Novel dysfunctional P2X7 binders

Abstract

The present invention relates to novel antigen binding proteins for binding to dysfunctional P2X7 receptors.

Description

[Technical field]

[0001] The present invention relates to antigen-binding proteins and related fragments that bind to dysfunctional P2X7 receptors, the production of said antigen-binding proteins and fragments, and the use of said antibodies and fragments for the detection and treatment of various conditions.

[0002] Related Applications

[0002] This application claims priority from Australian provisional application AU2021902832, the entire contents of which are incorporated herein by reference. [Background technology]

[0003]

[0003] Purinergic (P2X) receptors are ATP-gated cation-selective channels. Each receptor is composed of three protein subunits or monomers. To date, seven separate genes encoding P2X monomers have been identified: P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7.

[0004]

[0004] The P2X7 receptor is of particular interest because it is understood that expression of these receptors is restricted to cells that may undergo programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. In normal homeostasis, there is some expression of P2X7 receptors, such as on erythrocytes.

[0005] Interestingly, P2X7 receptors that contain one or more monomers with cis-isomerization at Pro210 and lack ATP-binding function are found in cells, such as pre-cancerous and neoplastic cells, that are understood to be unable to undergo programmed cell death. This conformational isoform of the receptor is called "non-functional" or "dysfunctional" receptor.

[0006]

[0006] Antibodies generated by immunization with peptides containing Pro210 in cis bind to dysfunctional P2X7 receptors. However, they do not bind to P2X7 receptors that can form apoptotic pores under normal physiological conditions in the presence of ATP. Therefore, these antibodies are useful for selective detection of many types of carcinomas and hematopoietic cancers and for the treatment of some of these conditions.

[0007]

[0007] Due to the nature of the conformational epitopes present in dysfunctional P2X7 receptors, it is not a simple task to identify and generate proteins that bind to this type of receptor with sufficient affinity. Higher affinity reagents are generally desired for cancer detection and treatment applications. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for improved reagents that bind to dysfunctional P2X7 receptors.

[0009] 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, regarded and / or combined with other pieces of prior art by a person skilled in the art. [Means for solving the problem]

[0009]

[0010] The present invention relates to an antigen-binding protein that comprises an antigen-binding domain that binds to P2X7 receptor that is not responsive to ATP (i.e., dysfunctional P2X7 receptor), which cannot form apoptotic pores under physiological conditions.Preferably, the antigen-binding protein of the present invention does not bind to the P2X7 receptor that responds to ATP and functions normally.

[0010]

[0011] The present invention provides an antigen binding protein that binds to a dysfunctional P2X7 receptor, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a (where: FR1, FR2, FR3 and FR4 are framework regions, CDR1, CDR2 and CDR3 are complementarity determining regions, FR1a, FR2a, FR3a and FR4a are framework regions, CDR1a, CDR2a and CDR3a are each a complementarity determining region, Antigen binding proteins are provided, the sequences of any of the framework regions or complementarity determining regions being described herein.

[0011]

[0012] The present invention provides an antigen-binding protein that binds to the nfP2X7 receptor, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a (where: FR1, FR2, FR3 and FR4 are framework regions, CDR1, CDR2 and CDR3 are complementarity determining regions, FR1a, FR2a, FR3a and FR4a are framework regions, CDR1a, CDR2a and CDR3a are each a complementarity determining region, The present invention provides an antigen binding protein in which the sequence of any of the complementarity determining regions has the amino acid sequence set out in Table 1 below. Preferably, the framework regions have the amino acid sequences also set out in the table below, including amino acid mutations of specific residues that can be determined by aligning the various framework regions from each antibody. The present invention also includes that when CDR1, CDR2 and CDR3 are sequences from the variable heavy chain (VH) of an antibody, CDR1a, CDR2a and CDR3a are sequences from the variable light chain (VL) of an antibody, or when CDR1, CDR2 and CDR3 are sequences from VL, CDR1a, CDR2a and CDR3a are sequences from VH.

[0012]

[0013] In any embodiment, the antigen binding protein described herein is: Includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a.

[0013]

[0014] As defined herein, a linker may be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0015] In certain preferred embodiments, the present invention provides antigen binding proteins comprising, consisting essentially of, or consisting of the amino acid sequences of SEQ ID NOs: 4 and 12 (in N-terminal to C-terminal or C-terminal to N-terminal order).

[0014]

[0016] In a particularly preferred embodiment, the antigen binding protein comprises, consists essentially of, or consists of, in N-terminal to C-terminal order, SEQ ID NO: 12 and SEQ ID NO: 4 (i.e., VL to VH). Optionally, the antigen binding protein comprises SEQ ID NO: 12 (VL)-linker-SEQ ID NO: 4 (VH).

[0015]

[0017] In any embodiment, the present invention provides an antigen binding protein that binds or specifically binds to the nfP2X7 receptor and competitively inhibits binding of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO:4, and a VL comprising the sequence set forth in SEQ ID NO:12.

[0016]

[0018] The present invention also provides an antigen binding protein that binds or specifically binds to the nfP2X7 receptor, comprising an antigen binding domain of an antibody, the antigen binding domain comprising: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 3, 31, 38 or 45; (ii) a VH comprising a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO:4; (iii) a VL comprising a CDR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 9, 50, 57 or 64, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 10, 51, 58 or 65, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 11, 52, 59 or 66; (iv) a VL comprising a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 12; (v) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising the sequence shown in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 comprising the sequence shown in SEQ ID NO: 3, 31, 38 or 45; (vi) a VH comprising the sequence shown in SEQ ID NO: 4; (vii) a VL comprising a CDR1 comprising a sequence as set forth in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising a sequence as set forth in SEQ ID NO: 10, 51, 58 or 65, and a CDR3 comprising a sequence as set forth in SEQ ID NO: 11, 52, 59 or 66; (viii) a VL comprising the sequence set forth in SEQ ID NO: 12; (ix) a VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising the sequence shown in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 comprising the sequence shown in SEQ ID NO: 3, 31, 38 or 45, and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising the sequence shown in SEQ ID NO: 10, 51, 58 or 65, and a CDR3 comprising the sequence shown in SEQ ID NO: 11, 52, 59 or 66; or (x) a VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 12 Includes at least one of the following:

[0017]

[0019] In any aspect of the invention, the antigen binding domain comprises: (i) a VH comprising a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 5, 32, 39, or 46; a FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 6, 33, 40, or 47; a FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 7, 34, 41, or 48; and a FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 8, 35, 42, or 49; (ii) a VL comprising an FR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 13, 53, 60 or 67; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 14, 54, 61 or 68; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 15, 55, 62 or 69; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO: 16, 56, 63 or 70; (iii) VH comprising FR1 comprising the sequence shown in SEQ ID NO: 5, 32, 39, or 46, FR2 comprising the sequence shown in SEQ ID NO: 6, 33, 40, or 47, FR3 comprising the sequence shown in SEQ ID NO: 7, 34, 41, or 48, and FR4 comprising the sequence shown in SEQ ID NO: 8, 35, 42, or 49; (iv) a VL comprising an FR1 comprising the sequence set forth in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising the sequence set forth in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising the sequence set forth in SEQ ID NO: 15, 55, 62 or 69, and an FR4 comprising the sequence set forth in SEQ ID NO: 16, 56, 63 or 70; or (v) a VH comprising an FR1 having a sequence as set forth in SEQ ID NO: 5, 32, 39, or 46, an FR2 having a sequence as set forth in SEQ ID NO: 6, 33, 40, or 47, an FR3 having a sequence as set forth in SEQ ID NO: 7, 34, 41, or 48, and an FR4 having a sequence as set forth in SEQ ID NO: 8, 35, 42, or 49, and a VL comprising an FR1 having a sequence as set forth in SEQ ID NO: 13, 53, 60, or 67, an FR2 having a sequence as set forth in SEQ ID NO: 14, 54, 61, or 68, an FR3 having a sequence as set forth in SEQ ID NO: 15, 55, 62, or 69, and an FR4 having a sequence as set forth in SEQ ID NO: 16, 56, 63, or 70; The present invention further includes at least one of the following:

