2007 / 001541 (or in the corresponding US publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under the accession number 06080101.

[0267] In a further embodiment, the binding polypeptide of the CAR comprises the V of the antibodies described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584 or US10,450,380), PCT / AU2007 / 001540 (or in the corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in the corresponding US publication US2010-0036101), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181,320, US 9,944,701 or US10,597,451), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US10,053,508) and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609). H and / or V LThe amino acid sequences of the chains, the entire contents of these documents are hereby incorporated 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 US patents US 9,127,059, US 9,688,771 or US 10,053,508) or the CDR amino acid sequences of BPM09 as described in PCT / AU2007 / 001541 (or in the corresponding US publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under the accession number 06080101.

[0268] In additional embodiments, the binding polypeptide of the CAR comprises the amino acid sequence of an antibody or fragment thereof as described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584 or US 10,450,380), PCT / AU2007 / 001540 (or in the corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in the corresponding US publication US2010-0036101), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 or US 10,053,508) and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609), the entire contents of these documents being incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 as described in PCT / AU2010 / 001070 (or in any one of the corresponding US patents US 9,127,059, US 9,688,771 or US 10,053,508) or the CDR amino acid sequence of BPM09 as described in PCT / AU2007 / 001541 (or in the corresponding US publication US2010-0036101) and produced by the hybridoma AB253 deposited at the European Collection of Cell Cultures (ECACC) under accession number 06080101.

[0269] A "signal peptide" refers to a peptide sequence that directs the trafficking and localization of a protein within a cell, such as to a particular organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0270] Generally, an "antigen-binding domain" (or antigen recognition domain) refers to the region of a CAR that specifically binds to an antigen (and thus is capable of targeting cells containing that antigen). A CAR can comprise one or more antigen-binding domains. Generally, the targeting region on a CAR is extracellular. The antigen-binding domain can comprise an antibody or an antibody-binding fragment thereof. The antigen-binding domain can comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a diabody, or a bispecific antibody. Any molecule that specifically binds to a given antigen, such as an affibody or a ligand-binding domain from a naturally occurring receptor, can be used as an antigen-binding domain. Generally, the antigen-binding domain is an scFv. Generally, in an scFv, the variable regions of the immunoglobulin heavy and light chains are fused via a flexible linker to form the scFv. Such linkers can be, for example, a "(G4 / S1)3 linker" and its variants, but those skilled in the art will understand that various linker sequences and forms can be used.

[0271] A CAR can also comprise a "hinge" region (sometimes referred to as a spacer or linker) that connects the antigen-binding domain to the transmembrane domain. This is generally a hydrophilic region between the antigen-binding domain and the transmembrane domain. A CAR can comprise an extracellular hinge domain, but it can also be without such a hinge. The hinge region can comprise, for example, an Fc fragment of an antibody or a fragment thereof, a hinge region of an antibody or a fragment thereof, a CH2 or CH3 region of an antibody, an accessory protein, an artificial hinge sequence, or a combination thereof. An example of a hinge region is the CD8α hinge.

[0272] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source of such a domain. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8α or CD28. When the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides, a CAR can have two (or more) transmembrane domains. Due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR, the splitting of the key signaling and antigen recognition modules enables small molecule-dependent, titratable, and reversible control of CAR cell expression (Wu et al., Science, 2015, Vol. 350: pp. 293-303).

[0273] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of immune cells expressing the CAR. "Effector function" means the specialized function of a cell. For example, in T cells, effector functions can be cytolytic activity or helper cell activity, including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein that transduces effector function signals and directs the cells expressing the CAR to perform specialized functions. The intracellular signaling domain can include any intact, mutated, or truncated part of an intracellular signaling domain of a given protein sufficient to transduce signals that initiate or block immune cell effector functions.

[0274] The function of the intracellular domain can be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0275] Notable examples of intracellular signaling domains for CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction upon antigen receptor engagement.

[0276] Typically, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences. The first class is the sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and the second class are the sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences, co-stimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can contain one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0277] Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0278] Examples of primary cytoplasmic signaling sequences containing ITAM commonly used for CARs are the signaling sequences derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. The most prominent is the sequence derived from CD3ζ.

