In vivo detection of immune cells

Abstract

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

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 This application claims priority to Australian Provisional Application No. 2022902654, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Cancer immunotherapy is a rapidly growing field, and the development of T cells expressing chimeric antigen receptors (CARs) has revolutionized adoptive cell therapy.

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

[0005] Several challenges remain before CAR T-cell therapy can be applied clinically. Given the challenges associated with CAR T-cell therapy, there is interest in developing approaches for imaging and tracking CAR T cells in vivo to gain further insight into their biological functions. These approaches may also be useful for monitoring and adjusting treatment involving CAR T-cell therapy.

[0006] Current methods for monitoring infused CAR T cells include serum profiling of cytokines related to T cell activation, direct enumeration of tumor-specific T cells in peripheral blood, and (repeated) tumor biopsies. However, these methods do not allow real-time or quantitative monitoring of infused CAR T cells in vivo. Labeled antibodies have also been developed to track T cells. However, a drawback associated with this method is that the antigens expressed on target cells are typically also expressed to some extent in other tissues, which may result in nonspecific background uptake of labeled antibodies.

[0007] Therefore, there is a need for alternative approaches to imaging and tracking immune cells, such as CAR T cells, in vivo.

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

[0009] The present invention provides (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by an antigen recognition domain of a receptor expressed on an immune cell, wherein the receptor is for binding the dysfunctional P2X7 receptor and comprises a signal transduction domain; (ii) a radionuclide linked directly or indirectly to an epitope moiety; or a salt or solvate thereof.

[0010] The present invention provides (i) a peptide; (ii) a radionuclide directly or indirectly linked to a peptide; or a salt or solvate thereof, Provided is a radiolabeled molecule, or a salt or solvate thereof, 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 amino acid sequence 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 of SEQ ID NOs: 2-69 and 122.

[0011] Preferably, the peptide is capable of being recognized by or bound by the antigen recognition domain of an exogenous cell surface receptor that includes an intracellular signaling domain (e.g., a chimeric antigen receptor that includes expression on T cells).

[0012] Preferably, the radionuclide may be a beta-emitting radioisotope (such as positron or beta-plus decay) or a gamma-emitting radioisotope.

[0013] In any embodiment, the radionuclide is 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, 125I, 131 I, 177 Lu, and 213 Bi.

[0014] The radionuclide may be directly linked to the epitope moiety, e.g., directly linked to an amino acid side chain of the epitope moiety. Alternatively, the radionuclide may be indirectly linked to the epitope moiety, e.g., the radionuclide may be included in a radiolabeling moiety that is conjugated to the epitope moiety. In some embodiments, the radiolabeling moiety comprises a covalently bound radionuclide. In other embodiments, the radiolabeling moiety comprises a chelator moiety capable of chelating the radionuclide, wherein the radionuclide is complexed with the chelator moiety. The chelator moiety may be selected from TMT, DOTA, TCMC, DO3A, CB-DO2A, NOTA, NETA, diamsar, DTPA, CHX-A"-DTPA, TETA, 11-tetraacetic acid, Te2A, HBED, 5HBED, HYBIC, DFO, DFOsq, and HOPO.

[0015] In any embodiment, the radiolabeled moiety is conjugated to a further peptide (e.g., a dysfunctional P2X7 receptor epitope moiety) that can be recognized or bound by the antigen recognition domain of a receptor expressed on an immune cell, preferably the receptor is for binding the dysfunctional P2X7 receptor and comprises a signal transduction domain.

[0016] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by an antigen recognition domain of a receptor expressed on an immune cell, wherein the antigen recognition domain is for binding the dysfunctional P2X7 receptor, and the receptor comprises a signal transduction domain; (ii) (A) an atom or functional group that can be converted into a radionuclide; (B) a reactive functional group capable of being conjugated to a radiolabeled prosthetic group, or (C) a chelator moiety capable of chelating a radionuclide; Alternatively, a salt or solvate thereof is provided.

[0017] In any embodiment, the atom or functional group can be any suitable atom or functional group that can be 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 that contains the radionuclide. In some embodiments, the atom or functional group is 18 It may be substituted with F. In some embodiments, the atom or functional group is 125 It can undergo isotopic exchange with I.

[0018] In any embodiment, the reactive functional group can be any suitable reactive functional group capable of conjugating a radiolabeled prosthetic group. It will be understood that a radiolabeled prosthetic group includes a radionuclide that can be covalently or non-covalently linked (e.g., by coordination) to the radiolabeled prosthetic group. In certain embodiments, the reactive functional group can react with the radiolabeled prosthetic group via click chemistry.

[0019] 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, 11-tetraacetic acid, Te2A, HBED, 5HBED, HYBIC, DFO, DFOsq, and HOPO.

[0020] In certain embodiments, the radiolabeled prosthetic group may be conjugated (or further conjugated) to a further dysfunctional P2X7 receptor epitope moiety that can be recognized or bound by the antigen recognition domain of a receptor expressed on an immune cell, the receptor being for binding the dysfunctional P2X7 receptor and comprising a signaling domain.

[0021] In a further aspect, the present invention provides a method for preparing a radiolabeled molecule, comprising the steps of: - providing a radiolabeled precursor molecule as defined herein; - a radiolabeled precursor molecule, (i) To convert the atom or functional group of (A) into a radionuclide, (ii) to conjugate the reactive functional group of (B) to a radiolabeled prosthetic group; or (ii) reacting under suitable conditions to chelate the chelator moiety of (C) to the radionuclide; Thereby, a method is provided for providing a radiolabeled molecule.

[0022] In a further aspect, the present invention provides the use of a radiolabeled molecule as described herein to detect immune cells expressing a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signal transduction domain.

[0023] In another aspect, the present invention provides a method for detecting immune cells in a subject that express a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signaling domain, the method comprising: - administering a radiolabeled molecule as described herein to a subject who has received immune cells expressing a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signal transduction domain; - detecting the radiolabeled molecule in the subject.

[0024] In any embodiment of this aspect, the radiolabeled molecule is detected by performing a radionuclide scan, which may be a positron emission tomography (PET) scan or a single photon emission computed tomography (SPECT) scan.

[0025] In some embodiments of this aspect, the method further comprises, prior to the step of detecting the radiolabeled molecule, concentrating the radiolabeled molecule at a site in the subject where the immune cells are found.

[0026] In some embodiments of this aspect, the method further comprises administering to the subject, before the step of administering the radiolabeled molecule, an immune cell expressing a receptor comprising an antigen recognition domain for binding the dysfunctional P2X7 receptor and the signal transduction domain.

[0027] In another aspect, the invention provides a composition comprising a radiolabeled molecule as described herein, or a salt or solvate thereof, or a radiolabeled precursor molecule as described herein, or a salt or solvate thereof.

[0028] The present invention provides (i) a peptide; (ii) (A) an atom or functional group that can be converted into a radionuclide; (B) a reactive functional group capable of conjugating to a radiolabeled prosthetic group, or (C) a chelator moiety capable of chelating a radionuclide; or a salt or solvate thereof, Provided is a radiolabeled precursor molecule, or a salt or solvate thereof, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 14 (preferably, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 7). Optionally, the peptide comprises or consists of the amino acid sequence 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 of SEQ ID NOs: 2-69 and 122.

[0029] Preferably, the peptide is capable of being recognized by or bound by the antigen recognition domain of an exogenous cell surface receptor that includes an intracellular signaling domain (e.g., a chimeric antigen receptor that includes expression on T cells).

[0030] The present invention provides a method for detecting immune cells expressing an exogenous cell surface receptor comprising an intracellular signaling domain (e.g., chimeric antigen receptor, including expression 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); administering a radiolabeled molecule as described herein to a subject receiving immune cells that express the receptor; - detecting the radiolabeled molecule in the subject.

[0031] Preferably, the radiolabeled molecule is detected by performing a radionuclide scan, which may be a positron emission tomography (PET) scan or a single photon emission computed tomography (SPECT) scan.

[0032] Optionally, the antigen recognition domain of the receptor is for binding to a peptide comprising or consisting of the amino acid sequence 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 of SEQ ID NOs: 2-69 and 122.

[0033] In any embodiment of the present invention, dysfunctional P2X7 receptor epitope part can be provided in the form of dysfunctional P2X7 receptor or a fragment of dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers, which cannot bind ATP.It is generally understood that such receptor cannot widen the opening of non-selective calcium channel to apoptotic pore.

[0034] In any embodiment, the dysfunctional P2X7 receptor epitope portion comprises or consists of a fragment of the dysfunctional P2X7 receptor. Exemplary fragments include peptides comprising the amino acid sequence 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 included in Table 1 herein, including any of SEQ ID NOs: 2-69 and 122. Preferably, the dysfunctional P2X7 receptor epitope portion comprises at least the sequence of SEQ ID NO: 7.

[0035] The dysfunctional P2X7 receptor epitope moiety may additionally comprise a spacer region linking the moiety to the radiolabel. Examples of such spacer sequences are further provided herein.

[0036] In a preferred embodiment of any aspect of the present invention, the architecture and spatial arrangement of the radiolabeled molecule of the present invention, or radiolabeled precursor molecule, is such that the minimal sequence of SEQ ID NO: 14, more preferably SEQ ID NO: 7, is available to bind to the antigen-binding domain of a receptor expressed on an immune cell (e.g., a chimeric antigen receptor for binding a dysfunctional P2X7 receptor), as described herein. In other words, the architecture is such that there is no steric hindrance that would prevent the antigen-binding domain from binding to the peptide.

[0037] In a preferred embodiment, the architecture of the radiolabeled molecule or radiolabeled precursor molecule of the present invention provides for a radionuclide that is directly or indirectly linked to the epitopic moiety via the C-terminal region of the epitopic moiety.

[0038] Alternatively, the architecture of the radiolabeled molecule or radiolabeled precursor molecule of the present invention provides for the radionuclide to be linked directly or indirectly to the epitope moiety via the N-terminal region of the epitope moiety.

[0039] In a preferred embodiment, the architecture of the radiolabeled precursor molecule of the present invention provides an atom or functional group that can be converted into a radionuclide that is directly or indirectly linked to the epitope moiety via the C-terminal region of the epitope moiety.

[0040] In a further embodiment, the architecture of the radiolabeled precursor molecules of the invention provides a reactive functional group that can be conjugated to a radiolabeled prosthetic group that is linked directly or indirectly to the epitope moiety via the C-terminal region of the epitope moiety.

[0041] In a further embodiment, the architecture of the radiolabeled precursor molecules of the present invention provides a chelator moiety capable of chelating a radionuclide that is linked directly or indirectly to the epitope moiety via the C-terminal region of the epitope moiety.

[0042] Alternatively, the architecture of the radiolabeled precursor molecules of the present invention provides (A) an atom or functional group that can be converted into a radionuclide, (B) a reactive functional group that can be conjugated to a radiolabeled prosthetic group, or (C) a chelator moiety that can chelate a radionuclide, linked directly or indirectly to the epitope moiety via the N-terminal region of the epitope moiety.