[0018]

[0020] As described herein, an antigen binding protein is (i) single chain Fv fragment (scFv), (ii) a dimeric scFv (di-scFv), or (iii) one of (i) or (ii) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; The compound may be in the form:

[0019]

[0021] Further, as described herein, the antigen binding protein may be (i) diabody, (ii) triabodies, (iii) tetrabodies, (iv) Fab, (v) F(ab')2, (vi) Fv, (vii) bispecific antibodies or other forms of multispecific antibodies (including BiTEs); or (viii) one of (i) to (vii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; The compound may be in the form:

[0020]

[0022] In a particularly preferred embodiment, the antigen-binding protein of the present invention is a protein that does not contain a constant region derived from an immunoglobulin. For example, the antigen-binding protein is preferably an scFv, a dimeric scFv, an Fv fragment, a single domain antibody (dAb), a diabody, or a fusion protein or a conjugate comprising a fusion protein.

[0021]

[0023] Such antigen-binding proteins may also be referred to as antigen-binding domains of antibodies.

[0024] In certain embodiments, the complementarity determining region sequences (CDRs) of the antigen binding proteins of the invention can be defined according to the IMGT numbering system, the Kabat or the Chothia system.

[0022]

[0025] When referring to a protein or antibody that "binds" to a dysfunctional P2X7 receptor (nfP2X7 receptor) in this specification, we are referring to a protein or antibody that "binds specifically to" or "specifically binds to" the nfP2X7 receptor.

[0023]

[0026] Preferably, the antigen-binding protein described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding protein is an antibody, such as a monoclonal antibody. The antigen-binding protein may be in the form of a recombinant or modified antibody (e.g., a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a primatized antibody, a deimmunized antibody, a synhumanised antibody, a half antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody). The antibody may further comprise a chemical modification, such as conjugation to an active agent or a radioactive label, or an agent that improves solubility, or other modification as described herein.

[0024]

[0027] As used herein, an antigen binding protein can be a variable domain.

[0028] The present invention also provides an anti-nfP2X7 receptor antibody or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, The heavy chain variable region comprises: - comprising a CDR H1 as depicted in SEQ ID NO: 1, 29, 36 or 43, a CDR H2 as depicted in SEQ ID NO: 2, 30, 37 or 44, and a CDR H3 as depicted in SEQ ID NO: 3, 31, 38 or 45, The light chain variable region comprises: - comprising a CDR L1 as depicted in SEQ ID NO: 9, 50, 57 or 64, a CDR L2 as depicted in SEQ ID NO: 10, 51, 58 or 65, and a CDR L3 as depicted in SEQ ID NO: 11, 52, 59 or 66.

[0025]

[0029] In any embodiment, the anti-nfP2X7 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:12.

[0030] In any embodiment, the anti-nfP2X7 receptor antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:4.

[0026]

[0031] In any embodiment, the anti-nfP2X7 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:12 and a heavy chain variable region comprising the sequence of SEQ ID NO:4.

[0027]

[0032] In any embodiment, the anti-nfP2X7 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising FR L1 set forth in SEQ ID NO: 13, 53, 60 or 67, FR L2 set forth in SEQ ID NO: 14, 54, 61 or 68, FR L3 set forth in SEQ ID NO: 15, 55, 62 or 69, and FR L4 set forth in SEQ ID NO: 16, 56, 63 or 70.

[0028]

[0033] In any embodiment of the invention, the anti-nfP2X7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising FR H1 as set forth in SEQ ID NO:5, 32, 39 or 46, FR H2 as set forth in SEQ ID NO:6, 33, 40 or 47, FR H3 as set forth in SEQ ID NO:7, 34, 41 or 48, and FR H4 as set forth in SEQ ID NO:8, 35, 42 or 49.

[0029]

[0034] In any embodiment of the invention, any antigen binding protein described herein further comprises an Fc region that has been modified to reduce its ability to induce antibody-dependent cellular cytotoxicity (ADCC). Preferably, the reduced ability to induce ADCC is conferred by mutation, deletion or modification of amino acids in the Fc region that interact with an Fc receptor.

[0030]

[0035] The present invention provides an antigen binding protein as described herein, wherein the amino acid sequences forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are human sequences.

[0031]

[0036] The present invention provides anti-nfP2X7 antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies comprising antigen binding proteins having sequences as described herein or comprising the CDR and / or FR sequences as described herein.

[0032]

[0037] The antigen binding proteins described herein may comprise a human constant region, e.g., an IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region or a mixture thereof. H and V L For antibodies or proteins containing V H may be linked to a heavy chain constant region, L may be linked to a light chain constant region.

[0033]

[0038] In one example, the antigen binding protein described herein comprises a constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In one example, the protein or antibody comprises an IgG4 constant region that includes a proline at position 241 (according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991)).

[0034]

[0039] In one example, an antigen binding protein as described herein or a composition of antigen binding proteins as described herein comprises a heavy chain constant region, including a stabilized heavy chain constant region, that comprises a mixture of sequences that fully, partially or completely lack a C-terminal lysine residue.

[0035]

[0040] In one example, the antigen binding protein comprises a VLK antibody as disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as discussed above).H Including V L is linked or fused to a kappa light chain constant region.

[0036]

[0041] In any aspect of the invention, the antibody is a naked antibody, specifically, the antibody is in unconjugated form and is not suitable for forming a conjugate.

[0042] The invention also provides conjugates in the form of antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies or fusion proteins as described herein that are conjugated to a label or a cytotoxic agent.

[0037]

[0043] In embodiments of the invention relating to multiple polypeptide chains forming an antigen binding protein, an expression construct comprises a nucleic acid encoding a polypeptide comprising, e.g., a VH operably linked to a promoter, and a nucleic acid encoding a polypeptide comprising, e.g., a VL operably linked to a promoter.

[0038]

[0044] In another example, an expression construct can include, for example, the following operably linked components: (i) a promoter; (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site, and (iv) a nucleic acid encoding a second polypeptide. in 5' to 3' order, where the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0039]

[0045] The present invention also contemplates separate expression constructs, where one expression construct encodes a first polypeptide comprising a VH and another expression construct encodes a second polypeptide comprising a VL. For example, the present invention provides (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter; Also provided is a composition comprising:

[0040]

[0046] The invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified, or recombinant. In one example, the cell comprises an expression construct of the invention or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter; wherein the first and second polypeptides combine to form the antigen binding protein of the invention.

[0041]

[0047] Examples of cells of the invention include bacterial cells, yeast cells, insect cells, or mammalian cells.

[0048] The invention provides nucleic acids encoding an antigen binding protein, immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein.

[0042]

[0049] The invention provides vectors comprising the nucleic acids described herein.

[0050] The invention provides a cell comprising a vector or nucleic acid described herein.

[0051] In any embodiment, the nucleic acid may comprise the nucleotide sequence set forth in any of SEQ ID NOs: 71-86, preferably SEQ ID NOs: 74 and / or 82, or a sequence at least 80% identical thereto.

[0043]

[0052] The present invention provides a pharmaceutical composition comprising an antigen binding protein, or an antigen binding protein comprising the CDR and / or FR sequences described herein, or an immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single chain antibody molecule, or multispecific antibody, fusion protein, or conjugate described herein, and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0044]

[0053] The present invention provides diagnostic compositions comprising an antigen binding protein (or comprising the CDR and / or FR sequences described herein), or an antigen binding protein as described herein, an immunoglobulin variable domain, an antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single chain antibody molecule, or multispecific antibody, a fusion protein or conjugate, a diluent, and optionally a label.