[0279] The cytoplasmic domain of the CAR can be designed to contain the CD3-ζ signaling domain itself or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of the CAR can contain a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for an effective response of lymphocytes to an antigen. Examples of co-stimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0280] In some embodiments, the activating receptor (a portion of the signaling domain is derived from this activating receptor) is a CD3 co-receptor complex or an Fc receptor.

[0281] In some embodiments, the co-stimulatory receptor (a portion of the signaling domain is derived from this co-stimulatory receptor) is selected from CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0282] In some embodiments, the co-stimulatory receptor (a portion of the signaling domain is derived from this co-stimulatory receptor) is selected from CD28, OX40, or 4-1BB.

[0283] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be connected to each other with or without a linker in a random or specified order. Short oligopeptide or polypeptide linkers (preferably between 2 and 10 amino acids in length) can form the connection. A prominent linker is the glycine-serine doublet.

[0284] As an example, the cytoplasmic domain can contain the signaling domain of CD3-ζ and the signaling domain of CD28. In another example, the cytoplasmic domain can contain the signaling domain of CD3-ζ and the signaling domain of CD27. In another example, the cytoplasmic domain can contain the signaling domain of CD3-ζ, the signaling domain of CD28, and the signaling domain of CD27.

[0285] As described above, the extracellular portion or transmembrane domain or cytoplasmic domain of the CAR can also contain a heterodimerization domain for the splitting of the key signaling and antigen recognition modules of the CAR.

[0286] CARs that bind to the radiolabeled molecule of the invention (such as CARs comprising the nfP2X7 E200 binding domain) can be designed to comprise any part or parts of the above-described domains as herein described in any order and / or combination, thereby generating a functional CAR.

[0287] The affinity of the dysfunctional P2X7 receptor binding domain of the CAR for the nfP2X7 recognition site E200 of the radiolabeled molecule of the invention can vary, but generally the binding affinity can be in the range of about 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM or about 1 nM, preferably at least about 10 pM or 1 pM. In a preferred embodiment, the binding affinity is at least about 1 nM or at least about 10 nM.

[0288] Receptors such as CARs, their variants, or TCRs or their variants are generally expressed by immune cells.

[0289] The immune cells can be "engineered cells", "genetically modified cells" or "immune effector cells" as herein described. Additionally, the immune cells can be immune cell precursors capable of differentiating into immune cells. Cells capable of differentiating into immune cells (e.g., T cells that will express a dysfunctional P2X7 CAR) can be stem cells, multi-lineage progenitors or induced pluripotent stem cells.

[0290] The immune cells can be white blood cells, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells (including CD4+ T cells, CD8+ T cells), natural killer cells, natural killer T cells or γδ T cells.

[0291] In any embodiment, the immune cells can be T cells, where optionally the T cells do not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic level or functional level).

[0292] In any embodiment, the immune cells optionally do not express co-stimulatory molecules, which can be checkpoint, exhaustion or apoptosis-related signaling receptors and ligands, such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R (e.g., by knocking out one of these genes at the genetic level or functional level).

[0293] In some embodiments, the genetically modified cell comprises two or more different receptors (e.g., two or more CARs, or variants thereof). The CARs can bind to different epitopes on the same target molecule (e.g., different epitopes on a dysfunctional P2X7 receptor). Alternatively, the CARs can bind to different target molecules such that only one of the CARs binds to the dysfunctional P2X7 receptor.

[0294] As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs having non-identical antigen recognition domains and / or non-identical signal transduction domains. In one example, "different CARs" includes two CARs having the same antigen recognition domain (e.g., both CARs can recognize a dysfunctional P2X7 receptor), but having different signal transduction domains (such as one CAR having a signal transduction domain with a portion of an activating receptor and the other CAR having a signal transduction domain with a portion of a co-stimulatory receptor). As will be appreciated, at least one of the two or more CARs within this embodiment will have an antigen recognition domain that recognizes the dysfunctional P2X7 receptor, and the other CARs can take any suitable form and can be directed against any suitable antigen.

[0295] Method for preparing a radiolabeled molecule

[0296] The present invention also provides a method for preparing a radiolabeled molecule, the method comprising:

[0297] - providing a radiolabeled precursor molecule as defined herein; and

[0298] - reacting the radiolabeled precursor molecule to provide a radiolabeled molecule as defined herein,

[0299] thereby providing a radiolabeled molecule.