[0043] According to any aspect of the present invention, the radiolabeled molecule or radiolabeled precursor molecule may be in the form of a fusion protein. Optionally, the fusion protein comprises a peptide (e.g., a linear epitope of the P2X7 receptor, such as SEQ ID NO: 7 or 14, or as defined in any of SEQ ID NOs: 2-69 and 122) linked to the Fc region of an antibody, as further described herein. Thus, the present invention provides a fusion protein comprising a peptide derived from the P2X7 receptor (e.g., a dysfunctional P2X7 receptor epitope portion) and the Fc region of an antibody, and optionally, the fusion protein comprises a radiolabel or a moiety that can be radiolabeled, or the fusion protein comprises (A) an atom or functional group that can be converted into a radionuclide; (B) a reactive functional group capable of conjugating to a radiolabeled prosthetic group, or (C) a chelator moiety capable of chelating a radionuclide; or a salt or solvate thereof.

[0044] Examples of such fusion proteins are provided herein in SEQ ID NOs: 145-158, 160, and 161.

[0045] In a preferred embodiment, the fusion protein comprises a dysfunctional P2X7 receptor epitope portion and the Fc region of an antibody, and preferably comprises one or more modifications to the Fc region, for example, 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), to reduce serum half-life (by attenuating or reducing the ability to bind to FcRN receptors), and / or by reducing the tendency of the Fc region to aggregate and dimerize.Relevant amino acid substitutions for altering effector function, serum half-life, and aggregation are well known to those skilled in the art and are further described herein, as exemplified in Table 1.

[0046] The present invention also provides heterodimeric asymmetric molecules comprising a fusion protein described herein (e.g., comprising a peptide of SEQ ID NO: 7 or 14, or a variant thereof exemplified by any of SEQ ID NOs: 2-69) and an antibody Fc region, and further comprising an antibody Fc region that does not contain the peptide. Such asymmetric heterodimeric molecules can be obtained using knobs-in-hole technology, as further described herein, to promote dimerization of non-identical Fc regions.

[0047] Preferably, the fusion protein or heterodimeric asymmetric molecule consists of or essentially consists of the peptide and the Fc region of an antibody, such that the fusion protein or heterodimeric asymmetric molecule does not contain the antigen-binding domain of an antibody (i.e., the fusion protein does not contain a VH, VL, Fab, Fv, or scFv derived from an antibody).

[0048] It will be understood that the radiolabel, atom or functional group capable of being converted into a radionuclide, reactive functional group capable of being conjugated to a radiolabeled prosthetic group, or chelator moiety may be linked to or be part of the dysfunctional P2X7 receptor epitope portion of the fusion protein, or may be linked to a portion of the Fc region of the fusion protein.

[0049] In any aspect, the dysfunctional P2X7 receptor epitope portion is bound or capable of being bound to an antigen binding protein or antigen binding fragment thereof that binds to the dysfunctional P2X7 receptor, but is not bound or capable of being bound to an antigen binding protein or antigen binding fragment thereof that binds to the functional P2X7 receptor. Examples of suitable antigen binding proteins, or fragments thereof, are further described herein.

[0050] A radiolabeled molecule or a radiolabeled precursor molecule may comprise two or more peptides (e.g., two or more dysfunctional P2X7 receptor epitope portions) described herein. The two or more peptides may comprise or consist of the same sequence or different sequences. For example, in any embodiment, a radiolabeled molecule or a radiolabeled precursor molecule may comprise a peptide in the form of an E200 epitope and an additional peptide in the form of an E300 epitope. Alternatively, in any embodiment, a radiolabeled molecule may comprise a peptide in the form of an E200 epitope and an additional peptide in the form of a composite epitope. Still further, in any embodiment, a radiolabeled molecule or a radiolabeled precursor molecule may comprise a first peptide in the form of an E200 epitope and an additional peptide in the form of an E200 epitope.

[0051] In any aspect of the invention, the receptor comprising an antigen recognition domain and a signaling domain can be a chimeric antigen receptor (CAR), or a variant thereof, including a ligand-based CAR, or a modified T cell receptor (TCR), etc.

[0052] In any aspect of the invention, the immune cell can be a leukocyte, peripheral blood mononuclear cell (PBMC), lymphocyte, T cell, CD4+ T cell, CD8+ T cell, natural killer cell, natural killer T cell, or γδ T cell. In any embodiment, the cell can be a T cell, optionally the T cell does not express TcRαβ, PD1, CD3, or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level). In any embodiment, the cell can be an immune cell, optionally the cell does not express an accessory molecule that may be a checkpoint, exhaustion, or apoptosis-related signaling receptor, and a ligand such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L, and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0053] In a preferred embodiment of any aspect of the invention, the immune cell is a T cell or other effector cell expressing a CAR comprising an antigen binding domain for binding a dysfunctional P2X7 receptor.

[0054] In another aspect, the invention provides a formulation comprising a radiolabeled molecule as described herein, or a salt or solvate thereof, or a radiolabeled precursor molecule as described herein, or a salt or solvate thereof.

[0055] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a radiolabeled molecule described herein, or a salt or solvate thereof; (ii) a radiolabeled precursor molecule described herein, or a salt or solvate thereof; (iii) a composition described herein; or (iv) Kits are provided that include one or more of the formulations described herein.

[0056] Optionally, the kit includes instructions for use or one or more reagents for use with the radiolabeled molecule or precursor molecule.

[0057] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and referring to the accompanying drawings, in which: [Brief explanation of the drawings]

[0058] [Figure 1] 1 shows a schematic diagram of an exemplary use of a radiolabeled molecule of the present invention to detect immune cell enrichment at the site of a tumor expressing a dysfunctional P2X7 receptor. [Figure 2] a) Flow cytometry using an anti-His antibody to detect CAR-expressing immune cells bound by a monomeric E200-Fc fusion protein. X-axis: His. b) Flow cytometry using anti-biotin antibody staining from mouse blood and bone marrow via ex vivo incubation of biotinylated monomeric molecules identified CAR-expressing cell subsets detectable via anti-biotin antibody. X-axis: monomeric fusion protein (DetR1, SEQ ID NO: 158). [Figure 3] Percentage of CD25+ / CD69+ and PD-L1+ cells 72 hours after contact with monomeric or dimeric fusion proteins (having the amino acid sequences of SEQ ID NOs: 158 and 149, respectively).

[0059] Sequence information [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

[0060] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0061] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0062] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0063] All patents and publications referenced herein are incorporated by reference in their entirety.

[0064] The present invention provides radiolabeled molecules, compositions and kits comprising same that may be useful for detecting immune cells that express a receptor and a signaling domain, such as CAR T cells. Additionally, the present invention provides radiolabeled precursor molecules that can be used to generate the radiolabeled molecules.

[0065] The radiolabeled molecule comprises (i) a dysfunctional P2X7 receptor epitope moiety that can be recognized or bound by the 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 signal transduction domain, and (ii) a radionuclide 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 the radiolabeled molecule to be detected, thereby allowing the location and / or distribution of the immune cell expressing the receptor.

[0066] Thus, radiolabeled molecules may advantageously provide a new approach for real-time monitoring of the distribution and / or quantification of immune cells, such as CAR T cells, to bind dysfunctional P2X7 receptors in vivo.

[0067] In a particularly preferred embodiment of the present invention, a fusion protein or heterodimeric molecule is provided, comprising a linear epitope derived from P2X7 receptor (for example, as exemplified by any of SEQ ID NO: 14 or 7) and an Fc region of an antibody.The presence of an Fc region in a fusion protein provides certain advantages, including preventing the loss of reagent from circulation due to kidney filtration.Therefore, the molecule of the present invention preferably comprises an Fc region from an antibody, to aid the stability of the protein in the circulation of the subject for which the detection of CAR T cells is determined.

[0068] In a particularly preferred embodiment, the Fc fusion protein is designed to contain only a single copy of a linear epitope derived from the P2X7 receptor. This can be achieved by introducing amino acid substitutions into the Fc region to prevent homodimerization, as described further herein, or alternatively, by using well-known knob-into-hole technology to ensure the formation of an asymmetric heterodimeric molecule (e.g., comprising an E200 peptide-Fc fusion protein and an Fc region that does not contain the E200 peptide). Such monomeric or asymmetric heterodimeric molecules have the advantage of reducing activation of target immune cells and preventing unnecessary exhaustion of target immune cells (as described further herein in the Examples). Without wishing to be bound by theory, the inventors believe this is due to a reduced ability of the molecule to cross-link either two different CAR receptors on one cell or two different CAR receptors on two separate CAR-expressing cells.

[0069] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0070] For purposes of interpreting this specification, the following definitions shall generally apply and, whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0071] As used herein, the term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings, or for either meaning.

[0072] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "a dysfunctional P2X7 receptor epitope portion" means one dysfunctional P2X7 receptor epitope portion or two or more dysfunctional P2X7 receptor epitope portions.

[0073] As used herein, unless the context requires otherwise, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, ingredients, elements, or steps.

[0074] "Purinergic receptor" generally refers to a receptor that uses a purine (such as ATP) as a ligand.

[0075] "P2X7 receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, at least one of which has the amino acid sequence substantially as set forth in SEQ ID NO: 1 of Table 1 herein.

[0076] Insofar as P2X7 receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X7 receptor" encompasses naturally occurring variants of P2X7 receptor, for example, P2X7 monomers are isoforms (e.g., forms consisting of extracellular domain sequence or truncated forms thereof), naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants, including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of the monomers that form P2X7 receptor. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, P2X7 receptor may have a modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO: 1 may be substituted, deleted, or residues may be inserted.

[0077] "Functional P2X7 receptor" generally refers to the form of P2X7 receptor that has three intact binding sites or clefts for binding to ATP.When bound to ATP, functional receptor forms a non-selective sodium / calcium channel that converts into a pore-like structure that allows calcium ions and molecules up to 900 Da to enter the cell matrix, one of the consequences of which may be the induction of programmed cell death.In normal homeostasis, the expression of functional P2X7 receptor is generally limited to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes.In addition, there may be some expression of functional P2X7 receptor on erythrocytes and other cell types.

[0078] "Dysfunctional P2X7 receptor" (also called "non-functional" or (nf)P2X7) is a P2X7 receptor that has a defective response to ATP, such that it cannot form an apoptotic pore under physiological conditions. Dysfunctional P2X7 receptor or (nfP2X7 receptor) generally refers to a form of P2X7 receptor that has a different conformation from functional P2X7, whereby the receptor cannot form an apoptotic pore, but can still act as a non-selective channel by maintaining a single functional ATP-binding site located between adjacent monomers. One example occurs when one or more monomers have cis-isomerization at Pro210 (according to SEQ ID NO: 1). Isomerization can result from any molecular event that leads to misfolding of the monomer, including, for example, mutation of the monomer primary sequence or abnormal post-translational processing. One consequence of this isomerization is that the receptor cannot bind ATP at one, or more specifically, two, of the ATP-binding sites on the trimer, and as a result, cannot extend the opening of the channel. In such a situation, receptor cannot form pore, which limits the degree that calcium ions can enter into the cytosol.Dysfunctional P2X7 receptor is expressed on a wide range of epithelial cancers and hematopoietic cancers.As used herein, term " dysfunctional P2X7 receptor " can be used interchangeably with term " non-functional P2X7 receptor " or " nfP2X7 " receptor.

[0079] A "cancer-associated P2X7 receptor" is a P2X7 receptor that is generally found on cancer cells (including pre-neoplastic cells, neoplastic cells, malignant cells, benign cells, or metastatic cells) but not on non-cancerous or normal cells.