[0045]

[0054] The invention provides kits or articles of manufacture comprising an antigen binding protein (or comprising the CDR and / or FR sequences described herein), or an immunoglobulin variable domain, antibody, dab, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single chain antibody molecule, or multispecific antibody, fusion protein or conjugate described herein.

[0046]

[0055] The present invention provides for the use of a sequence according to one or more of CDR1, CDR2, FR1, FR2, FR3 and FR4 described herein to produce an antigen binding protein that binds to the nfP2X7 receptor.

[0047]

[0056] The present invention provides for the use of the antigen binding proteins or CDR and / or FR sequences described herein to produce anti-nfP2X7 receptor antigen binding proteins with increased affinity for the nfP2X7 receptor.

[0048]

[0057] The present invention provides a library of nucleic acid molecules produced from mutations in the antigen binding proteins or CDR and / or FR sequences described herein, wherein at least one nucleic acid molecule of the library encodes an antigen binding protein that binds to the nfP2X7 receptor.

[0049]

[0058] The invention provides a method for producing an antigen binding protein that binds to the nfP2X7 receptor described herein, comprising the step of expressing a nucleic acid described herein in a cell or animal described herein.

[0050]

[0059] The functional properties of the antigen binding proteins of the invention apply mutatis mutandis to the antibodies of the invention.

[0060] The antigen binding proteins described herein may be purified, substantially purified, isolated and / or recombinant.

[0051]

[0061] The antigen binding protein of the invention may be part of the supernatant harvested from the culture in which a hybridoma expressing the antigen binding protein of the invention is grown.

[0062] The present invention provides a method for preventing or treating a condition or disease associated with the expression of nfP2X7 in an individual, comprising providing an antigen binding protein, an immunoglobulin variable domain, an antibody, a dab, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule, or a multispecific antibody, a fusion protein, a conjugate or a pharmaceutical composition described herein to an individual in need of said condition or disease treatment.The disease or condition associated with the expression of nfP2X7 is preferably cancer.

[0052]

[0063] In another aspect, the invention also provides a method of treating or preventing cancer in a subject, comprising administering to the subject an antigen binding protein of the invention, thereby treating or preventing cancer in the subject. As used herein, a method of treating cancer includes a method of inhibiting, preventing, or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer.

[0053]

[0064] In another aspect, the present invention also provides the use of an antigen binding protein of the present invention in the manufacture of a medicament for the treatment or prevention of cancer in a subject.

[0065] In another aspect, the present invention provides an antigen binding protein or a pharmaceutical composition comprising an antigen binding protein of the invention for use in the treatment or prevention of cancer in a subject.

[0054]

[0066] Unless the context otherwise requires, as used herein, the term "comprise" and variations of that term, such as "comprising", "comprises" and "comprised", do not exclude further appended elements, components, integers or steps. The terms "comprising" and "including" are used interchangeably.

[0055]

[0067] 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 with reference to the accompanying drawings, in which:

[0056] Sequence information

[0068]

[0057] [Table 1-1]

[0058] [Table 1-2]

[0059] [Table 1-3]

[0060] [Table 1-4]

[0061] [Table 1-5]

[0062] [Table 1-6] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063]

[0069] 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.

[0064]

[0070] 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 with reference to the accompanying drawings, in which:

[0065]

[0071] Reference will now be made in detail to certain 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. Rather, 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.

[0066]

[0072] A variety of antigen-binding proteins, including single-domain heavy chain antibodies, are known that bind to dysfunctional P2X7 receptors. Although such antibodies are useful for binding to dysfunctional P2X7 receptors, they are often difficult to express, and even if they are successfully expressed, they typically aggregate, making these molecules difficult to use in clinical applications. One solution to this problem is to pair a single heavy chain with a light chain counterpart. However, it is difficult to identify the appropriate light chain pair for a given heavy chain without affecting the binding affinity to target antigen. The present inventors have identified suitable heavy-light chain combinations that have particular utility in binding to dysfunctional P2X7 receptor antigens and peptide antigens derived therefrom.

[0067] Overview and Definitions

[0073] Throughout this specification, unless specifically stated otherwise or unless the context otherwise requires, reference to a single step, composition, step, or composition shall be understood to encompass both one and more (i.e., one or more) of that step, composition, step, or composition. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to "a" includes both the singular and two or more, reference to "an" includes both the singular and two or more, reference to "the" includes both the singular and two or more, etc.

[0068]

[0074] Those skilled in the art will understand that the present invention is subject to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions, and compounds referred to or shown herein, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0069]

[0075] 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, and the present invention is in no way limited to the methods and materials described.

[0070]

[0076] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0077] The present invention should not be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention.

[0071]

[0078] For purposes of interpreting this specification, the following definitions shall apply and whenever necessary, terms used in the singular shall also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0072]

[0079] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0073]

[0080] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors), Current Protocols in It is described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all current editions).

[0074]

[0081] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989, Martin ("enhanced Chothia"; Mol Immunol. (2008) 45:3832-9; and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0075]

[0082] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as expressly endorsing both meanings or either meaning.

[0076]

[0083] As used herein, the term "derived from" shall be understood to indicate that the specified integer may be obtained from a particular source, but not necessarily directly from that source.

[0077]

[0084] "Purinergic receptor" generally refers to a receptor that uses purines (such as ATP) as a ligand.

[0085] "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: 17: MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVYEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNFRYAKYYKENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY

[0086] "P2X7 receptor" may be a functional receptor as described below, or may be a non-functional receptor. "P2X7 receptor" encompasses naturally occurring variants of P2X7 receptor. For example, P2X7 monomers are splice variants, allelic variants, and isoforms, including naturally occurring truncated or secreted forms (e.g., forms consisting of extracellular domain sequence or truncated forms thereof) of monomers forming P2X7 receptor, naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO: 17. 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: 17 may be substituted, deleted, or residues may be inserted.

[0078]

[0087] "Functional P2X7 receptor" generally refers to the type of P2X7 receptor that has a binding site or binding groove that binds ATP.When bound to ATP, the receptor forms a pore-like structure that allows calcium ions to enter cytosol, one result of which can be programmed cell death.In normal homeostasis, the expression of functional P2X7 receptor is mostly limited to cells that undergo programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages and monocytes.There can also be some expression of functional P2X7 receptor in erythrocytes.

[0079]

[0088] "Dysfunctional P2X7 receptor" (also called "non-functional" or (nf)P2X7) is a P2X7 receptor that does not respond to ATP, such that it cannot form an apoptotic pore under physiological conditions. Dysfunctional P2X7 receptor (or nfP2X7 receptor) generally refers to a type of P2X7 receptor in which one or more of the monomers have cis isomerization at Pro210 (according to SEQ ID NO: 17). 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 result of isomerization is that the receptor cannot bind ATP or binds ATP with a lower affinity than that otherwise observed between the receptor and ATP that does not contain isomerization at Pro210. Under such circumstances, the receptor cannot form a pore, which limits the extent to which calcium ions can enter the cytosol. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial, mesenchymal, neural, embryonic and hematopoietic cancers. As used herein, dysfunctional P2X7 is preferably understood to be P2X7 receptor that does not respond to ATP, such that it cannot form apoptotic pore under normal physiological conditions.As used herein, the term "dysfunctional P2X7 receptor" can be used interchangeably with the term "non-functional P2X7 receptor" or "nfP2X7 receptor".

[0080]

[0089] A "cancer-associated P2X7 receptor" is a P2X7 receptor that is generally found on cancer cells (including precancerous, neoplastic, malignant, benign, or metastatic cells) but not on non-cancerous or normal cells.