[0300] The radiolabeled precursor molecule can be reacted appropriately to provide a radiolabeled molecule according to the nature of the radiolabeled precursor moiety.

[0301] In some embodiments, the method comprises:

[0302] - providing a radiolabeled precursor molecule comprising:

[0303] (i) an epitope portion of a dysfunctional P2X7 receptor, the epitope portion of the dysfunctional P2X7 receptor being recognized or capable of being bound by an antigen binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding to the dysfunctional P2X7 receptor and comprises a signal transduction domain; and

[0304] (ii) An atom or functional group that can be converted into a radionuclide,

[0305] or a salt or solvate thereof, and

[0306] - converting the atom or functional group into a radionuclide;

[0307] thereby providing a radiolabeled molecule.

[0308] In some embodiments, the method comprises:

[0309] - providing a radiolabeled precursor molecule comprising:

[0310] (i) A dysfunctional P2X7 receptor epitope portion that is recognized or capable of being bound by the antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding to the dysfunctional P2X7 receptor and comprises a signaling domain; and

[0311] (ii) A reactive functional group that can be conjugated to a radiolabeled cofactor,

[0312] or a salt or solvate thereof, and

[0313] - conjugating the radiolabeled cofactor via the reactive functional group;

[0314] thereby providing a radiolabeled molecule.

[0315] In some embodiments, the method comprises:

[0316] - providing a radiolabeled precursor molecule comprising:

[0317] (i) A dysfunctional P2X7 receptor epitope portion that is recognized or capable of being bound by the antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding to the dysfunctional P2X7 receptor and comprises a signaling domain; and

[0318] (ii) A chelator moiety that can chelate a radionuclide,

[0319] or a salt or solvate thereof, and

[0320] - chelating the radionuclide with the chelator moiety;

[0321] thereby providing a radiolabeled molecule.

[0322] In the above method, atoms or functional groups that can be transformed into radionuclides, reactive functional groups that can conjugate with radiolabeled auxiliaries, and chelator moieties that can chelate radionuclides may be present in the epitope moiety or in the radiolabeled precursor moiety conjugated to the epitope moiety, as described herein.

[0323] Radiolabeled precursor molecules can be appropriately prepared and reacted by methods known in the art (including the methods described herein) to provide radiolabeled molecules. Suitable methods for obtaining radiolabeled peptides (such as peptides conjugated for coupling 68 Ga with DOTA-like conjugates) are described, for example, in Mueller et al., 2011, Nature Protocols, Vol. 11: pp. 1057-1066, which is incorporated herein by reference.

[0324] The epitope moiety of the radiolabeled molecule or radiolabeled precursor molecule of the present invention can be prepared by known chemical methods, including solid-phase and solution-phase peptide synthesis using Fmoc or Boc-protected amino acid residues. The epitope moiety can also be prepared by known recombinant DNA techniques.

[0325] The radiolabeling of peptides can be through one or more histidine residues present in the peptide. Examples of the radiolabeling of histidine residues are well known in the art and are described, for example, in Ibrahim et al., 2016, Radiochemistry, Vol. 58: pp. 521-527, which is incorporated herein by reference. In such cases, the peptide may contain a biotin label or amide at the C-terminus.

[0326] In additional examples, tyrosine residues in the peptide can be labeled using standard techniques known to those skilled in the art.

[0327] Alternatively, the radiolabeling can be the labeling of cysteine residues using F18 compounds such as N-[N-(S)-1,3-dicarboxypropyl]carbamoyl]-4- 18 F]fluorobenzyl-L-cysteine( 18 F-DCFBC). In this case, the peptide will preferably contain an amino acid sequence as shown in any one of SEQ ID NO:2, SEQ ID NO:10, or SEQ ID NO:13 (for example: GHNYTTRNILPGLNITSTFHKTC-amide). Methods for 18F labeling are described in David et al., 2019, RSC Adv. Vol. 15: pp. 8638-8649, which is incorporated herein by reference.