[0080] "E200 epitope" generally refers to the epitope having the sequence GHNYTTRNILPGLNITC (SEQ ID NO: 2), variants of which are exemplified in Table 1, including any of SEQ ID NOs: 3, or 7-69, or 155.

[0081] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO: 4) or a variant thereof, as defined in SEQ ID NO: 5.

[0082] "Composite epitope" generally refers to an epitope formed from the juxtaposition of the E200 epitope and the E300 epitope, or portions of these epitopes. An example of a composite epitope comprising the E200 epitope and the E300 epitope is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 6).

[0083] As used herein, the term "antigen" is intended to include a substance that binds to or induces the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and recognized by the adaptive immune system, including, but not limited to, antibodies or TCRs, or engineered molecules, including, but not limited to, transgenic TCRs, CARs, scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single-chain antibodies or multimers thereof, or any other molecule that can achieve high affinity binding to a structure.

[0084] "Epitope" generally refers to the portion of an antigen bound by the antigen-binding site of an antibody. An epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to a sequence of amino acids as in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope," i.e., an epitope in which the hypervariable loops of the CDRs bind to residues as they appear in the tertiary or quaternary protein structure.

[0085] With respect to a receptor that refers to an antigen-binding domain that recognizes and binds to a dysfunctional P2X7 receptor, the terms "binds to," "specifically binds to," or "specific for" are intended to mean that the receptor does not substantially recognize or bind to other antigens in a sample.

[0086] The term "immune cell" or "immune effector cell" refers to a cell that can be part of the immune system and performs a specific effector function, e.g., α-β T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γ-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic progenitor cell, such as an early progenitor cell subset that can mature or differentiate into pluripotent stem cells and somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (iPSCs and other artificial cell types). Preferred immune cells are cells with cytotoxic effector function, e.g., α-β T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or γ-delta T cells. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0087] As used herein, the term "amino acid" refers to a compound having an amino group and a carboxylic acid group. Amino acids can be L- or D-isomers, or mixtures thereof. Amino acids can have naturally occurring side chains (see Table 1) or non-proteinaceous side chains. Amino acids can also have, at the α-position, -C 1-6 Alkyl, -(CH2)nCOR a , -(CH2) n Rb and -PO3H, n is an integer selected from 1 to 8, and R a -OH, -NH2, -NHC 1-3 Alkyl, -OC 1-3 Alkyl or -C 1-3 alkyl, and R b -OH, -SH, -SC 1-3 Alkyl, -OC 1-3 Alkyl, -NH2, -NHC 1-3 alkyl, or -NHC(C=NH)NH2, where each alkyl group is -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 It may be substituted with one or more groups selected from alkyl.

[0088] The amino acid structures and single and three letter abbreviations used throughout this specification are defined in Table 2, which lists the 20 naturally occurring proteinogenic amino acids that occur in proteins as the L-isomer. Table 2 [ka] [Table 2]

[0089] As used herein, the term "non-proteinogenic amino acid" refers to an amino acid having a side chain that does not occur in the naturally occurring L-α-amino acids listed in Table 2. Examples of non-proteinogenic amino acids and derivatives include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, citrulline, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of the natural amino acids.

[0090] As used herein, the term "α-amino acid" refers to an amino acid having a single carbon atom (α-carbon atom) separating its carboxyl terminus (C-terminus) and amino terminus (N-terminus). α-Amino acids include naturally occurring and non-naturally occurring L-amino acids, including their D-isomers and derivatives thereof, such as salts or derivatives in which functional groups are protected by suitable protecting groups. Unless otherwise specified, the term "amino acid" as used herein refers to an α-amino acid.

[0091] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 6 carbon atoms. Where appropriate, alkyl groups include alkyl groups having the specified number of carbon atoms, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. 1-6 Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 5-methylpentyl.

[0092] 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.

[0093] Basic salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, and alkylammonium; alkoxyammonium salts such as those formed with triethylamine, ethanolamine, and salts formed from ethylenediamine, choline, or amino acids such as arginine, lysine, or histidine.

[0094] Basic nitrogen-containing groups may be quaternized with lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate and diethyl sulfate; and the like.

[0095] Salts or other derivatives of the compounds of the present invention may be provided in the form of solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent, and may be formed during the crystallization process with pharmaceutically acceptable solvents such as water, alcohols such as methanol, ethanol, or isopropyl alcohol, DMSO, acetonitrile, dimethylformamide (DMF), etc., with the solvate forming part of the crystalline lattice either by non-covalent bonding or by occupying holes in the crystalline lattice. Hydrates are formed when the solvent is water, and alcoholates are formed when the solvent is alcohol. Solvates of the compounds of the present invention can be conveniently prepared or formed during the processes described herein. In general, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0096] 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, although the subject can also be a healthy subject.

[0097] radiolabeled molecules It will be understood that the radiolabeled molecule of the present invention may be in any form, provided that it comprises (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by the antigen binding domain of a receptor for binding a dysfunctional P2X7 receptor expressed on an immune cell, the receptor comprising a signal transduction domain, and (ii) a radionuclide linked directly or indirectly to the epitope portion.

[0098] Typically, the dysfunctional P2X7 receptor epitope portion is in the form of a peptide.Dysfunctional P2X7 receptor epitope portions are further described herein.

[0099] It will be appreciated that the presence of a radionuclide can affect the local charge fields of nearby atoms, and therefore it may be preferable to attach the radionuclide at a location within the radiolabeled molecule such that it does not affect binding and recognition of the epitope moiety by the antigen-binding domain of the receptor.

[0100] Therefore, in some embodiments, epitope moiety comprises one or more spacers between dysfunctional P2X7 receptor epitope moiety and radionuclide.Spacer is the amino acid sequence in N-terminal or C-terminal of epitope moiety, which may exist, to the recognition sequence of epitope moiety.When radionuclide is directly or indirectly linked to the spacer of epitope moiety, spacer may sufficiently separate radionuclide from the recognition sequence so as not to affect the binding of epitope moiety by antigen binding domain of immune cell receptor.

[0101] 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 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer amino acids, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, or 4 or fewer amino acids. Minimum and maximum amounts can be combined to form ranges, provided that the range is 1 to 20 amino acids, e.g., 3 to 15 amino acids, or 5 to 11 amino acids, or 2 to 6 amino acids.

[0102] In certain examples, the spacer comprises amino acid residues derived from the dysfunctional P2X7 receptor sequence, whether located at the N-terminal or C-terminal to the epitope sequence.For example, in some embodiments, the spacer is selected from the amino acid sequence C-terminal to the core E200 epitope sequence of the receptor, such as the sequence TFHKT (SEQ ID NO: 138) and exemplified by the peptide defined in SEQ ID NO: 9.Alternatively or additionally, the spacer may comprise the amino acid sequence N-terminal to the core E200 epitope sequence of the receptor, such as the amino acid sequence DFP (SEQ ID NO: 139) and exemplified by the peptide of SEQ ID NO: 140.

[0103] It should be understood that in a further embodiment, the radiolabeled molecule or the molecule to be radiolabeled according to the present invention may be in the form of a fusion protein, and the fusion protein comprises a first amino acid sequence comprising an epitope of a dysfunctional P2X7 receptor and a second amino acid sequence. In certain embodiments, the second amino acid sequence may comprise the "spacer" sequence outlined above. Alternatively, the second amino acid sequence may comprise a protein amino acid sequence to increase the solubility or stability of the molecule. Thus, the second amino acid sequence may comprise an amino acid sequence derived from immunoglobulins, or other proteins such as serum albumin, transferrin, the carboxy-terminal peptide of chorionic gonadotropin (CG) beta chain, imprecise repeat peptide sequences, polypeptide sequences composed of proline-alanine-serine polymers, elastin-like peptide (ELP) repeat sequences, homopolymers of glycine residues, or gelatin-like proteins.

[0104] In certain embodiments, the second amino acid sequence may comprise an amino acid sequence from an immunoglobulin, such as an Fc region (e.g., comprising a CH2 and / or CH3 region) or a variant thereof. Thus, the radiolabeled molecule or molecule to be radiolabeled may be an Fc fusion protein consisting of an amino acid sequence of an epitope of a dysfunctional P2X7 receptor linked to the Fc region of an antibody.

[0105] In any embodiment, the amino acid sequence of the epitope of dysfunctional P2X7 receptor can be fused to the N-terminal region of the Fc region of antibody or its variant via its C-terminal region.In any embodiment, the amino acid sequence of the epitope of dysfunctional P2X7 receptor can be fused to the C-terminal region of the Fc region of antibody or its variant via its N-terminal region.

[0106] Preferably, the Fc region of the fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chains.

[0107] The Fc region may contain one or more amino acid sequence modifications compared to the naturally occurring Fc sequence.The Fc region may contain one or more amino acid substitutions, such as substitution of one or more cysteine ​​residues, to prevent dimerization of the molecule into the same molecule.It will be understood that any amino acid substitution that prevents dimerization of the Fc region may be used.Therefore, in vivo, the Fc fusion protein described herein may be a monomeric protein.

[0108] Thus, the Fc region of the fusion protein contains one or more amino acid substitutions, relative to a naturally occurring Fc sequence, that prevent or reduce the ability of the Fc region to homodimerize. Preferably, the amino acid substitutions include one or more substitutions of cysteine ​​residues to prevent disulfide bond formation between Fc molecules. The cysteine ​​residues in the Fc region may optionally be substituted with any other amino acid residue, including glycine, serine, alanine, lysine, and glutamic acid, preferably glycine or serine.

[0109] The cysteine ​​residues for substitution are preferably one or more of the cysteine ​​residues located in the region of the Fc region corresponding to the hinge region of an immunoglobulin. An example of an IgG1 hinge region and examples of variations thereof, including cysteine-to-serine substitutions, are provided in Table 3 herein. The hinge region of an immunoglobulin (e.g., of an IgG1) contains three cysteine ​​residues, numbered C220, C226, and C229 (according to EU numbering). Thus, in any embodiment, at least one, at least two, or all three of the cysteine ​​residues in an immunoglobulin hinge region are substituted. Preferably, at least two or all three of the cysteine ​​residues are substituted. More preferably, all cysteine ​​residues in the Fc region, such as the hinge region, are substituted. In a particularly preferred embodiment, at least one of C226 and C229 is substituted, and preferably both C226 and C229 are substituted.

[0110] Therefore, in a preferred embodiment, the fusion protein comprises a hinge region for linking the dysfunctional P2X7 receptor epitope portion and an Fc region of an antibody, wherein the hinge region comprises an amino acid sequence corresponding to any of the sequences set forth in SEQ ID NOs: 76 to 113, or 136 to 137, or 141, or 142.

[0111] In further embodiments, the fusion protein region may comprise an Fc region corresponding to the Fc "hole" or "knob" for use in "knob-in-hole" heterodimers. The use of such Fc sequences is known in the art and provides asymmetric heterodimeric molecules that bind to an additional Fc region that does not contain the epitope moiety, including fusion proteins having a single copy of the epitope moiety and an Fc region described herein.