[0081]

[0090] The terms "E200", "E300", and "composite" epitopes refer to specific epitopes present in dysfunctional P2X7 receptors, one or more of which are bound by the antigen binding proteins of the present invention. "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NO: 18). "E300 epitope" generally refers to an epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO: 19). "Composite epitope" generally refers to an epitope formed from the juxtaposition of E200 and E300 epitopes or portions of these epitopes. An example of a composite epitope that includes E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 20).

[0082]

[0091] The term "anti-Ρ2X7 receptor antibody" or "antibody that binds to the Ρ2X7 receptor" refers to an antibody that can bind to the Ρ2X7 receptor with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting the Ρ2X7 receptor, typically a non-functional Ρ2X7 receptor or a cancer-associated Ρ2X7 receptor. Preferably, the degree of binding of the Ρ2X7 receptor antibody to an unrelated protein is less than about 10% of the binding of the antibody to the Ρ2X7 receptor, as measured, for example, by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Biacore, or flow cytometry. In certain embodiments, the antibody that binds to the Ρ2X7 receptor has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. Anti-nfP2X7 receptor antibodies generally have some or all of these serological characteristics and bind to dysfunctional but not functional P2X7 receptors.

[0083]

[0092] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that is not associated with the natural binding components associated with it in its natural state based on its origin or source of origin and is substantially free of other proteins from the same source. The protein can be rendered substantially free of natural binding components or can be substantially purified by isolation using protein purification methods known in the art. "Substantially purified" means that the protein is substantially free of contaminants, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminants.

[0084]

[0093] The term "recombinant" shall be understood to mean a product of artificial genetic recombination. Thus, in the context of recombinant proteins containing an antibody antigen-binding domain, the term does not encompass naturally occurring antibodies in a subject's body that are the product of natural recombination occurring during B-cell maturation. However, if such antibodies are isolated, such antibodies should be considered as isolated proteins that contain an antibody antigen-binding domain. Similarly, if a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein that contains an antibody antigen-binding domain. Recombinant proteins also encompass proteins expressed by artificial recombinant means, for example, when the protein expressed by artificial recombinant means is within the cell, tissue, or subject in which it is expressed.

[0085]

[0094] The term "protein" shall be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains that are covalently or non-covalently bonded to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bonded using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. A protein may include one or more unnatural amino acids.

[0086]

[0095] It will be understood from the previous paragraph that the term "polypeptide" or "polypeptide chain" means a series of consecutive amino acids linked by peptide bonds.

[0096] As used herein, the term "antigen-binding domain" refers to the region of an antibody that is capable of specifically binding to an antigen, i.e., the V H Or V L or an Fv comprising both a VH and a VL. The antigen-binding domain need not be in the context of a whole antibody, but may for example be in isolated form (e.g., a domain antibody) or in another form such as, for example, an scFv, as described herein.

[0087]

[0097] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens via the antigen-binding domain contained within the Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, analogous humanized antibodies, half antibodies, bispecific antibodies).

[0088]

[0098] Antibodies generally contain a constant domain, which may be arranged in a constant region or constant fragment or fragment crystallizable (Fc). An exemplary form of an antibody contains a four-chain structure as its basic unit. A full-length antibody contains two covalently linked heavy chains (about 50-70 kDa) and two light chains (about 23 kDa each). The light chains generally contain a variable region (if present) and a constant domain, and in mammals are either kappa or lambda light chains. The heavy chains generally contain a variable region and one or two constant domains linked to an additional constant domain by a hinge region. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains as well as the heavy and light chains are linked by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds may vary depending on the type of antibody. Each chain contains an N-terminal variable region (V, each about 110 amino acids long). H or V L ) and one or more constant domains at the C-terminus. L ) is the first constant domain of the heavy chain (C H1 ) and are disulfide bonded. The light chain variable region is aligned with the heavy chain variable region. Antibody heavy chains have two or more additional C H domains (e.g., CH2, CH3, etc.), H1 Constant domain and C H2 The constant domains may include a hinge region between them. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as human) antibody. In one example, the antibody heavy chain is missing a C-terminal lysine residue. In one example, the antibody is humanized, near-humanized, chimerized, CDR-grafted, or deimmunized.

[0089]

[0099] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment of the antibody. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domain may be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0090]

[0100] As used herein, "variable region" refers to a portion of the light and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen and includes the amino acid sequences of the complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) together with three CDRs. H V refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0091]

[0101] As used herein, the term "complementarity determining region" (synonym CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in an antibody variable region whose presence is a major contributor to specific antigen binding. Each variable region domain (V H or VL) typically have three CDRs identified as CDR1, CDR2 and CDR3. H The CDRs of are also referred to herein as CDR H1, CDR H2 and CDR H3, respectively, and CDR H1 is H CDR1 corresponds to V H CDR2 corresponds to V H Similarly, V L The CDRs of are referred to herein as CDR L1, CDR L2 and CDR L3, respectively, and CDR L1 is V L CDR1 corresponds to V L CDR2 corresponds to V LIn one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to the CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but includes all numbering systems, including the standard numbering system, or the numbering system of Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; the numbering system of Honnegher and Plukthun J. Mol. Biol. 309:657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25:206-211, 1997.

[0092]

[0102] In one example, CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or any one or more of these amino acids are replaced with another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9:133-139, 1995, established that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0093]

[0103] "Framework regions" (FR) are those variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, and FR H1 is V H FR1 corresponds to V, FR H2 corresponds to V H FR2 corresponds to V, FR H3 corresponds to V H FR3 corresponds to V, FR H4 corresponds to V H Similarly, V L The FRs are referred to herein as FR L1, FR L2, FR L3 and FR L4, respectively, and FR L1 is V L FR1 corresponds to V, and FR L2 corresponds to V L FR2 corresponds to V, and FR L3 corresponds to V L FR3 corresponds to V, FR L4 corresponds to V L Compatible with FR4.

[0094]

[0104] As used herein, the term "Fv" refers to a V polypeptide, whether composed of multiple polypeptides or a single polypeptide. L and V H The term "antigen-binding domain" should be understood to mean any protein to which V binds and forms a complex having an antigen-binding domain, i.e. capable of specifically binding to an antigen. H and V L The V may be a single polypeptide chain or different polypeptide chains. Furthermore, an Fv of the invention (and any protein of the invention) may have multiple antigen binding domains that may or may not bind to the same antigen. This term shall be understood to encompass fragments derived directly from antibodies, and proteins corresponding to such fragments produced using recombinant means. In some instances, the V H is the heavy chain constant domain (C H ) 1 and / or V L is the light chain constant domain (C LExemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, an scFv, a diabody, a triabody, a tetrabody, or a higher order complex, or any of the foregoing linked to a constant region or a domain thereof, e.g., a CH2 or CH3 domain, e.g., a minibody.

[0095]

[0105] A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of whole antibody with the enzyme papain to produce a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be produced by treating whole antibody with pepsin, followed by reduction, to produce a fragment consisting of an intact light chain and a portion of the heavy chain. H and a portion of the heavy chain containing a single constant domain. Two Fab' fragments are obtained for each antibody treated in this way. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked by two disulfide bonds and can be obtained by treating the whole antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing two Fab fragments linked, for example, using a leucine zipper or CH3 domain. A "single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0096]

[0106] As used herein, the term "bind" in reference to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the antigen. For example, an antibody recognizes and binds to a particular protein structure rather than the entire protein. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free unlabeled "A") will reduce the amount of labeled "A" bound to the antibody in a reaction containing labeled "A" and the protein.

[0097]

[0107] As used herein, the term "specifically bind" or "bind specifically" shall be understood to mean that the antigen-binding protein of the present invention reacts with or binds to a particular antigen or a cell expressing the same more frequently, more rapidly, for a longer period of time, and / or with greater affinity than another antigen or cell. For example, the antigen-binding protein binds to a dysfunctional P2X7 receptor with significantly greater affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than it binds to other related molecules, such as other purinergic receptors, and in particular than it binds to a functional P2X7 receptor. In an example of the present invention, the antigen-binding protein "specifically binds" to a dysfunctional P2X7 receptor with at least 1.5-fold or 2-fold or more (e.g., 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold) affinity than it binds to a functional P2X7 receptor. Generally, but not necessarily, reference to binding refers to specific binding, and each term will be understood to expressly support the other.