[0328] Applications

[0329] The radiolabeled molecule of the present invention can be used to detect immune cells expressing a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor. The radiolabeled molecule comprises an epitope portion of a dysfunctional P2X7 receptor that is recognized or capable of being bound by the receptor expressed on the immune cells. Thus, the presence of the epitope portion of the dysfunctional P2X7 receptor can allow the molecule of the present invention to bind to the immune cells.

[0330] Accordingly, the present invention provides the use of the radiolabeled molecule described herein for detecting immune cells expressing a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor.

[0331] The present invention also provides a method for detecting immune cells expressing a receptor comprising an antigen recognition domain and a signaling domain for binding to a dysfunctional P2X7 receptor in a subject, the method comprising:

[0332] - administering to the subject a radiolabeled molecule as described herein, the subject having been administered immune cells expressing a receptor comprising an antigen recognition domain for binding to a dysfunctional P2X7 receptor; and

[0333] - detecting the radiolabeled compound in the subject, wherein the presence of the radiolabeled compound indicates the presence of immune cells.

[0334] In some embodiments, the radiolabeled molecule is detected by performing a radionuclide scan. The radionuclide scan can be appropriately selected according to the radionuclide present in the radiolabeled molecule. In some embodiments, the radionuclide scan can be positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0335] In some embodiments, the method further comprises imaging the detected radiolabeled molecule.

[0336] In some embodiments, the method further comprises allowing the radiolabeled molecule to accumulate at the site in the subject where immune cells are found, prior to the step of detecting the radiolabeled molecule.

[0337] In some embodiments, the method further comprises administering to the subject immune cells expressing a receptor comprising an antigen recognition domain for binding to a dysfunctional P2X7 receptor, prior to the step of administering the radiolabeled molecule to the subject.

[0338] The methods and uses of radiolabeled molecules described herein can advantageously permit (i) determination of whether immunocytes expressing a receptor comprising an antigen recognition domain and a signaling domain for binding a dysfunctional P2X7 receptor are present in a subject, (ii) identification of the location of said immunocytes, including determination of the distribution of populations of said immunocytes in the subject, and (iii) quantification of the number of said immunocytes in a subject or at a specific location / site within a subject. This information can assist in the development or adjustment of treatment regimens using said immunocytes.

[0339] Compositions and formulations

[0340] The radiolabeled molecule can be provided or formulated in a suitable form for administration to a subject.

[0341] Accordingly, the present invention provides a composition comprising a radiolabeled molecule of the present invention or a salt or solvate thereof.

[0342] The composition can be a pharmaceutical composition. In the case of a pharmaceutical composition, the composition can comprise a pharmaceutically acceptable carrier, such as an aqueous carrier.

[0343] The present invention further provides a formulation comprising a radiolabeled molecule of the present invention or a salt or solvate thereof. The formulation of the radiolabeled molecule can comprise a pharmaceutically acceptable excipient (carrier or diluent). Examples of commonly used excipients include, but are not limited to: saline, buffered saline, glucose, water for injection, glycerol, ethanol, and combinations thereof; stabilizers, solubilizers, and surfactants; buffers and preservatives; tonicity agents, fillers, and lubricants.

[0344] The compositions and formulations of the present invention can comprise one type of radiolabeled molecule, or more than one type of radiolabeled molecule (e.g., where the radiolabeled molecules can have the same or different epitope moieties of the dysfunctional P2X7 receptor).

[0345] These compositions and formulations can be applicable to the methods and uses for detecting immunocytes expressing a receptor comprising an antigen recognition domain and a signaling domain for binding a dysfunctional P2X7 receptor as described herein.

[0346] The radiolabeled molecule that can be in the composition or formulation of the present invention can be administered to a subject using modes and techniques known to those skilled in the art. Exemplary modes include, but are not limited to: intravenous, intraperitoneal, and intratumoral injection. Other modes include, but are not limited to: intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intra-arterial, intramedullary, intracavitary, intracardiac, intra-articular (joint), intra-synovial (joint fluid area), intracranial, intraspinal, and intrathecal (cerebrospinal fluid).

[0347] Compositions and formulations comprising a radiolabeled molecule can be administered to a subject in an amount effective to detect, for example, by radionuclide scanning, the radiolabeled molecule. The dose can be appropriately selected according to the radionuclide present in the radiolabeled molecule. The dose can be further appropriately selected according to the intended use and the particular mode of administration, according to fluid volume, viscosity, body weight, etc. The physician can ultimately determine the appropriate dose to be used.