[0112] Those skilled in the art will be familiar with techniques and Fc sequences that allow for the formation of so-called monomeric fusion proteins, including, but not limited to, the use of the "knob-into-hole" IgG1 format (Ridgway et al., (1996), Protein Eng, 9:617-621). Such an approach in the context of the present invention allows for the expression and purification of heterodimeric fusion proteins with only one copy of a peptide epitope (e.g., an epitope portion derived from the E200 epitope described herein) per molecule. Examples of "knob-into-hole" Fc pairings are provided herein in SEQ ID NOS: 157 and 159 (knob and hole, respectively), 158 and 159, respectively, 160 and 162 (hole and knob, respectively), and 161 and 162, respectively. Thus, in any embodiment, the present invention provides a fusion protein comprising the amino acid sequence of any of SEQ ID NOs: 2-69 and 122 linked to an Fc region defined in SEQ ID NO: 160 or 162, wherein the fusion protein can form a heterodimer with an Fc region that does not include an E200 peptide portion.

[0113] Therefore, the fusion proteins of the present invention are preferably capable of forming heterodimeric molecules containing a single E200-containing amino acid sequence (in other words, the Fc portion of the fusion protein can heterodimerize with the Fc region of an antibody that does not contain an E200 peptide fused thereto).

[0114] In further embodiments, the Fc region may comprise one or more substitutions to eliminate or reduce effector function, such as reducing FcR-mediated binding and activation, as further described below.

[0115] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region is the C-terminal region of an antibody. H 2 domain and C HIn the context of the present invention, the Fc region comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and by the C H The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called delta, epsilon, gamma, and mu, respectively, and are held together by hydrophobic interactions of the three domains.

[0116] In some embodiments, the fusion protein does not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that an antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

[0117] Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be measured in vivo as described, e.g., in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Also, a C1q binding assay can be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006), WO2013 / 120929A1).

[0118] Fc regions with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutation, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). For example, an antibody variant can include an Fc region with one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, the substitutions are L234A and L235A (LALA) (see, e.g., WO2012 / 130831). Additionally, changes can be made in the Fc region that result in altered (i.e., reduced) C1q binding and / or complement dependent cytotoxicity (CDC), as described, for example, in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000) (e.g., G236R).

[0119] Further examples of modified Fc regions include those containing "LALALS" (amino acid substitutions L234A / L235A / M428L / N434S, as described in Zalevsky et al., (2010) Nat. Biotechnol. 28:157-159), and LALAPG (amino acid substitutions L234A / L235A / P329G, as described in Gunn et al., (2021, Immunity 54:815)).

[0120] In some embodiments, the Fc region of the Fc fusion proteins of the invention may contain at least the "LALA" mutation (L234A and L235A) to reduce binding to FcR. The fusion protein may additionally or alternatively contain the mutation G346R to inhibit recruitment of complement C1q.

[0121] Other Fc modifications for use in the present invention include variants that reduce or eliminate binding to FcγRs and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Such variants are also referred to herein as "knockout variants" or "KO variants." Variants that reduce binding to FcγRs and complement are useful for reducing undesired interactions mediated by the Fc region. Preferred knockout variants are those referred to as "Fc Variants with Optimized

[0042] Preferred modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, numbering according to the EU index. Preferred modifications include 234G, 235E, 235G, 236R, 237K, 238E, 238F, 238G, 238H ... Examples of variants include, but are not limited to, 267R, 269R, 325L, and 328R, where numbering is according to the EU index. A preferred variant includes 236R / 328R. The variants may be used in the context of any IgG isotype or IgG isotype Fc region, including, but not limited to, human IgG1, IgG2, IgG3, and / or IgG4. Preferred IgG Fc regions for reducing FcγR and complement binding and reducing Fc-mediated effector functions are IgG2 and IgG4 Fc regions. Hybrid isotypes, such as the hybrid IgG1 / IgG2 isotype described in U.S. Patent Application No. 11 / 256,060, may be useful.Other modifications to reduce FcγR and complement interactions include, but are not limited to, substitutions 297A, 297D, 234A, 235A, 237A, 318A, 228P, 236E, ΔG236, 265G, 268Q, 297Q, 309L, 330S, 331S, 327Q, 220S, 226S, 229S, 238S, 233P, 234A, and 234V, as well as removal of glycosylation at position 297 by mutation or enzymatic means, or by production in an organism such as a bacterium that does not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691, which is incorporated by reference in its entirety.

[0122] In some embodiments, the Fc region comprises mutations to the complement (C1q) and / or Fc gamma receptor (FcγR) binding sites, and in some embodiments, such mutations can render the fusion protein incapable of antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC).

[0123] The Fc region used in the context of the present invention preferably does not induce cytotoxicity, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0124] In some embodiments, the Fc region may contain one or more substitutions to reduce affinity for FcRn, thereby decreasing the serum or circulating half-life of the fusion protein. Substitutions to reduce affinity for FcRn are known in the art and are described, for example, in Ward et al., (2015), Mol. Immunol., 67:131-141, and Grevys et al., (2015), 194:5497-5508. Exemplary substitutions include substitutions at one or more of Ile253, His310, and His435, such as I253A, H310A, and H435A.

[0125] The term "Fc region" also includes native-sequence Fc regions and variant Fc regions. The Fc region may include the carboxyl terminus of the heavy chain. Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain the full-length heavy chain or a truncated variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to EU numbering, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the Fc region of an antibody may be contemplated. Amino acid sequence variants of the Fc region of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues in the amino acid sequence of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., inducing or supporting an anti-inflammatory response.

[0126] The Fc region of an antibody can be the Fc region of any of the antibody classes, such as IgA, IgD, IgE, IgG, and IgM. The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, when used in the context of the present invention, an antibody can be the Fc region of an IgG. For example, the Fc region of an antibody can be the Fc region of an IgG1, IgG2, IgG2b, IgG3, or IgG4. In some embodiments, the fusion protein of the present invention comprises the Fc region of an IgG. In the context of the present invention, the Fc region of an antibody is the Fc region of an IgG, preferably an IgG1.

[0127] The dysfunctional P2X7 receptor epitope 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 as exemplified in Table 1 or Table 3. Alternatively, the linker sequence can be any amino acid-based linker sequence commonly used in the art.

[0128] The linker is usually a peptide having a length of up to 20 amino acids, but may be up to 50 amino acids in length. The term "linked to" or "fused to" refers to a covalent bond, such as a peptide bond, formed between two moieties. Thus, in the context of the present invention, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the fusion protein provided herein may comprise a linker between the epitope of a dysfunctional P2X7 receptor and the Fc region of an antibody, for example, between the N-terminus of the Fc region and the C-terminus of the dysfunctional P2X7 receptor epitope. As another example, the fusion protein provided herein may comprise a linker between the epitope of a dysfunctional P2X7 receptor and the Fc region of an antibody, for example, between the C-terminus of the Fc region and the N-terminus of the dysfunctional P2X7 receptor epitope moiety. In particular, dysfunctional P2X7 receptor epitope portion can be fused to the N-terminus of Fc region via a C-terminal linker.This linker has the advantage that different polypeptides of fusion protein can be independently folded and behave as expected.Therefore, in the context of the present invention, dysfunctional P2X7 receptor epitope portion and antibody Fc region can be comprised in a single chain multifunctional polypeptide.

[0129] In some embodiments, the fusion protein of the present invention comprises a peptide linker. In some embodiments, the peptide linker connects the dysfunctional P2X7 receptor epitope portion to the Fc region of the antibody. In some embodiments, the peptide linker can comprise the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Ser (GGGGS). In some embodiments, the peptide linker can comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length) or more. The linker can be of different lengths, i.e., a (GS)n series of repeated glycine and serine residues (GS), where n is any number from 1 to 15 or more. For example, the linker can be (GS)3 (i.e., GSGSGS) or longer, or (GS)11 or longer. It will be understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or longer. Fusion proteins with linkers of such lengths are included within the scope of the present invention. Preferably, n is 3 or less (i.e., when n is equal to 3, the linker is GSGSGS).

[0130] In further embodiments, the linker may include the inclusion of amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linker region may also include the sequence GSGK.

[0131] The peptide linker may consist of a repeating series of Thr-Pro (TP) with one or more additional amino acids N- and C-terminal to the repeat sequence. For example, the linker may comprise or consist of the sequence GTPTPTPTPTGEF (also known as a TP5 linker). In further embodiments, the linker may be a short and / or alpha-helical rigid linker (e.g., A(EAAAK)3A, PAPAP, or a dipeptide such as LE or CC).

[0132] In a further embodiment, instead of or in addition to the above-mentioned glycine-serine-based linker region, the fusion protein may comprise a dysfunctional P2X7 receptor epitope moiety linked to the Fc region of an antibody via a hinge region. The link between the dysfunctional P2X7 receptor epitope moiety and the Fc region may comprise a combination of a hinge region and a linker region.

[0133] Examples of suitable hinge regions include those derived from immunoglobulins. The hinge region may be derived from IgG1, IgG2, IgG3, or IgG4 and may contain one or more amino acid substitutions (e.g., to prevent or reduce the possibility of disulfide bridge formation). Alternative hinge sequences may be derived from alternative immunoglobulin domains: CD8A, CD8B, CD4, or CD28, TRAC, TRBC, TRGC, TRDC.

[0134] Table 3 below provides non-limiting examples of suitable hinge regions for use in linking dysfunctional P2X7 receptor epitope moieties and Fc regions in the molecules of the invention.

[0135] It will be understood that the dysfunctional P2X7 receptor epitope portion can be linked to the Fc region by more than one linker and / or more than one hinge region. For example, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion directly conjugated (N-terminus to C-terminus) to the Fc region. Alternatively, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, and then an Fc region. Still further, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, and then an Fc region. In yet another embodiment, the fusion protein can comprise a dysfunctional P2X7 receptor epitope portion, followed by a linker region, then a hinge region, another linker region, and then an Fc region. Of course, one skilled in the art will understand that alternative configurations are possible (i.e., the dysfunctional P2X7 receptor epitope portion is linked to the C-terminus of the Fc region via one or more linkers and / or hinge regions). [Table 3-1] [Table 3-2]

[0136] In certain embodiments, the dysfunctional P2X7 receptor epitope portion is fused directly to the Fc region of the antibody, such that there is no linker between the two regions of the fusion protein.

[0137] The radionuclide may be any radionuclide suitable for use in nuclear medicine, such as nuclear medicine tomography imaging.The radionuclide may, for example, allow the radiolabeled compound of the present invention to be detected by radionuclide scanning.In some embodiments, the radionuclide is a positron-emitting radioisotope, which can be detected by positron emission tomography (PET).In some embodiments, the radionuclide is a gamma-emitting isotope, which can be detected by single-photon emission computed tomography (SPECT).

[0138] The radionuclide may be linked to the radiolabeled compound of the invention by a covalent or non-covalent bond (e.g., coordination). In some embodiments, the radionuclide is carbon-11 ( 11 C), fluorine-18( 18 F), scandium-44( 44 Sc), copper-62, -64 and -67 ( 62 Cu, 64 Cu, 67 Cu), gallium-67 and -68 ( 67 Ga, 68 Ga), yttrium-86 and -90 ( 86 Y, 90 Y), Zirconium-89( 89 Zr), Niobium-90( 90 Nb), technetium-94 and -99 ( 94m Tc, 99m Tc), Indium-111( 111 In), iodine-123, -124, -125 and -131 ( 123 I,124 I, 125 I, 131 I), lutetium-177( 177 Lu), and bismuth-123( 213 In some embodiments, the radionuclide is selected from radioactive isotopes of C, F, Sc, Cu, Ga, Y, Zr, Nb, Tc, In, I, Lu, and Bi.