[0098]

[0108] As used herein, the term "not detectably bind" shall be understood to mean that an antigen-binding protein, e.g., an antibody, binds to a candidate antigen at a level that is 10%, or less than 8%, or 6%, or 5% above background. Background may be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen-binding protein is immobilized and contacted with the antigen.

[0099]

[0109] As used herein, the term "does not significantly bind" shall be understood to mean that the level of binding of the antigen binding protein of the invention to the polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the antigen binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control polypeptide. The level of binding is detected using a biosensor assay (e.g., Biacore or Blitz) in which the antigen binding protein is immobilized and contacted with the antigen.

[0100]

[0110] As used herein, the term "epitope" (synonym "antigenic determinant") shall be understood to mean the region of a dysfunctional P2X7 receptor to which an antigen binding protein, including the antigen binding domain of an antibody, binds. Unless otherwise defined, the term is not necessarily limited to the particular residues or structures that the antigen binding protein contacts. For example, the term includes the region spanning the amino acids that the antigen binding protein contacts, and 5-10 (or more) or 2-5 or 1-3 amino acids outside of this region. In some instances, an epitope includes a series of discontinuous amino acids that are located close to each other when the antigen binding protein is folded, i.e., a "conformational epitope." One of skill in the art will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groups of molecules, such as sugar, phosphoryl, or sulfonyl side chains, and in certain instances may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0101]

[0111] As used herein, the terms "preventing," "prevent" or "prevention" include administering an antigen binding protein of the invention to thereby stop or delay the onset of at least one symptom of a condition. The term also encompasses treatment of a subject in remission to prevent or delay recurrence.

[0102]

[0112] As used herein, the terms "treating," "treat," or "treatment" include administering an antigen binding protein described herein to thereby reduce or eliminate at least one symptom of a particular disease or condition.

[0103]

[0113] As used herein, the term "subject" shall be understood to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0104]

[0114] The terms "modified cell" and "genetically modified cell" as used herein may be used interchangeably to denote a cell that contains and / or expresses an exogenous gene or nucleic acid sequence that in turn modifies the genotype or phenotype of the cell or its progeny.

[0105] antibody

[0115] In one example, the antigen binding protein according to any of the examples described herein is an antibody.

[0106]

[0116] The method of generating antibody is known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).Generally, in such a method, dysfunctional P2X7 receptor or its region (e.g., extracellular region) or its immunogenic fragment or epitope or the cell expressing and presenting them (i.e., immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to non-human animals, such as mice, chickens, rats, rabbits, guinea pigs, dogs, horses, cows, goats, or pigs.Immunogens can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0107]

[0117] The production of polyclonal antibodies can be monitored by taking blood from the immunized animal at various times after immunization. One or more additional immunizations may be performed if necessary to achieve the desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. Once the desired level of immunogenicity is obtained, the immunized animal is bled, and serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0108]

[0118] Monoclonal antibody is one exemplary form of antibody contemplated by the present invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population that can bind to the same antigen, for example, the same epitope within the antigen. This term is not intended to be limiting with respect to the source of the antibody or the way the antibody is made.

[0109]

[0119] For the production of mAbs, any one of a number of known techniques may be used, eg, the procedures exemplified in US 4,196,265 or Harlow and Lane (1988), supra.

[0110]

[0120] For example, suitable animal is immunized with immunogen under sufficient conditions to stimulate antibody-producing cells.Exemplary animals include rodents such as rabbits, mice and rats.For example, mice genetically modified to express human antibodies that do not express mouse antibodies can also be used to generate the antibody of the present invention (for example, as described in WO2002 / 066630).

[0111]

[0121] After immunization, somatic cells that have the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in mAb generation protocols. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from peripheral blood samples. The B cells from the immunized animal are then fused with cells of immortal myeloma cells, generally derived from the same species as the animal that was immunized with the immunogen.

[0112]

[0122] The hybrids are amplified by culturing in selective medium containing drugs that block de novo synthesis of nucleotides in tissue culture medium. Exemplary drugs are aminopterin, methotrexate, and azaserine.

[0113]

[0123] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunoassays, etc.).

[0114]

[0124] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to generate cell lines secreting MAbs (eg as described in Largaespada et al., J. Immunol. Methods. 197:85-95, 1996).

[0115]

[0125] Antibodies can also be produced or isolated by screening display libraries, such as phage display libraries, for example as described in US6300064 and / or US5885793. For example, the present inventors have isolated fully human antibodies from phage display libraries.

[0116]

[0126] The antibody of the invention may be a synthetic antibody, for example, the antibody is a chimeric antibody, a humanized antibody, a human antibody, a similar humanized antibody, a primatized antibody, or a deimmunized antibody. Antibody-binding domain-containing proteins Single Domain Antibodies

[0127] In some examples, the protein of the present invention is or comprises a single domain antibody (used interchangeably with the term "domain antibody" or "sdAb"). A single domain antibody is a single polypeptide chain that includes all or a portion of the heavy chain variable region of an antibody. In certain examples, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA, see, e.g., US6248516).

[0117] Diabodies, triabodies, tetrabodies

[0128] In some examples, the proteins of the invention are or include diabodies, triabodies, tetrabodies, or higher order protein complexes, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0118]

[0129] For example, a diabody is a protein that includes two binding polypeptide chains, each of which has the structure V L -XV H or V H -XV L (where V L is the antibody light chain variable region, V H is an antibody heavy chain variable region, and X is the V H and V L a linker that contains insufficient residues to allow binding of the VH of one polypeptide chain (or formation of an Fv) or is absent; L to form an antigen-binding domain, i.e., an Fv molecule capable of specifically binding to one or more antigens. L and V H may be the same for each polypeptide chain, or V L and V H may be different on each polypeptide chain to form bispecific diabodies (i.e., containing two Fvs with different specificities).

[0119] Single chain Fc (scFv)

[0130] Those skilled in the art will appreciate that scFvs are VFs of a single polypeptide chain. H and V L domains for antigen binding (i.e., the V domains of a single polypeptide chain) H and V L V that allows the scFv to form the desired structure (for the scFvs to bind to each other to form the Fv) H and V LFor example, the linker may comprise more than 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFvs.

[0120]

[0131] The present invention relates to a method for treating a cancer H FR and V L Also contemplated are disulfide-stabilized Fvs (or diFvs or dsFvs) in which cysteine ​​residues are introduced into the FR of and linked by disulfide bonds to produce a stable Fv.

[0121]

[0132] Alternatively, or in addition, the invention encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by non-covalent or covalent bonds, e.g., by a leucine zipper domain (e.g., from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to form both scFvs and enable them to bind antigen, e.g., as described in US20060263367.

[0122] Heavy Chain Antibodies

[0133] Heavy chain antibodies are structurally different from many other forms of antibodies in that they contain heavy chains but no light chains.Therefore, these antibodies are also called "heavy chain-only antibodies".Heavy chain antibodies are found, for example, in camelids and cartilaginous fish (also called IgNAR).

[0123]

[0134] The heavy chain variable region (V H domains), and the light chain variable region (V L To distinguish them from the variable regions found in naturally occurring heavy chain antibodies, the variable regions are generally called "V domains" in camelid antibodies. HH domain', and in IgNARs they are called V-NARs.

[0124]

[0135] A general description of camelid heavy chain antibodies and their variable regions and methods for their production and / or isolation and / or use can be found, inter alia, in the following references: WO94 / 04678, WO97 / 49805 and WO97 / 49805.