[0348] Kit

[0349] The present invention further provides a kit comprising one or more of the following:

[0350] (i) The radiolabeled molecule of the present invention or a salt or solvate thereof;

[0351] (ii) The radiolabeled precursor molecule of the present invention or a salt or solvate thereof;

[0352] (iii) The composition of the present invention; or

[0353] (iv) The formulation of the present invention.

[0354] In the case where the kit comprises the radiolabeled precursor compound of the present invention (including the composition or formulation comprising the same), the kit can be used, for example, to prepare a radiolabeled molecule from the radiolabeled precursor molecule by the methods described herein.

[0355] The kit can be used to detect immunocytes in a subject that express a receptor comprising an antigen recognition domain and a signal transduction domain for binding a dysfunctional P2X7 receptor, the subject having been administered the immunocytes.

[0356] Optionally, the kit of the present invention may further comprise immunocytes that express a receptor comprising an antigen recognition domain and a signal transduction domain for binding a dysfunctional P2X7 receptor.

[0357] Optionally, the kit of the present invention is packaged with instructions for one or more of the methods described herein.

[0358] Examples

[0359] Example 1: Preparation of Exemplary Radioactively Labeled Molecules

[0360] A radiolabeled E200 peptide comprising the amino acid sequence GHNYTTRNILPGLNITSTFHKTSGSGK was prepared by combining a biotinylated peptide of approximately 2900 g / mol with Ga 68 (70 g / mol).

[0361] Simple conjugation of the radiolabel is through two histidine residues in the peptide using the method described in Mueller et al., 2016, Nature Protocols, Volume 11: pp. 1057-1066. Briefly, the peptide is conjugated with the chelator DOTA using standard techniques, followed by conjugation with 68 Ga.

[0362] In an alternative example, a radiolabel is conjugated to an Fc fusion protein containing an epitope of the nfP2X7 receptor (such as an amino acid sequence having SEQ ID NO: 145 (DetR1, monomer; Fc attenuated; or DetR2 SEQ ID NO: 146; or dimer Fc attenuated SEQ ID NO: 149)) using a similar method.

[0363] Example 2: For Binding to nfP2X7 Detection of radiolabeled molecules on receptor-binding CAR T cells: Imaging studies Study Design

[0364] The NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ mouse model was used, in which the breast cancer cell line MDA-MB-231 (ATCC HTB-26) was implanted in situ into the fourth mammary fat pad at 5×10 6 on day 0. On day 7 of the preclinical study, 5E0 6 nfP2X7-targeted CAR T cells were intravenously injected into the tail vein.

[0365] To detect the presence of CAR T cells in the mice, the radiolabeled molecule (i.e., peptide or fusion protein) prepared in Example 1 was administered to the mice by tail vein injection.

[0366] The mice were assigned to one of the following groups (n = 3 per group), each group representing a different time period (T) between administration of the radiolabeled molecule and detection using positron emission tomography:

[0367] Control (i.e., no radiolabeled molecule was administered);

[0368] T = 0

[0369] T = 5 minutes

[0370] T = 10 minutes

[0371] T = 15 minutes

[0372] T = 20 minutes

[0373] T = 60 minutes

[0374] T = 120 minutes

[0375] T = 240 minutes

[0376] At each time point, the distribution of the radiolabeled peptide or radiolabeled Fc fusion protein was evaluated by detecting the positron emission of gallium-68 via positron emission tomography (PET).

[0377] Whole-body static PET images were acquired, followed by whole-body CT scans for anatomical reference.

[0378] High positron emission was detected at the tumor site, indicating the enrichment of anti-dysfunctional P2X7 receptor CAR T cells and the localization of the cells at the tumor site.

[0379] A schematic diagram of the method is shown in Figure 1 .

[0380] Example 3: Demonstration of the Ability of the Detection Reagent to Bind to CAR T Cells In Vivo

[0381] On day 0, E200-targeted CAR T cells (i.e., CAR T cells capable of binding to the E200 epitope as described herein) were infused into mice bearing AsPC-1 tumors derived from a pancreatic cancer cell line (administered at a dose of 0.8E06 on day -7), intravenously injected into the tail vein.