[0139] The radionuclide may be linked directly to the epitope moiety, for example, to an amino acid side chain. If the epitope moiety includes a spacer, the radionuclide may be linked directly to an amino acid side chain of the spacer, which may space the radionuclide sufficiently from the recognition sequence so as not to affect binding of the epitope moiety. In embodiments in which the radionuclide is linked directly to the epitope moiety, the radionuclide may be 11 C. 18 F, and 99m An example of an amino acid directly linked to a radionuclide is fluorine-18 labeled tyrosine ( 18 F-Tyr) and technetium-99 labeled histidine ( 99m Tc-His).

[0140] Alternatively, the radionuclide may be indirectly linked to the epitopic moiety, e.g., the radionuclide may be contained in a radiolabeled moiety that is conjugated to the epitopic moiety. In some embodiments, the radiolabeled moiety is conjugated to an amino acid side chain of the epitopic moiety. In some embodiments, the radiolabeled moiety is conjugated to the N-terminus or C-terminus of the epitopic moiety.

[0141] The radionuclide may be linked to the radiolabeled moiety by a covalent bond. Thus, in some embodiments, the radiolabeled moiety comprises a covalently bound radionuclide. In these embodiments, the radionuclide is 11 C. 18 F, 123 I, 124 I, 125 I, and 131 It may be selected from I.

[0142] Alternatively, the radionuclide may be linked to the radiolabeling moiety by a non-covalent bond (e.g., coordination). Thus, in some embodiments, the radiolabeling moiety comprises a chelator moiety capable of chelating the radionuclide, and the radionuclide is complexed with the chelator moiety. In these embodiments, the radionuclide is 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.

[0143] The chelator moiety can be any suitable chelator capable of chelating a radionuclide. In some embodiments, the chelator moiety is TMT (6,6"-bis[N,N",N'"-tetra(carboxymethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine), DOTA (1,4,7,10-tetraazacyclododecane-N-N',N"(N'"-tetraacetic acid, also known as tetraxetane), TCMC (tetra primary amine of DOTA), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-tris(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]triazonan-1-yl}), Diamsal (3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), DTPA (pentaerythritol), tetraacetic acid or diethylenetriaminepentaacetic acid), CHX-A'''-DTPA ([(R)-2-amino-3-(4-isocyanatophenyl)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)-2-hydroxybenzyl)-2-hydroxybenzyl benzoate), Benzyl alcohol (Benzyl alcohol, ... ) ethylenediamine-N,N'-diacetic acid), 5HBED (3,3'-((ethane-1,2-diylbis((carboxymethyl)azanediyl))bis(methylene))bis(4-hydroxybenzenesulfinate)), HYBIC (6-hydrazinonicotinic acid), DFO (desferoxamine), DFOsq (DFO-squaramide), and HOPO (3,4,3-(LI-1,2-HOPO), or other chelating agents described herein.

[0144] The chelator moiety may be conjugated directly to the epitope moiety or indirectly to the epitope moiety via a linker, which may include a peptide or a chemical group. The linker may 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 may be conjugated to the epitope moiety or linker (if present) by any suitable means. As a non-limiting example, if the chelator moiety is DOTA, DOTA may be conjugated to the epitope moiety or linker through at least one of the carboxylic acid groups of DOTA, for example, by forming an amide or ester bond with a suitable functional group (e.g., an amine or hydroxyl group) on the epitope moiety or linker. DOTA may alternatively be conjugated to an epitope moiety or linker via at least one of the carbon atoms in the tetraazacyclododecane ring and / or via a methylene group in at least one of the four carboxylic acid groups of DOTA.

[0145] In any embodiment, the radiolabeled moiety can be further conjugated with another dysfunctional P2X7 receptor epitope moiety, which can be recognized or bound by the receptor expressed on immune cells, and the receptor comprises an antigen recognition domain for binding the dysfunctional P2X7 receptor and the signal transduction domain.That is, the radiolabeled compound of the present invention can comprise one or more dysfunctional P2X7 receptor epitope moieties, as described elsewhere herein.When the radiolabeled compound of the present invention comprises two or more dysfunctional P2X7 receptor epitope moieties, the epitope moieties can comprise or consist of the same sequence or different sequences.

[0146] Radiolabeled precursor molecules In molecules labeled with radionuclides, the radionuclides may be prone to decay and have a relatively short half-life. Therefore, it may be necessary to prepare the radiolabeled molecule immediately before its intended use (for example, before administration to a subject and in vivo detection via radionuclide scanning) so that the radiolabeled molecule can be used within the expected lifespan of the radionuclide. Ideally, the radiolabeled molecule is prepared from a precursor by a minimum number of reaction steps that can enable efficient preparation of the radiolabeled molecule. As a non-limiting example, the radiolabeled molecule can be prepared from a radiolabeled precursor compound within about 30 minutes.

[0147] Thus, the present invention also provides a radiolabeled precursor molecule comprising: (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by an antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding the dysfunctional P2X7 receptor and comprises a signal transduction domain; and (ii) a radionuclide precursor moiety, (A) an atom or functional group that can be converted into a radionuclide; (B) a reactive functional group capable of conjugating to a radiolabeled prosthetic group, or (C) a radionuclide precursor moiety selected from the group consisting of a chelator moiety capable of chelating a radionuclide; Alternatively, a salt or solvate thereof is provided.

[0148] The radiolabeled precursor molecules of the invention can be used to prepare the radiolabeled molecules described herein. Accordingly, the present invention also provides the use of radiolabeled precursor molecules to prepare the radiolabeled molecules described herein.

[0149] As will be apparent to those skilled in the art, the epitope moiety will typically be any suitable epitope moiety as defined herein for a radiolabeled molecule, however, rather than being linked directly or indirectly to a radionuclide, the epitope moiety will instead be linked directly or indirectly to a moiety (such as any of (A), (B), or (C) above) that can be converted into a radionuclide or conjugated or chelated to a radionuclide.

[0150] The radiolabeled precursor molecule may comprise a radionuclide precursor moiety conjugated to an epitope moiety, the radiolabeled precursor moiety comprising one of (A), (B), or (C) above. Thus, any of (A), (B), and (C) may be present in the epitope moiety or in the radiolabeled precursor moiety conjugated to the epitope moiety.

[0151] Any suitable atom or functional group that can be converted into a radionuclide known in the art 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 converted into a radionuclide (e.g., 18 F) can be any suitable leaving group that can be displaced by F). When an atom or functional group is present in the epitope moiety, the atom or functional group can be, for example, 18 Replaced by F 18 It may be a hydroxyl group of the tyrosine side chain, which may provide F-Tyr. When the atom or functional group is present in the radiolabel precursor moiety, the atom or functional group may be selected from the group consisting of, for example, the hydroxyl group of the tyrosine side chain, which may provide F-Tyr. 18 The leaving group may be any suitable leaving group, as described in F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry. 2017 Nov 7;23(62):15553-15577. As a further non-limiting example, the atom or functional group may be: 125 The iodine atom may be exchangeable with I or other iodine isotopes.

[0152] Any suitable reactive functional group capable of conjugating a radiolabeled prosthetic group known in the art can be used. It should be understood that the reactive functional group can be capable of forming a covalent bond with a complementary reactive functional group present on the radiolabeled prosthetic group. When 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). When the reactive group is present in the radiolabeled precursor moiety, the reactive group can be conjugated to the epitope moiety by any suitable linker, which can include a peptide or a chemical group.

[0153] In some embodiments, the reactive functional group is an amino group capable of forming an amide bond to a radiolabel prosthetic group comprising a carboxylic acid group. In some embodiments, the reactive functional group is a carboxylic acid group capable of forming an amide bond to a radiolabel comprising an amine group. In some embodiments, the reactive functional group is a hydroxyl group capable of forming an ester bond to a radiolabel prosthetic group comprising a carboxylic acid group. In some embodiments, the reactive functional group is a carboxylic acid group capable of forming an ester bond to a radiolabel prosthetic group comprising a hydroxyl group. In some embodiments, the reactive functional group comprises a leaving group (such as, but not limited to, a halogen, tosylate, mesylate, triflate, etc.) that can be coupled via nucleophilic substitution to a radiolabel prosthetic group comprising a nucleophilic group (such as, but not limited to, a thiol, hydroxyl, amine, or carboxylic acid). In some embodiments, the reactive functional group comprises a nucleophilic group (such as, but not limited to, a thiol, hydroxyl, amine, or carboxylic acid) that can be coupled via nucleophilic substitution to a radiolabel prosthetic group comprising a leaving group (such as, but not limited to, a halogen, tosylate, mesylate, triflate, etc.). In some embodiments, the reactive functional group comprises a group represented by an open valence (such as the generic alkyl group R-CH2-), which can be linked via a single covalent bond to a radiolabeled prosthetic group. This list is not intended to be exhaustive, but merely illustrative. Any other reactions and reagents known to promote intermolecular bonding are incorporated herein. Any of these compounds and corresponding conjugates are contemplated within the present invention.As further non-limiting examples, reactive functional groups (or complementary reactive functional groups) include, but are not limited to, -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 ketones , α,β-unsaturated esters, α,β-unsaturated amides, α,β-unsaturated sulfones, α,β-unsaturated sulfonamides, propargyl ketones, propargyl esters, propargyl amides, propargyl sulfones, propargyl sulfoxides, propargyl sulfonamides, maleimides, α-chloroamides, disulfides, 5-fluoro-2,4-dinitrobenzene, etc., or as described by Krishnan et al. (. 18 It may be possible to form a covalent bond with a nitrogen-containing functional group (e.g., an amine) or a sulfur-containing functional group (e.g., a thiol), such as any other amine- or thiol-modifying functional group known in the art, including those described in "F-Labeling of Sensitive Biomolecules for Positron Emission Tomography," Chemistry. 2017 Nov 7;23(62):15553-15577." It is understood that the reactive functional group present in the radiolabeled precursor compound and the complementary reactive functional group present in the radiolabeled prosthetic group may be reversed.

[0154] In some embodiments, the reactive functional group can be, for example, as described by Krishnan et al. 18The reactive functional group can react with a radiolabeled prosthetic group via click chemistry, as described in F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry. 2017 Nov 7;23(62):15553-15577. In some embodiments, the reactive functional group is an alkyne that can react with a radiolabeled prosthetic group containing an azide group via click chemistry. In some embodiments, the reactive functional group is an azide that can react with a radiolabeled prosthetic group containing an alkyne group via 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 that can react with a radiolabeled prosthetic group containing an alkene via click chemistry. In some embodiments, the reactive functional group is an alkene that can react with a radiolabeled prosthetic group containing a tetrazine via click chemistry. Suitable alkenes include strained alkenes such as transcyclooctene (TCO), cyclooctyne, and norbornene.