[0125]

[0136] An overview of elasmobranch heavy chain antibodies and their variable regions, as well as methods for their production and / or isolation and / or use, can be found, inter alia, in WO2005 / 118629.

[0126] Other antibodies and proteins containing their antigen-binding domains

[0137] The present invention relates to other antibodies and proteins comprising their antigen-binding domains: (i) "lock and key" bispecific proteins as described in US5731168; (ii) heteroconjugate proteins, e.g. as described in US4676980; (iii) Heteroconjugate proteins produced using chemical cross-linkers, e.g., as described in US4676980; (iv) Fab3 (e.g., as described in EP19930302894) We also plan to do the following:

[0127]

[0138] In any of the above antibody structures, the binding domains of the proteins may be linked via a linker. For example, in the context of an scFv, the linker between VH and VL may be a combination of one or more amino acid residues to provide a flexible linker. Those skilled in the art will be familiar with suitable linker sequences to utilize. In a typical example, the linker consists of one or more glycine and serine residues. In one example, the linker may include the sequence G4S (i.e., GGGGS), etc. It will be understood that the linker may also include repeats of glycine and serine residues, such as (G4S)3, although any modification thereof, such as (G4S)3T, may also be suitable.

[0128] Protein mutations

[0139] The present invention also provides antigen binding proteins or nucleic acids encoding antigen binding proteins having at least 80% identity to the sequences disclosed herein. In one example, an antigen binding protein or nucleic acid of the invention comprises a sequence that is at least about 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.

[0129]

[0140] Alternatively, or in addition, the antigen binding protein may be any of the V H or V L The CDRs (e.g., three CDRs) are at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the CDRs of

[0130]

[0141] In another example, a nucleic acid of the invention comprises a sequence that is at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence that encodes an antigen binding protein having a function according to any of the examples described herein. The invention also encompasses nucleic acids encoding antigen binding proteins of the invention that differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0131]

[0142] Percent identity of nucleic acids or polypeptides is determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.

[0132]

[0143] The present invention also contemplates nucleic acids that hybridize under stringent hybridization conditions to nucleic acids encoding antigen-binding sites as described herein. "Moderate stringency" is defined herein as hybridization and / or washing carried out at a temperature ranging from 45°C to 65°C in 2xSSC buffer, 0.1% (w / v) SDS, or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing carried out at 0.1xSSC buffer, 0.1% (w / v) SDS, or lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions. When a particular level of stringency is referred to herein, it encompasses equivalent conditions using washing / hybridization solutions other than SSC that are known to those of skill in the art. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid dissociate (also known as the melting temperature, or Tm) are known in the art. A temperature similar (e.g., within 5° C. or within 10° C.) or equal to the Tm of the nucleic acid is considered high stringency. Moderate stringency should be considered within 10° C.-20° C. or within 10° C.-15° C. of the calculated Tm of the nucleic acid.

[0133]

[0144] The present invention also contemplates variants of the antigen binding proteins of the present invention that contain one or more conservative amino acid substitutions compared to the sequences shown herein. In some examples, the antigen binding proteins contain 10 or fewer conservative amino acid substitutions, for example, 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydrophobicity and / or hydrophilicity.

[0134]

[0145] Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indexes are described, for example, in Kyte and Doolittle J. Mol. Biol., 157:105-132, 1982, and hydrophilic indexes are described, for example, in US4554101.

[0135]

[0146] The present invention also contemplates non-conservative amino acid changes.For example, of particular interest are the substitution of charged amino acid with another charged amino acid, and neutral or positively charged amino acid.In some examples, antigen binding protein comprises 10 or less non-conservative amino acid substitutions, for example, 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1.

[0136]

[0147] In one example, the mutations occur within the FRs of the antigen binding domain of the antigen binding protein of the invention, hi another example, the mutations occur within the CDRs of the antigen binding protein of the invention.

[0148] Exemplary methods for producing mutant versions of an antigen binding protein include: mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7:18, 2007; and WO1999 / 058661), introduction of a nucleic acid encoding the polypeptide into a mutator cell, e.g., XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling, as disclosed, for example, in Stemmer, Nature 370:389-91, 1994, and Site-directed mutagenesis, as described, for example, in Dieffenbach (Eds.) and Dveksler (Eds.) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995) Includes:

[0137]

[0149] Exemplary methods for determining the biological activity, e.g., antigen binding, of the variant antigen binding proteins of the invention will be apparent to one of skill in the art and / or are described herein. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0138] Constant region

[0150] The present invention encompasses antigen binding proteins and / or antibodies described herein that comprise an antibody constant region, including an antigen-binding fragment of an antibody fused to Fc.

[0139]

[0151] The sequence of the constant region useful for producing the protein of the present invention can be obtained from several different sources. In some examples, the constant region of the protein or a portion thereof is derived from human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is a human isotype IgG4 or stabilized IgG4 constant region.

[0140]

[0152] In one example, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to a native or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region-containing protein are known in the art and / or described herein.

[0141]

[0153] In one example, the Fc region is an IgG4 Fc region (i.e., derived from an IgG4 constant region), such as a human IgG4 Fc region. The sequences of suitable IgG4 Fc regions will be apparent to those of skill in the art and / or are available in public databases (e.g., available from the National Center for Biotechnology Information).

[0142]

[0154] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange or to form half antibodies or to form half antibodies. "Fab arm exchange" refers to a type of protein modification of human IgG4 in which the IgG4 heavy chain and associated light chain (half molecule) are exchanged with a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. "Half antibodies" occur when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0143]

[0155] In one example, the stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001, and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally serine. After replacement of the proline with serine, the IgG4 hinge region contains the sequence CPPC. In this regard, the skilled artisan will be aware that the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, which confers flexibility to the two Fab arms of the antibody. The hinge region contains the cysteine ​​residues involved in the inter-heavy chain disulfide bond. The hinge region is generally defined as extending from Glu226 to Pro243 of human IgG1 according to the Kabat numbering system. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond in the same position (see, for example, WO2010 / 080538).

[0144]

[0156] An additional example of a stabilized IgG4 antibody is an antibody in which the arginine at position 409 (according to the EU numbering system) in the heavy chain constant region of human IgG4 is replaced with lysine, threonine, methionine, or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., the CPPC sequence) (as described above).

[0145]

[0157] In another example, the Fc region is a region modified to reduce effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region that includes substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region that includes one or more of the following modifications E233P, L234V, L235A, and a deletion of G236, and / or one or more of the following modifications A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177:1129-1138, 2006; and / or Hezareh J Virol; 75:12161-12168, 2001.

[0146]

[0158] In another example, the Fc region is a chimeric Fc region comprising, for example, at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, and the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0147] Additional modifications

[0159] The present invention also contemplates additional modifications to antibodies or antigen binding proteins comprising the Fc region or constant region.

[0148]

[0160] For example, the antibody comprises one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody comprises an Fc region that comprises one or more amino acid substitutions that increase the affinity of the Fc region for neonatal Fc region (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to promote Fc / FcRn binding in endosomes. In one example, the Fc region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, to promote re-release of Fc into blood after cellular recycling. These amino acid substitutions are useful for extending the half-life of the protein by reducing clearance from blood.

[0149]

[0161] Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.

[0150] Protein production

[0162] In one example, an antigen binding protein described herein according to any of the examples is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or known in the art.

[0151] Recombinant expression

[0163] In another example, the antigen binding proteins described herein according to any of the examples are recombinant.

[0152]

[0164] In the case of recombinant proteins, the nucleic acid encoding the recombinant protein may be cloned into an expression construct or vector, and then transfected into a host cell that does not otherwise produce the protein, for example, E. coli cells, yeast cells, insect cells, or mammalian cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells. Exemplary cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning methods that achieve these goals are known in the art and are described, for example, in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions), or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art. See, for example, US4816567 or US5530101.