[0382] Subsequently, 50 μg of monomeric E200-Fc fusion protein (e.g., comprising the amino acid sequence of SEQ ID NO: 145) and containing a C-terminal His-tag were injected intraperitoneally into the mice. One hour was allowed for the fusion protein to bind to the CAR T cells in vivo.

[0383] Three samples of whole blood and bone marrow were collected from the mice and flow cytometry was performed using an anti-His-tag-FITC antibody (VioGreen).

[0384] Briefly, regarding Figure 2a the results shown (using the monomeric fusion protein of the His-tag of the present invention, e.g., having the amino acid sequence of SEQ ID NO: 158):

[0385] A) Whole blood was lysed and stained with an anti-HIS-tag antibody conjugated to FITC to detect the monomeric fusion protein reagent that binds to CAR-expressing cells.

[0386] B) Bone marrow was isolated from the femurs and stained with an anti-HIS antibody conjugated to FITC to detect the monomeric fusion protein that binds to CAR-expressing cells.

[0387] The anti-HIS-tag antibody was used according to the manufacturer's instructions. Data were acquired on a MACSQuant16 flow cytometer (Miltenyi).

[0388] Regarding Figure 2b the results shown (using the LCLC biotin-conjugated monomeric fusion protein of the present invention, such as having the amino acid sequence of SEQ ID NO: 158):

[0389] A) Blood was lysed and incubated with the LCLC biotin-conjugated monomeric fusion protein, and then stained with an anti-biotin antibody in commercial FITC (VioGreen) to detect the monomeric detection reagent that binds to the CAR-expressing cells.

[0390] B) Bone marrow was isolated, then incubated with the LCLC biotin-conjugated monomeric fusion protein, and then stained with a commercial anti-biotin antibody conjugated to FITC (VioGreen) or APC to detect the monomeric detection reagent that binds to the CAR-expressing cells.

[0391] The anti-biotin antibody was used according to the manufacturer's instructions. Data were collected on a MACSQuant 16 flow cytometer (Miltenyi).

[0392] Figure 2a The results shown indicate that the monomeric molecules bound in vivo are capable of binding to the CAR-expressing cells. The molecules were detected ex vivo by an anti-HIS antibody by flow cytometry.

[0393] Figure 2b The results shown demonstrate that by ex vivo incubation of the biotinylated monomeric molecules, PBMC staining from murine blood and bone marrow is detectable by an anti-biotin antibody to identify the CAR-expressing cell subset.

[0394] In summary, the results show that CAR T cells can bind in vivo using the monomeric fusion protein as described herein.

[0395] A series of in vitro experiments were conducted in parallel, wherein briefly, Jurkat cells and / or primary CD4+ T cells and CD8+ T cells (mixed at a ratio of 1:1 after enrichment) from healthy volunteer donors were stably transduced with lentivirus (3rd generation LV system) to express an anti-nfP2X7 chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain for binding to the E200 epitope of the P2X7 receptor.

[0396] The CAR T cells were contacted with monomeric fusion proteins or dimeric fusion proteins each comprising a peptide moiety capable of being bound by the CAR or a protein. The fusion proteins used in this experiment comprise the amino acid sequences of SEQ ID NO: 158 (monomer) and SEQ ID NO: 149 (dimer).

[0397] Figure 3Shows the levels of CD25+ / CD69+ expression (each a measure of T cell activation) and the level of PD-1 expression (a measure of T cell exhaustion) up to 72 hours after contact with fusion proteins at different concentrations (10 ng / mL to 400 ng / mL).

[0398] The results show that contacting CAR T cells with monomeric fusion proteins led to a significant reduction in T cell activation and a significant reduction in T cell exhaustion in a concentration-dependent manner compared to when using dimeric fusion proteins. These results suggest that for in vivo imaging of CAR T cells, it is preferable to use monomeric fusion proteins in order to minimize the unwanted activation and exhaustion of CAR T cells in patients.