[0155] The radiolabeled prosthetic group can be any suitable radiolabeled prosthetic group that can be conjugated to the epitopic moiety by reacting with a reactive functional group on the epitopic moiety. Non-limiting examples of radiolabeled prosthetic groups containing covalently bound radionuclides include those described by Krishnan et al. 18F-Labeling of Sensitive Biomolecules for Positron Emission Tomography, Chemistry. 2017 Nov 7;23(62):15553-15577). When the radionuclide is non-covalently linked to the radiolabeled prosthetic group, the radiolabeling moiety may be a chelator moiety capable of chelating the radionuclide, and the radionuclide is complexed with the chelator moiety. In this case, the chelator moiety may be the same chelator moiety as defined for the radiolabeled compounds described herein.

[0156] The radiolabeled prosthetic group may be further conjugated (or can be further conjugated) to a further dysfunctional P2X7 receptor epitope moiety that can be recognized or bound by a receptor expressed on an immune cell, the receptor comprising an antigen recognition domain for binding the dysfunctional P2X7 receptor to the signal transduction domain.

[0157] Any suitable chelator moiety capable of chelating a radionuclide may be used. The chelator moiety may be present in the epitope moiety. In this case, the chelator moiety may be, for example, a histidine residue (e.g., 99m for providing Tc-His 99m Tc). Alternatively, the chelator moiety may be present in a radiolabeled precursor conjugated to the epitope moiety. In this case, the chelator moiety may be the same chelator moiety as defined for the radiolabeled compounds described herein. Furthermore, the chelator moiety may be conjugated to the epitope moiety by any suitable linker described herein.

[0158] Dysfunctional P2X7 receptor epitope region The dysfunctional P2X7 receptor epitope portion can be provided in the form of dysfunctional P2X7 receptor or a fragment of dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP. Such receptor cannot widen the opening of non-selective calcium channel to apoptotic pore.

[0159] According to the present invention, the dysfunctional P2X7 receptor epitope portion is typically in the form of a peptide fragment of the dysfunctional P2X7 receptor. Thus, in a particularly preferred embodiment, the radiolabeled molecule of the present invention is (i) a peptide capable of being recognized or bound by an antigen recognition domain of a receptor expressed on an immune cell, wherein the receptor is for binding a dysfunctional P2X7 receptor and comprises a signal transduction domain; and (ii) a radionuclide linked directly or indirectly to an epitope moiety; or a salt or solvate thereof.

[0160] Furthermore, the present invention provides (i) a peptide capable of being recognized or bound by an antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding a dysfunctional P2X7 receptor and comprises a signal transduction domain; (ii) (A) an atom or functional group that can be converted into a radionuclide; (B) a reactive functional group capable of conjugating to a radiolabeled prosthetic group, or (C) a radionuclide precursor moiety selected from the group consisting of: Alternatively, a salt or solvate thereof is provided.

[0161] Typically, the peptide comprises an epitope that is not found on or available for binding to a functional P2X7 receptor.

[0162] In some embodiments, the peptide comprises a proline at amino acid 210 of the dysfunctional P2X7 receptor. In some embodiments, the peptide comprises one or more amino acid residues ranging from glycine at amino acid 200 to cysteine ​​at amino acid 216 of the dysfunctional P2X7 receptor.

[0163] A range of peptide fragments of dysfunctional P2X7 receptors are disclosed in PCT / AU2002 / 000061 (and corresponding publications WO2002 / 057306, and US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2008 / 001364 (and corresponding publications and PCT / AU2009 / 000869 (and corresponding publications WO2010 / 000041, and US8,597,643, US9,328,155, or US10,238,716), the contents of all of which are incorporated in their entirety. Exemplary peptides within these specifications that contain epitopes contemplated for use in the present invention are described below. [Table 4]

[0164] Non-limiting examples of variations of the E200 peptide sequence (including N-terminal and / or C-terminal extensions, and various linker, hinge, or spacer regions) are provided in Table 1.

[0165] The amino acid sequence of any one of SEQ ID NOs: 2 to 7 may comprise a portion of an epitope portion that can be recognized or bound by a receptor expressed on an immune cell (also referred to herein as the "recognition sequence" of the epitope portion).

[0166] In some embodiments, the epitope portion comprises or consists of an amino acid sequence selected from any of the peptide sequences listed in Table 1 above.

[0167] For peptides in Table 1 that have histidine residues, the radionuclide can be conjugated to the epitope moiety via the two histidine residues. For sequences that have cysteine ​​residues, conjugation can be via N-[N-(S)-1,3-dicarboxypropyl]carbamyl]-4-[ 18 F] Fluorobenzyl-L-cysteine ​​( 18 Labeling of cysteine ​​residues may be via labeling using F18 compounds described herein, such as F-DCFBC. For sequences with lysine residues, attachment of radionuclides may be via the lysine residues.

[0168] In some embodiments, the N-terminus of the epitope moiety is a free amine (-NH2).

[0169] In some embodiments, the C-terminus of the epitope moiety is a free acid (—COOH). In some embodiments, the C-terminus is a derivative or analog of the free acid group, such as an ester (—COOC alkyl) or a primary or secondary amide (—CONHR, where R is selected from H and C alkyl). Advantageously, having a C-terminus that is a derivative or analog of the free acid group may improve the biological stability of the peptide compared to the free acid. In some embodiments, the C-terminus is a derivative or analog of the free acid group that includes a functional moiety, such as biotin.

[0170] Receptors and immune cells that express them In any embodiment, the receptor that comprises the antigen binding domain for binding to the dysfunctional P2X7 receptor and the signal transduction domain is preferably chimeric antigen receptor (CAR) or its variant.The receptor can also be modified TCR.

[0171] Generally, CAR, its variant or TCR can comprise an extracellular domain (extracellular portion) comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain. The extracellular domain can be linked to the transmembrane domain by a linker. The extracellular domain can also comprise a signal peptide. Preferably, the extracellular portion of CAR, its variant or TCR comprises the nfP2X7 binding domain that recognizes the E200 (or E300, or E200-300 complex) epitope disclosed herein.

[0172] Typically, the antigen recognition domain comprises a binding polypeptide comprising amino acid sequence homology to one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide comprises one or more CDRs of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L It contains amino acid sequence homology to the CDR1, CDR2, and CDR3 domains of the chains.

[0173] In a preferred embodiment, the binding polypeptides are those described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,450,380). 0), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or 9,944,701). No. 10,597,451), PCT / AU2008 / 001365 (or corresponding U.S. Pat. No. 8,293,491, or corresponding U.S. Pat. No. 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Pat. No. 8,597,643, U.S. Pat. No. 9,328,155, or corresponding U.S. Pat. No. 10,238,716) in PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US9,127,059, US9,688,771, or US10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO2011 / 075789 or US8,835,609). H and / or V LThe amino acid sequences of the CDRs of the 2-2-1, 2-2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38, 2-39, 2-40, 2-41, 2-42, 2-43, 2-44, 2-45, 2-46, 2-47, 2-48, 2-49, 2-51, 2-52, 2-53, 2-54, 2-55, 2-56, 2-57, 2-58, 2-59, 2-60, 2-61, 2-62, 2-63, 2-64, 2-65, 2-66, 2-67, 2-68, 2-71, 2-72, 2-73, 2-74, 2-75, 2-76, 2-77, 2-77, 2-78, 2-79, 2-80, 2-81, 2-82, 2-83, 2-84, 2-85, 2-86, 2-87, 2-88,

[0174] In further embodiments, the CAR binding polypeptide is a polypeptide of interest described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,450). ,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or Nos. 8,293,491 or 8,658,385; PCT / AU2009 / 000869 (or any one of the corresponding U.S. Patents 8,597,643, 9,328,155, or 10,238,716); and the V of antibodies described in PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US9,127,059, US9,688,771, or US10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO2011 / 075789 or US8,835,609). H and / or V LThe entire contents of which are incorporated herein by reference. Preferably, the antibody comprises the CDR amino acid sequences of 2-2-1, as described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010-0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0175] In further embodiments, the CAR binding polypeptide is a polypeptide of interest described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or any of the corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,450,380). No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or 10,597,451). Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or corresponding U.S. Patent Nos. 8,597,643, 9,328,155, or 10,238,716), PCT / AU2010 / 001070 (or corresponding publications and PCT / AU2010 / 001741 (or any one of the corresponding publications WO2011 / 075789 or US8,835,609), the entire contents of which are incorporated herein by reference.Preferably, the antibody comprises the CDR amino acid sequences of 2-2-1, as described in PCT / AU2010 / 001070 (or in any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09, as described in PCT / AU2007 / 001541 (or in the corresponding U.S. Publication No. 2010-0036101), and is produced by hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under accession number 06080101.

[0176] "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a specific organelle (such as the endothelial endoplasmic reticulum) and / or to the cell surface.

[0177] Generally, the "antigen-binding domain" (or antigen-recognition domain) refers to the region of a CAR that specifically binds to an antigen (and thus can target cells containing the antigen). A CAR may contain one or more antigen-binding domains. Generally, the target region on a CAR is extracellular. The antigen-binding domain may comprise an antibody or an antibody-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a bispecific antibody. Any molecule that specifically binds to a given antigen, such as an affibody or ligand-binding domain from a naturally occurring receptor, may be used as the antigen-binding domain. Often, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of an immunoglobulin heavy chain and a light chain are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3-linker" and variations thereof, although one skilled in the art will understand that a variety of linker sequences and formats may be used.

[0178] CARs may also include a "hinge" region (sometimes called a spacer region or linker region) that links the antigen-binding domain to the transmembrane domain. This is typically a hydrophilic region located between the antigen-binding domain and the transmembrane domain. CARs may include an extracellular hinge domain, but it is also possible to exclude such a hinge. The hinge region may include, for example, an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial hinge sequence, or a combination thereof. One example of a hinge region is the CD8α hinge.

[0179] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source for such a domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8α or CD28. If the main signaling and antigen recognition modules (domains) are located on two (or more) polypeptides, the CAR can have two (or more) transmembrane domains. The division of the main signaling and antigen recognition modules allows for small molecule-dependent, titratable, and reversible control of CAR cell expression (Wu et al., 2015, Science 350:293-303), which is due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR.

[0180] The cytoplasmic domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to a specific function of a cell; for example, in T cells, effector function can be cytolytic activity or helper cell activity, including secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to perform a specific function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector function.

[0181] The function of the intracellular domain may be pro-inflammatory or anti-inflammatory and / or immunomodulatory, or a combination thereof.

[0182] Prominent examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor binding.

[0183] Generally, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: first, those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and second, those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0184] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0185] Examples of ITAMs containing primary cytoplasmic signaling sequences commonly used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most notably, the sequence derived from CD3 zeta.

[0186] The cytoplasmic domain of the CAR can be designed to contain a CD3-zeta signaling domain, either alone or in combination with any desired cytoplasmic domain. The cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for efficient and sufficient lymphocyte response to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0187] In some embodiments, the activating receptor (from which a portion of the signaling domain is derived) is a CD3 co-receptor complex or an Fc receptor.

[0188] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0189] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0190] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order, with or without a linker. A short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A prominent linker is a glycine-serine duplex.

[0191] As another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0192] As mentioned above, either the extracellular portion of the CAR, or the transmembrane domain, or the cytoplasmic domain may also contain a heterodimerization domain for the purpose of separating the main signaling and antigen recognition modules of the CAR.