[0153]

[0165] Once isolated, the nucleic acid operably linked to a promoter is inserted into an expression construct or vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0154]

[0166] As used herein, the term "promoter" should be understood in its broadest context, including the transcriptional control sequences of genomic genes, including the TATA box or initiation element required for accurate transcription initiation, with or without additional control elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers, and silencers) that alter the expression of the nucleic acid, for example, in response to developmental and / or external stimuli, or in a tissue-specific manner. In this context, the term "promoter" is also used to describe recombinant, synthetic, or fusion nucleic acids, or derivatives, that confer, activate, or enhance the expression of the nucleic acid to which the promoter is operably linked. Exemplary promoters may contain additional copies of one or more specific control elements to further enhance the expression of the nucleic acid and / or alter spatial and / or temporal expression.

[0155]

[0167] As used herein, the term "operably linked to" refers to positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0156]

[0168] Many vectors are available for expression in cells. Vector components generally include, but are not limited to, one or more of the following: signal sequence, protein coding sequence (e.g., obtained from the information provided herein), enhancer element, promoter, and transcription termination sequence. Those skilled in the art will be aware of suitable sequences for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, alpha-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0157]

[0169] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), alpha-myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, beta-actin promoter; hybrid control elements including CMV enhancer / beta-actin promoter, or immunoglobulin promoters or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0158]

[0170] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0159]

[0171] Means of introducing isolated nucleic acid or expression constructs containing it into cells for expression are known to those skilled in the art. The method used for a given cell depends on known successful methods. Means of introducing recombinant DNA into cells include microinjection, DEAE-dextran mediated transfection, liposome mediated transfection, such as by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microprojectile bombardment, such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0160]

[0172] The host cells used to produce the proteins can be cultured in a variety of media depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing the other cell types discussed herein are known in the art.

[0161] Protein isolation

[0173] Methods for isolating proteins are known in the art and / or described herein.

[0162]

[0174] If the antigen-binding protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, may be included in any of the foregoing steps to inhibit protein degradation, and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively, or in addition, the supernatant can be filtered and / or separated from the cells expressing the protein, e.g., using continuous centrifugation.

[0163]

[0175] Antigen binding proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., Protein A affinity chromatography or Protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example, in WO99 / 57134 or in Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0164]

[0176] Those skilled in the art will also be aware that proteins can be modified to include tags that facilitate purification or detection, such as polyhistidine tags, such as hexahistidine tags, or influenza virus hemagglutinin (HA) tags, or simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, proteins that include hexa-his tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively, or in addition, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0165] Analysis of antigen-binding protein binding

[0177] It will be clear to those skilled in the art that the antigen-binding protein of the present invention binds to dysfunctional P2X7 receptor. Methods for evaluating binding to proteins are known in the art, for example as described in Scopes (Protein purification: principles and practice, 3rd edition, Springer Verlag, 1994). Such methods generally involve immobilizing an antigen-binding protein and contacting it with a labeled antigen (dysfunctional P2X7 receptor). After washing to remove non-specific binding proteins, the amount of label and, as a result, bound antigen is detected. Of course, the antigen-binding protein may be labeled and the antigen may be immobilized. Panning-type assays may also be used. Alternatively, or in addition, surface plasmon resonance assays may be used.

[0166]

[0178] In some cases, the dissociation constant (Kd), binding constant (Ka) and / or affinity constant (KD) of the antigen-binding protein immobilized to dysfunctional P2X7 receptor or its epitope is determined. In one example, the "Kd" or "Ka" or "KD" of dysfunctional P2X7 receptor-binding protein is measured by radioactive or fluorescently labeled dysfunctional P2X7 receptor ligand binding assay. For "Kd", this assay equilibrates antigen-binding protein with a minimum concentration of labeled dysfunctional P2X7 receptor or its epitope in the presence of a series of titrations of unlabeled dysfunctional P2X7 receptor. After washing to remove unbound dysfunctional P2X7 receptor or its epitope, the amount of label is determined. The amount of label indicates the Kd of the protein.

[0167]

[0179] According to another example, K d , K a or K D is measured using a surface plasmon resonance assay with immobilized dysfunctional P2X7 receptor or a region thereof or immobilized antigen binding protein, for example, using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ).

[0168]

[0180] In some embodiments, the function of the P2X7 receptor may be reduced by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 99% when bound by an antigen binding protein, modified receptor or immunogen containing the same of the present invention.

[0169] Condition to be treated

[0181] The antigen binding proteins of the present invention are particularly useful in the manufacture of medicaments (eg, antibodies, antibody-drug conjugates) for the treatment of cancers which have dysfunctional P2X7 receptors on their cell surface.

[0170]

[0182] Examples of cancers that can be treated by the methods of the present invention include precancerous and neoplastic diseases. Broad examples include breast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, hepatocellular / cholangiocarcinomas, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinomas, melanomas, atypical fibroxanthomas, seminomas, non-seminomas, stromal Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumors.

[0171]

[0183] Examples of specific cancers include adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDS-related cancer, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adrenal cortical carcinoma, primary myelofibrosis, alopecia, alveolar soft part sarcoma, ameloblastoma, angiokeratoma, eosinophilic angiolymphocytosis, sclerosing hemangioma, hemangiomatosis, apdoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestinal cancer, brain stem glioma, brain and CNS tumors, breast cancer, branchiomas, CNS tumors, Carcinoid tumor, cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinoma (e.g., Walker's carcinoma, basal cell carcinoma, basosquamous cell carcinoma, Brown-Pierce carcinoma, ductal carcinoma, Ehrlich's carcinoma, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, oat cell carcinoma, papillary carcinoma, scirrhous adenocarcinoma, bronchiolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma, and transitional cell carcinoma), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodes phyllodies), cementoma, chordoma, schistoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangiocarcinoma, cholangiocarcinoma, cholesteatoma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine carcinoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancercancer), gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioneuroma, glioma, glomus hemangioma, granulosa cell tumor, hemispheric ovarian tumor, hematologic malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, angiosarcoma, histiocytic disorder, malignant histiocytosis, histiocytoma, hepatoma, hidradenoma, hondrosarcoma, immunoproliferative small), opoma, ontraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leigomyosarcoma, leukemia (e.g., B cell, mixed cell, null cell, T cell, chronic T cell, HTLV-II-associated, lymphoma) lymphangiosarcoma, acute lymphocytic, chronic lymphocytic, mast cell, and myeloid), leukemia, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangiomas, lymphangiocytomas, lymphangiomas, lymphangiomas, lymphangiosarcomas, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer cancer), multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, malignant carcinoid syndrome, carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesothelioma, myoblastoma, fibroid, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen chromosomal instability syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g., bone, breast, digestive system, colorectal, liver), eye cancercancer, oesophageal cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, paraganglioma, nonchromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogenes, rare cancers and related diseases, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thompson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), stomach cancer These include, but are not limited to, synovial sarcoma, sarcoma (e.g., Ewing experimental sarcoma, Kaposi's sarcoma, and mast cell sarcoma), Sertoli cell tumor, synovium, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), choriocarcinoma, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary tract cancer, uroplakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0172]

[0184] Other diseases and conditions include various inflammatory conditions, such as proliferative factors. Specific examples include acne, angina, arthritis, aspiration pneumonia, disease, empyema, gastroenteritis, inflammation, intestinal flu, nee, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, pelvic inflammatory disease (pid), pleurisy, raw throat, flushing, redness, sore throat, stomach flu and urinary tract infection, chronic inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyneuropathy, or chronic inflammatory demyelinating polyradiculoneuropathy.