[0399] Similar experiments were performed using heterodimeric asymmetric molecules as described herein (e.g., such that the molecule comprises dimerization between an E200 peptide-Fc fusion protein and a non-identical Fc region of an antibody; using the KIH technique). The results similarly showed that contacting CAR T cells with heterodimeric asymmetric molecules containing a single copy of the E200 peptide sequence led to a significant reduction in T cell activation and a significant reduction in T cell exhaustion in a concentration-dependent manner compared to when using dimeric fusion proteins containing two copies of the E200 peptide (e.g., where the dimer is a homodimer of the E200-Fc fusion protein). These results suggest that for in vivo imaging of CAR T cells, it is preferable to use asymmetric heterodimeric molecules or monomeric fusion proteins (i.e., containing a single E200 peptide sequence) to minimize the unwanted activation and exhaustion of CAR T cells in patients.

Claims

1. A radiolabeled molecule, the radiolabeled molecule comprising: (i) A dysfunctional P2X7 receptor epitope portion that is recognized or capable of being bound by an antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding to a dysfunctional P2X7 receptor and comprises a signaling domain; and (ii) A radionuclide that is directly or indirectly linked to the epitope portion, or a salt or solvate thereof.

2. The radiolabeled molecule according to claim 1, wherein the radionuclide is a positron-emitting radioisotope or a gamma-emitting isotope.

3. The radiolabeled molecule according to claim 1 or 2, wherein the radionuclide is selected from 11 C, 18 F, 44 Sc, 62 Cu, 64 Cu, 68 Ga, 86 Y, 89 Zr, 90 Nb, 99m Tc, 111 In, 124 I, 125 I, 131 I, 177 Lu and 213 Bi.

4. The radiolabeled molecule according to any one of claims 1 to 3, wherein the radionuclide is directly linked to an amino acid side chain of the epitope portion.

5. The radiolabeled molecule according to any one of claims 1 to 4, wherein the molecule comprises a radiolabeled moiety conjugated to the epitope portion.

6. The radiolabeled molecule according to claim 5, wherein the radiolabeled moiety comprises a covalently bound radionuclide.

7. The radiolabeled molecule according to claim 5, wherein the radiolabeled moiety comprises a chelator moiety capable of chelating a radionuclide, wherein the radionuclide is complexed with the chelator moiety.

8. The radiolabeled compound according to claim 7, wherein the chelator moiety is selected from TMT, DOTA, TCMC, DO3A, CB-DO2A, NOTA, NETA, diamsar, DTPA, CHX-A”-DTPA, TETA, HBED, 5HBED, HYBIC, DFO, DFOsq and HOPO.

9. A radiolabeled compound according to any one of claims 5 to 8, wherein the radiolabeled moiety is conjugated to another dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by the antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor comprises an antigen recognition domain for binding to the dysfunctional P2X7 receptor and a signal transduction domain.

10. A radiolabeled precursor molecule, the radiolabeled precursor molecule comprising: (i) A dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by the antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding to the dysfunctional P2X7 receptor and comprises a signal transduction domain; and (ii) A radionuclide precursor moiety selected from: (A) An atom or functional group capable of being converted into a radionuclide; (B) A reactive functional group capable of being conjugated to a radiolabeled cofactor; or (C) A chelator moiety capable of chelating a radionuclide, or a salt or solvate thereof.

11. A radiolabeled precursor molecule according to claim 10, wherein the radionuclide precursor moiety is conjugated to the epitope moiety.

12. A radiolabeled precursor molecule according to claim 11, wherein the radiolabeled precursor moiety comprises a chelator moiety selected from TMT, DOTA, TCMC, DO3A, CB-DO2A, NOTA, NETA, diamsar, DTPA, CHX-A”-DTPA, TETA, HBED, 5HBED, HYBIC, DFO, DFOsq, and HOPO.

13. A radiolabeled precursor molecule according to claim 11 or claim 12, wherein the radiolabeled precursor moiety is further conjugated to another dysfunctional P2X7 receptor epitope moiety that is recognized or capable of being bound by a receptor expressed on an immune cell, wherein the receptor comprises an antigen recognition domain for binding to the dysfunctional P2X7 receptor and a signal transduction domain.

14. A radiolabeled molecule according to any one of claims 1 to 9, or a radiolabeled precursor molecule according to any one of claims 10 to 13, wherein the epitope moiety comprises or consists of an amino acid sequence selected from any one of SEQ ID NO:2 to SEQ ID NO:69 or SEQ ID NO:

122.