[0193] A CAR that binds a radiolabeled molecule of the present invention, for example a CAR that comprises an nfP2X7E200 binding domain, may be designed to include any portion or part of the above-mentioned domains described herein in any order and / or combination that results in a functional CAR.

[0194] The affinity that dysfunctional P2X7 receptor binding domain of CAR binds to nfP2X7 recognition site E200 of radiolabeled molecule of the present invention can vary, but generally, binding affinity can be within the range of about 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM or about 1 nM, preferably at least about 10 pM or 1 pM.In preferred embodiments, binding affinity is at least about 1 nM or at least about 10 nM.

[0195] The receptor (e.g., a CAR, a variant thereof, or a TCR, or a variant thereof) is typically expressed by an immune cell.

[0196] The immune cells may be "engineered cells," "genetically modified cells," or "immune effector cells" as described herein. Furthermore, the immune cells may be immune cell precursors that can differentiate into immune cells. Cells that can differentiate into immune cells (e.g., T cells expressing a dysfunctional P2X7CAR) may be stem cells, multilineage progenitor cells, or induced pluripotent stem cells.

[0197] The immune cells can be leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells (including CD4+ T cells or CD8+ T cells), natural killer cells, natural killer T cells, or γδ T cells.

[0198] In any embodiment, the immune cell may be a T cell, and optionally the T cell does not express TcRαβ, PD1, CD3, or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0199] In any embodiment, the immune cells optionally do not express accessory molecules, which may be checkpoint, exhaustion, or apoptosis-related signaling receptors, and ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L, and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0200] 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 the dysfunctional P2X7 receptor). Alternatively, the CARs can bind to different target molecules, so that only one of the CARs binds to the dysfunctional P2X7 receptor.

[0201] As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs that have either non-identical antigen recognition and / or non-identical signal transduction domains.In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize dysfunctional P2X7 receptors), but have different signal transduction domains, such as one CAR with a signal transduction domain that has a part of an activating receptor and the other CAR with a signal transduction domain that has a part of a costimulatory receptor.As will be understood, at least one of the two or more CARs in this embodiment has an antigen recognition domain that recognizes dysfunctional P2X7 receptors, and the other CAR can take any suitable form and be directed against any suitable antigen.

[0202] Methods for preparing radiolabeled molecules The present invention provides a method for preparing a radiolabeled molecule, comprising the steps of: - providing a radiolabeled precursor molecule as defined herein; - reacting a radiolabeled precursor molecule to provide a radiolabeled molecule as defined herein; There is further provided a method whereby a radiolabeled molecule is provided.

[0203] The radiolabeled precursor molecule may be reacted appropriately to provide the radiolabeled molecule depending on the nature of the radiolabeled precursor moiety.

[0204] In some embodiments, the method comprises: - (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by an antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding the dysfunctional P2X7 receptor and comprises a signal transduction domain; and (ii) a radioactive precursor molecule containing an atom or functional group that can be converted into a radionuclide; or a salt or solvate thereof; - converting the atom or functional group into a radionuclide, Thereby providing a radiolabeled molecule.

[0205] In some embodiments, the method comprises: - (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by an antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding the dysfunctional P2X7 receptor and comprises a signal transduction domain; and (ii) a reactive functional group capable of conjugating to a radiolabeled prosthetic group; or a salt or solvate thereof; - conjugating a radiolabeled prosthetic group via a reactive functional group, Thereby providing a radiolabeled molecule.

[0206] In some embodiments, the method comprises: - (i) a dysfunctional P2X7 receptor epitope portion capable of being recognized or bound by an antigen-binding domain of a receptor expressed on an immune cell, wherein the receptor is for binding the dysfunctional P2X7 receptor and comprises a signal transduction domain; and (ii) a radiolabeled precursor molecule comprising a chelator moiety capable of chelating a radionuclide; or a salt or solvate thereof; chelating the radionuclide to a chelating agent moiety, Thereby providing a radiolabeled molecule.

[0207] In the above methods, the atom or functional group capable of being converted to a radionuclide, the reactive functional group capable of being conjugated to a radiolabeled prosthetic group, and the chelator moiety capable of chelating a radionuclide can be present in the epitope moiety or the radiolabeled precursor moiety conjugated to the epitope moiety, as described herein.

[0208] The radiolabeled precursor molecule may be suitably prepared and reacted to provide the radiolabeled molecule by methods known in the art, including those described herein. Suitable methods for obtaining radiolabeled peptides ( 68 Methods for attaching Ga to DOTA-like conjugate peptides are described, for example, in Mueller et al., (2011) Nature Protocols, 11:1057-1066, which is incorporated herein by reference.

[0209] The epitope portion of the radiolabeled molecules or radiolabeled precursor molecules 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 portion can also be prepared by known recombinant DNA techniques.

[0210] Radiolabeling of the peptide can be via one or more histidine residues present in the peptide. Examples of radiolabeling of histidine residues are well known in the art, for example, as described in Ibrahim et al., (2016) Radiochemistry, 58:521-527, which is incorporated herein by reference. In such cases, the peptide can include a biotin label or amide at the C-terminus.

[0211] In a further example, tyrosine residues in the peptide can be labeled using standard techniques known to those skilled in the art.

[0212] Still further, the radiolabel may be N-[N-(S)-1,3-dicarboxypropyl]carbamyl]-4-[ 18 F] Fluorobenzyl-L-cysteine ​​(18 The peptide may be a cysteine ​​residue using an F-labeling compound such as F-DCFBC. In such cases, the peptide preferably comprises the amino acid sequence set forth in any of SEQ ID NOs: 2, 10, or 13 (e.g., GHNYTTRNILPGLNITSTFHKTC-amide). Methods for F-labeling are described in David et al., (2019) RSC Adv. 15:8638-8649, incorporated herein by reference.

[0213] Purpose The radiolabeled molecule of the present invention can be useful for detecting immune cells that express a receptor that comprises an antigen recognition domain for binding dysfunctional P2X7 receptor and signal transduction domain.The radiolabeled molecule comprises a dysfunctional P2X7 receptor epitope portion that can be recognized or bound by the receptor expressed on the immune cell.Therefore, the presence of dysfunctional P2X7 receptor epitope portion can enable the molecule of the present invention to bind to the immune cell.

[0214] Thus, the present invention provides the use of the radiolabeled molecules described herein to detect immune cells expressing a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signal transduction domain.

[0215] The present invention also provides a method for detecting immune cells in a subject that express a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signal transduction domain, the method comprising: - administering a radiolabeled molecule as described herein to a subject who has received immune cells that express a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor; - detecting a radiolabeled compound in the subject, wherein the presence of the radiolabeled compound indicates the presence of immune cells.

[0216] In some embodiments, the radiolabeled molecule is detected by performing a radionuclide scan. The radionuclide scan may be suitably selected depending on the radionuclide present in the radiolabeled molecule. In some embodiments, the radionuclide scan may be a positron emission tomography (PET) scan or a single photon emission computed tomography (SPECT) scan.

[0217] In some embodiments, the method further comprises imaging the detected radiolabeled molecule.

[0218] In some embodiments, the method further comprises, prior to the step of detecting the radiolabeled molecule, allowing the radiolabeled molecule to concentrate at a site in the subject where immune cells are found.

[0219] In some embodiments, the method further comprises administering to the subject an immune cell that expresses a receptor comprising an antigen recognition domain for binding the dysfunctional P2X7 receptor before administering to the subject the radiolabeled molecule.

[0220] The method and use of radiolabeled molecule described herein can advantageously allow (i) determining whether immune cells expressing receptors that comprise antigen recognition domains for binding dysfunctional P2X7 receptors and signal transduction domains are present in a subject, (ii) identifying the location of the immune cells, including determining the distribution of the population of the immune cells in a subject, and (iii) quantifying the number of the immune cells in a subject or at a specific location / site within a subject.This information can be useful for providing information on the development or adjustment of therapeutic regimens using the immune cells.

[0221] Compositions and Formulations The radiolabeled molecule may be provided in a suitable form or formulated for administration to a subject.

[0222] Thus, the present invention provides a composition comprising a radiolabeled molecule of the present invention, or a salt or solvate thereof.

[0223] The composition may be a pharmaceutical composition. In the case of a pharmaceutical composition, the composition may include a pharmaceutically acceptable carrier, for example, an aqueous carrier.

[0224] The present invention additionally provides a formulation comprising the radiolabeled molecule of the present invention, or a salt or solvate thereof. The formulation of the radiolabeled molecule may include 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, bulking agents, and lubricants.

[0225] The compositions and formulations of the present invention may comprise one type of radiolabeled molecule, or two or more types of radiolabeled molecules (e.g., the radiolabeled molecules may have the same or different dysfunctional P2X7 receptor epitope moieties).

[0226] The compositions and formulations may be suitable for use in methods and applications for detecting immune cells expressing a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signal transduction domain, as described herein.

[0227] The radiolabeled molecules that may be in the compositions or formulations of the present invention may 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 (sc, sq, sub-Q, hypo), intramuscular (im), intraarterial, intramedullary, intracavitary, intracardiac, intraarticular (joint), bursa (synovial area), intracranial, intraspinal, and intrathecal (spinal fluid) administration.

[0228] Compositions and formulations containing radiolabeled molecules can be administered to a subject in an amount effective to detect the radiolabeled molecule, for example, by radionuclide scanning. The dose can be suitably selected depending on the radionuclide present in the radiolabeled molecule. The dose can further be suitably selected depending on fluid volume, viscosity, body weight, etc., according to the intended use and the particular mode of administration. A physician can determine the appropriate dose to ultimately use.

[0229] kit The present invention provides the following: (i) a radiolabeled molecule of the present invention, or a salt or solvate thereof; (ii) a radiolabeled precursor molecule of the present invention, or a salt or solvate thereof; (iii) a composition described herein; or (iv) Kits are additionally provided that include one or more of the formulations of the present invention.

[0230] In the case of kits comprising radiolabeled precursor compounds of the invention (including compositions or formulations comprising same), the kits can be used to prepare radiolabeled molecules from the radiolabeled precursor molecules, for example, via the methods described herein.

[0231] This kit can be used to detect immune cells expressing a receptor comprising an antigen recognition domain for binding a dysfunctional P2X7 receptor and a signal transduction domain in a subject to which the immune cells have been administered.

[0232] Optionally, the kit of the present invention may further comprise an immune cell expressing a receptor comprising an antigen recognition domain for binding the dysfunctional P2X7 receptor and the signal transduction domain.

[0233] Optionally, the kits of the invention are packaged with instructions for use in one or more of the methods described herein. [Example]

[0234] Example 1: Preparation of exemplary radiolabeled molecules Radiolabeled E200 peptide containing the amino acid sequence GHNYTTRNILPGLNITSTFHKTSGSGK is made by combining approximately 2900 g / mol of biotinylated peptide with Ga68 (70 g / mol).

[0235] A simple conjugation of the radiolabel is via two histidine residues in the peptide using the method described by Mueller et al. (2016) Nature Protocols, 11:1057-1066. Briefly, the peptide is conjugated to the chelator DOTA using standard techniques, followed by cleavage. 68 It is conjugated to Ga.