15. A radiolabeled molecule according to any one of claims 1 to 9, or a radiolabeled precursor molecule according to any one of claims 10 to 13, wherein the epitope portion comprises at least the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:14 or consists thereof.

16. A radiolabeled molecule according to any one of claims 1 to 9, or a radiolabeled precursor molecule according to any one of claims 10 to 13, wherein the molecule is in the form of a fusion protein.

17. A fusion protein comprising a radiolabeled molecule according to any one of claims 1 to 9 or a radiolabeled precursor molecule according to any one of claims 10 to 13.

18. The fusion protein according to claim 17, wherein the fusion protein comprises a dysfunctional P2X7 receptor epitope portion and the Fc region of an antibody.

19. The fusion protein according to claim 18, wherein the Fc region of the antibody comprises at least the CH2 and CH3 domains of an immunoglobulin.

20. The fusion protein according to claim 18 or 19, wherein the Fc region comprises one or more amino acid substitutions for preventing homodimerization of the Fc region.

21. The fusion protein according to claim 20, wherein the amino acid substitutions include one or more substitutions of cysteine residues.

22. The fusion protein according to any one of claims 18 to 21, wherein the Fc region further comprises one or more amino acid substitutions for reducing binding to any one of FcγRI, FcγRII, and FcγRIII and / or one or more substitutions for reducing binding to FcRn.

23. The fusion protein according to any one of claims 18 to 22, wherein the protein comprises the amino acid sequence shown in any one of SEQ ID NO:145 to SEQ ID NO:158, SEQ ID NO:160, and SEQ ID NO:161 or consists thereof.

24. A method for preparing a radiolabeled molecule, the method comprising: - Provide a radiolabeled precursor molecule as defined in any one of claims 10 to 16 or a fusion protein as described in any one of claims 17 to 23; And - React the radiolabeled precursor molecule under conditions suitable for: Converting the atom or functional group of (A) into a radionuclide; Conjugating the reactive functional group of (B) with a radiolabeled cofactor; or Chelating the chelator moiety of (C) with a radionuclide.

25. A radiolabeled molecule, said radiolabeled molecule being obtained by the method according to claim 24.

26. A radiolabeled asymmetric heterodimeric molecule, said radiolabeled asymmetric heterodimeric molecule comprising a fusion protein according to any one of claims 18 to 23.

27. Use of a radiolabeled molecule according to any one of claims 1 to 9 or 25 or 26 for detecting immune cells or a population of immune cells expressing a receptor comprising an antigen recognition domain and a signal transduction domain for binding to a dysfunctional P2X7 receptor.

28. A method for detecting immune cells or a population of immune cells expressing a receptor comprising an antigen recognition domain and a signal transduction domain for binding to a dysfunctional P2X7 receptor in a subject, said method comprising: - Administer to a subject a radiolabeled molecule as described in any one of claims 1 to 9 or 25 or 26, wherein the subject has been administered immune cells expressing a receptor comprising an antigen recognition domain and a signal transduction domain for binding a dysfunctional P2X7 receptor; and - Detect the radiolabeled molecule in the subject, wherein the presence of the radiolabeled molecule indicates the presence of the immune cells.

29. The method according to claim 28, wherein the radiolabeled molecule is detected by performing a radionuclide scan.

30. A composition, said composition comprising a radiolabeled molecule or radiolabeled precursor molecule according to any one of claims 1 to 16, a radiolabeled molecule according to claim 25 or 26, or a fusion protein according to any one of claims 17 to 23, or a salt or solvate thereof.

31. A preparation, said preparation comprising a radiolabeled molecule or radiolabeled precursor molecule according to any one of claims 1 to 16, a radiolabeled molecule according to claim 25 or 26, or a fusion protein according to any one of claims 17 to 23, or a salt or solvate thereof.

32. A kit, said kit comprising one or more of the following: (i) A radiolabeled molecule or a salt or solvate thereof according to any one of claims 1 to 9 or 25 or 26; (ii) A radiolabeled precursor molecule or a salt or solvate thereof according to any one of claims 10 to 15; (iii) A fusion protein according to any one of claims 17 to 23; (iv) The composition according to claim 30; or (v) The preparation according to claim 31.

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