[0236] In an alternative example, an Fc fusion protein containing an epitope of the nfP2X7 receptor (such as having the amino acid sequence of SEQ ID NO: 145 (DetR1, monomeric; Fc attenuated; or DetR2 SEQ ID NO: 146; or dimeric Fc attenuated SEQ ID NO: 149)) is conjugated to a radiolabel using a similar approach.

[0237] Example 2: Detection of radiolabeled molecules for binding nfP2X7 receptor-binding CAR T cells: Imaging study design. The NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ mouse model was cultured in a 5 × 10 6 The test was performed by orthopedic application to the fourth mammary fat pad of the 1000-kJ / kg / day-1000 mice on day 0. On day 7 of the preclinical study, 5E0 6 nfP2X7-targeted CAR T cells are injected intravenously into the tail vein.

[0238] To detect the presence of CAR T cells in mice, mice are administered the radiolabeled molecule (i.e., peptide or fusion protein) produced in Example 1 via tail vein injection.

[0239] Mice are assigned to one of the following groups (n=3 per group), each representing a different time period (T) between administration of the radiolabeled molecule and detection using positron emission tomography: • Control (i.e., no radiolabeled molecule administered), ●T=0 ●T=5 minutes ●T=10 minutes ●T=15 minutes ●T=20 minutes ●T=60 minutes ●T=120 minutes ●T=240 minutes

[0240] The distribution of radiolabeled peptide or radiolabeled Fc fusion protein is assessed at each time point via positron emission tomography (PET) scans to detect positron emission of gallium-68.

[0241] A whole-body static PET image is acquired, followed by a whole-body CT scan for anatomical reference.

[0242] High positron emission was detected at the tumor site, indicating enrichment of anti-dysfunctional P2X7 receptor CAR T cells and cellular localization at the tumor site.

[0243] This approach is outlined in Figure 1.

[0244] Example 3: Demonstration of the ability of detection reagents to bind to CAR T cells in vivo Mice bearing AsPC-1 tumors derived from a pancreatic cancer cell line (administered at a dose of 0.8E06 on day 7) were infused on day 0 with E200-targeting CAR T cells (i.e., CAR T cells capable of binding to the E200 epitope as described herein), injected intravenously into the tail vein.

[0245] Mice were then injected intraperitoneally with 50 μg of a monomeric E200-Fc fusion protein (e.g., comprising the amino acid sequence of SEQ ID NO: 145) containing a C-terminal His-tag. One hour was allowed to elapse to allow the fusion protein to bind to the CAR T cells in vivo.

[0246] Three samples of whole blood and bone marrow were collected from the mice and subjected to flow cytometry using anti-His-tag FITC antibody (VioGreen).

[0247] Briefly, for the results shown in Figure 2a (e.g., using a His-tagged monomeric fusion protein of the invention having the amino acid sequence of SEQ ID NO: 158):

[0248] A) Blood was lysed and stained with an anti-HIS-tag antibody conjugated to FITC to detect monomeric fusion protein reagent bound to CAR-expressing cells.

[0249] B) Bone marrow was isolated from femurs and stained with FITC-conjugated anti-HIS antibody to detect monomeric fusion protein bound to CAR-expressing cells.

[0250] Anti-HIS-tag antibody was used according to the manufacturer's instructions. Data were acquired on a MACSQuant16 flow cytometer (Miltenyi).

[0251] For the results shown in Figure 2b (e.g., using an LCLC-biotin conjugate monomer fusion protein of the invention having the amino acid sequence of SEQ ID NO: 158):

[0252] A) Blood was lysed and incubated with LCLC biotin-conjugated monomeric fusion protein, then stained with a commercially available anti-biotin antibody in FITC (VioGreen) to detect monomeric detection reagent bound to CAR-expressing cells.

[0253] B) Bone marrow was isolated and then incubated with LCLC biotin-conjugated monomeric fusion protein, then stained with a commercially available anti-biotin antibody conjugated with FITC (VioGreen) or APC to detect the monomeric detection reagent bound to the CAR-expressing cells.

[0254] Anti-biotin antibody was used according to the manufacturer's instructions. Data were acquired on a MACSQuant16 flow cytometer (Miltenyi).

[0255] The results shown in Figure 2a demonstrate that the in vivo binding monomeric molecules are capable of binding to CAR-expressing cells, which were detected by flow cytometry via an anti-HIS antibody ex vivo.

[0256] The results shown in Figure 2b demonstrate that flow cytometry staining of PBMCs from mouse blood and bone marrow via ex vivo incubation of biotinylated monomeric molecules identified a CAR-expressing cell subset detectable via anti-biotin antibodies.

[0257] The overall results demonstrate that CAR T cells can bind in vivo using the monomeric fusion proteins described herein.

[0258] In parallel, a series of in vitro experiments were performed. Briefly, Jurkat cells and / or primary CD4+ T cells and CD8+ T cells (mixed at a 1:1 ratio after enrichment) were stably transduced with lentivirus (third generation LV system) to express anti-nfP2X7 chimeric antigen receptor (CAR), which contained an antigen-binding domain for binding to the E200 epitope of the P2X7 receptor.

[0259] CAR T cells were contacted with either a monomeric or dimeric fusion protein, each containing a peptide moiety that can be bound by the CAR or protein. The fusion proteins used in this experiment comprise the amino acid sequences of SEQ ID NOs: 158 (monomer) and 149 (dimer).

[0260] Figure 3 shows the levels of CD25+ / CD69+ expression (measures of T cell activation, respectively) and PD-1 expression (measures of T cell exhaustion) up to 72 hours after exposure with various concentrations of fusion protein (10 ng / ml to 400 ng / ml).

[0261] The results show that contacting CAR T cells with monomeric fusion proteins leads to significantly less T cell activation and significantly less T cell exhaustion in a concentration-dependent manner compared to using dimeric fusion proteins. These results indicate that for the purposes of in vivo imaging of CAR T cells, it is preferable to use monomeric fusion proteins to minimize unwanted activation and exhaustion of CAR T cells in patients.

[0262] Similar experiments are performed using the heterodimeric asymmetric molecules described herein (e.g., such that the molecules comprise dimerization between an E200 peptide-Fc fusion protein and a non-identical Fc region of an antibody using KIH technology). The results similarly show that contacting CAR T cells with heterodimeric asymmetric molecules comprising a single copy of the E200 peptide sequence leads to a significant decrease in T cell activation and T cell exhaustion in a concentration-dependent manner compared to using a homodimeric fusion protein comprising two copies of the E200 peptide (e.g., the dimer is a homodimer of the E200-Fc fusion protein). These results indicate that for the purposes of in vivo imaging of CAR T cells, it is preferable to use asymmetric heterodimeric molecules or monomeric fusion proteins (i.e., comprising a single E200 peptide sequence) to minimize undesired activation and exhaustion of CAR T cells in patients.

Claims

1. A radiolabeled molecule comprising: (i) a dysfunctional P2X that can be recognized or bound by the antigen-binding domain of a receptor expressed on an immune cell ７ A receptor epitope portion, wherein the receptor is a dysfunctional P2X ７ Dysfunctional P2X receptors that bind to the receptor and contain signaling domains ７ a receptor epitope portion; (ii) a radionuclide linked directly or indirectly to said epitope moiety; or a salt or solvate thereof.

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

3. The radionuclide is 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 3. The radiolabeled molecule of claim 1, wherein the radiolabeled molecule is selected from Bi.

4. 4. The radiolabeled molecule of claim 1, wherein the radionuclide is directly linked to an amino acid side chain of the epitope moiety.

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

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

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

8. 8. The radiolabeled compound of 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. The radiolabeled moiety is capable of being recognized or bound by the antigen-binding domain of a receptor expressed on an immune cell. ７ conjugated to a receptor epitope moiety, wherein the receptor is a dysfunctional P2X ７ The radiolabeled compound of any one of claims 5 to 8, which is for binding a receptor and comprises a signaling domain.

10. A radiolabeled precursor molecule comprising: (i) a dysfunctional P2X that can be recognized or bound by the antigen-binding domain of a receptor expressed on an immune cell ７ A receptor epitope portion, wherein the receptor is a dysfunctional P2X ７ Dysfunctional P2X receptors that bind to the receptor and contain signaling domains ７ a receptor epitope portion; (ii) (A) an atom or functional group that can be converted into a radionuclide; (B) a reactive functional group capable of conjugating to a radiolabeled prosthetic group; or (C) a radionuclide precursor moiety selected from the group consisting of: or a salt or solvate thereof.

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

12. 12. The radiolabeled precursor molecule of 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. The radiolabeled precursor moiety is capable of being recognized by or bound to a receptor expressed on immune cells. ７ and a receptor epitope moiety, wherein the receptor is a dysfunctional P2X ７ 13. A radiolabeled precursor molecule according to claim 11 or claim 12, comprising an antigen recognition domain for binding a receptor and a signalling domain.

14. The radiolabeled molecule of any one of claims 1 to 9, or the radiolabeled precursor molecule of any one of claims 10 to 13, wherein the epitope portion comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 2 to 69 or 122.

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

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 comprises a functionally impaired P2X ７ The fusion protein of claim 17 , comprising a receptor epitope portion and an antibody Fc region.

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

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

21. 21. The fusion protein of claim 20, wherein the amino acid substitutions comprise one or more substitutions of cysteine ​​residues.

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

23. 23. The fusion protein of any one of claims 18 to 22, wherein the protein comprises or consists of an amino acid sequence set forth in any of SEQ ID NOs: 145 to 158, 160, and 161.

24. 1. A method for preparing a radiolabeled molecule, comprising: - providing a radiolabeled precursor molecule as defined in any one of claims 10 to 16 or a fusion protein as defined in any one of claims 17 to 23; - said radiolabeled precursor molecule, (i) converting said atom or functional group of (A) into a radionuclide; (ii) to conjugate the reactive functional group of (B) to a radiolabeled prosthetic group; or (iii) reacting under suitable conditions to chelate said chelator moiety of (C) to a radionuclide.

25. 25. A radiolabeled molecule obtained according to the method of claim 24.

26. A radiolabeled asymmetric heterodimeric molecule comprising the fusion protein of any one of claims 18 to 23.

27. Dysfunctional P2X ７ 27. Use of a radiolabeled molecule according to any one of claims 1 to 9, or 25, or 26 for detecting an immune cell or population of immune cells expressing a receptor comprising an antigen recognition domain for linking the receptor and a signalling domain.

28. Dysfunctional P2X in a subject ７ 1. A method for detecting an immune cell or population of immune cells that expresses a receptor comprising an antigen recognition domain for linking the receptor to a signaling domain, comprising: -Dysfunctional P2X ７ administering a radiolabeled molecule of any one of claims 1 to 9, 25, or 26 to a subject receiving immune cells expressing a receptor comprising an antigen recognition domain for binding the receptor to a signaling domain; - detecting said radiolabeled molecule in said subject, wherein the presence of said radiolabeled molecule indicates the presence of said immune cell.

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

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

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

32. below: (i) a radiolabeled molecule according to any one of claims 1 to 9, or 25, or 26, or a salt or solvate thereof; (ii) a radiolabeled precursor molecule according to any one of claims 10 to 15, or a salt or solvate thereof; (iii) a fusion protein according to any one of claims 17 to 23; (iv) a composition according to claim 30; or (v) a formulation according to claim 31.

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