[0173] composition

[0185] In some examples, the antigen binding proteins described herein can be administered orally, parenterally, by inhalation spray, by adsorption, by absorption, topically, rectally, nasally, buccally, vaginally, intracerebroventricularly, via an implanted reservoir of a dosage formulation containing a conventional non-toxic pharma- ceutical acceptable carrier, or by any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0174]

[0186] Methods for preparing antigen binding proteins into a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the United States Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0175]

[0187] The pharmaceutical compositions of the invention are particularly useful for parenteral administration, such as intravenous administration or administration into a cavity or joint of a body cavity or organ. A composition for administration usually comprises a solution of the antigen-binding protein dissolved in a pharma- ceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers, such as buffered saline, may be used. The composition may contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., as necessary to approximate physiological conditions. The concentration of the antigen-binding protein of the invention in these formulations may vary widely and is selected primarily based on fluid volume, viscosity, weight, etc., according to the particular method of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous solvents, such as mixed oils and ethyl oleate, may also be used. Liposomes may also be used as carriers. The solvent may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0176]

[0188] The antigen binding proteins of the present invention can be formulated for local or topical administration, for example, for topical application to the skin or tissue in need of treatment. Formulations for topical administration typically include a topical solvent combined with an active agent, with or without additional optional ingredients. The pharmaceutical compositions of the present invention may be in the form of a spray, cream, gel, lotion, etc. for topical administration.

[0177]

[0189] Suitable topical solvents and additional components are well known in the art, and it will be clear that the choice of solvent depends on the specific physical form and delivery method.Topical solvents include organic solvents such as alcohol (e.g., ethanol, isopropyl alcohol, or glycerin), glycols such as butylene, isoprene, or propylene glycol, fatty alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin, lipid-based materials such as fatty acids, acylglycerols (including oils such as mineral oils, and fats of natural or synthetic origin), phosphoglycerides, sphingolipids, and waxes, protein-based materials such as collagen and gelatin, silicone-based materials (both non-volatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

[0178]

[0190] The composition may further comprise one or more components suitable for improving the stability or effectiveness of the applied formulation, such as stabilizers, suspending agents, emulsifiers, viscosity adjusting agents, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizing agents, and sustained release materials.Examples of such components are described in Martindale-The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.The formulation may comprise microcapsules such as hydroxymethylcellulose microcapsules or gelatin microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

[0179]

[0191] Topical formulations can be prepared in various physical forms, including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays, and skin patches. The physical appearance and viscosity of such forms can be influenced by the presence and amount of emulsifiers and viscosity modifiers present in the formulation. Solids are generally hard and cannot be poured, and are usually formulated as bars or sticks, or in particulate form. Solids can be opaque or transparent, and may optionally contain solvents, emulsifiers, moisturizers, emollients, fragrances, pigments / colorants, preservatives, and other active ingredients that enhance or strengthen the effectiveness of the final product. Creams and lotions are often similar to each other, differing mainly in their viscosities. Both lotions and creams can be opaque, translucent, or transparent, and often contain emulsifiers, solvents, and viscosity modifiers, as well as moisturizers, emollients, fragrances, pigments / colorants, preservatives, and other active ingredients that enhance or strengthen the effectiveness of the final product.

[0180]

[0192] The viscosity of gels can be prepared ranging from thick or high to thin or low. Like lotion and cream formulations, these formulations may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, pigments / colorants, preservatives, and other active ingredients that enhance or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, pigments / colorants, preservatives, and other active ingredients that enhance or enhance the efficacy of the final product.

[0181]

[0193] Emulsifiers used in topical formulations include, but are not limited to, ionic emulsifiers, non-ionic emulsifiers such as cetearyl alcohol, polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate, and glyceryl stearate. Suitable viscosity modifiers include, but are not limited to, protective colloids or non-ionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. Gel compositions can be formed by the addition of gelling agents such as chitosan, methylcellulose, ethylcellulose, polyvinyl alcohol, polyquaternium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer, or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants.For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used in topical formulations.

[0182]

[0194] Preservatives include, but are not limited to, antimicrobial agents such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate / ascorbic acid, and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate, and mineral oil. Suitable fragrances and colorants include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in the topical formulations include, but are not limited to, abrasives, absorbents, anti-caking agents, anti-foaming agents, anti-static agents, astringents (such as American witch hazel), alcohols, and herbal extracts such as chamomile extract, binders / excipients, buffers, chelating agents, film formers, conditioning agents, propellants, opacifying agents, pH adjusters, and protectants.

[0183]

[0195] Typical delivery methods for topical compositions include application using the fingers, application using a physical applicator such as a cloth, tissue, swab, stick, or brush, spraying, including mist, aerosol, or foam spray, drop application, sprinkling, dipping, and rinsing. Controlled release vehicles can also be used, and the compositions can be formulated for transdermal administration (e.g., as a transdermal patch).

[0184]

[0196] Once formulated, the antigen-binding proteins of the present invention are administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically / prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although other pharma- ceutically acceptable forms are also contemplated (e.g., tablets, pills, capsules, or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms, etc.). Pharmaceutical "slow-release" capsules or compositions can also be used. Slow-release formulations are generally designed to provide a constant drug level over an extended period of time and can be used to deliver the antigen-binding proteins of the present invention.

[0185]

[0197] WO2002 / 080967 describes compositions and methods for administering aerosolized compositions which are also suitable for administration of antigen binding proteins of the invention, eg containing antibodies for the treatment of asthma.

[0186] Dosage and timing

[0198] The appropriate dosage of the antigen-binding protein of the present invention will vary depending on the specific antigen-binding protein, the condition being treated, and / or the subject being treated. It is within the ability of a skilled physician to determine the appropriate dosage, for example, by starting with a suboptimal dosage and incrementally modifying the dosage to determine the optimal or useful dosage. Alternatively, to determine the dosage appropriate for treatment / prophylaxis, it may be desirable to determine whether the appropriate dose is the ED of the active compound with little or no toxicity. 50 Data from cell culture assays or animal studies that are within a circulating concentration range including the IC50, IC60, IC70, IC80, IC90, IC100, IC110, IC120, IC130, IC240, IC250, IC260, IC310, IC420, IC50, IC60, IC80, IC110, IC120, IC250, IC130, IC260, IC140, IC250, IC150, IC260, IC160, IC270, IC170, IC280, IC310, IC270, IC180, IC280, IC190, IC290, IC290, IC310, IC420, IC50, IC60, IC100, IC250, IC120, IC260, IC130, IC270, IC140, IC280, IC150, IC290, IC290, IC310, IC280, IC130, IC290, IC290, IC310, IC420, IC50, IC60, IC140, IC290, IC290, IC150, IC290, IC290, IC310, IC290, IC100, IC290, IC310, IC420, IC50, IC60, IC100, IC290, IC290, IC100, IC290, IC100, IC290, IC100, IC290, IC310, IC420, IC50, IC60, IC100, IC290, IC290, IC100, IC290, IC310, IC420, IC50, IC60, IC100, IC290, IC290, IC310, IC420 50 Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration or amount of compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0187]

[0199] In some examples, the methods of the present invention comprise the step of administering a prophylactically or therapeutically effective amount of a protein described herein.

[0200] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces or suppresses one or more symptoms of a clinical condition described herein below the level observed and recognized as a clinical diagnostic or clinical feature of the condition. The amount administered to a subject will depend on the specific characteristics of the condition being treated, the type and stage of the condition being treated, the method of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and weight. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. Thus, the term should not be construed to limit the invention to a specific amount, such as weight or amount of protein, but rather the invention encompasses any amount of antigen binding protein sufficient to achieve the stated result in a subject.

[0188]

[0201] As used herein, the term "prophylactically effective amount" shall be understood to mean a sufficient amount of protein to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. One of skill in the art will be aware that such amounts will vary depending, for example, on the particular antigen binding protein administered, and / or the particular subject, and / or the type or severity or level of the condition, and / or predisposition (genetic or otherwise) to the condition. Thus, this term should not be construed to limit the invention to a particular amount, e.g., weight or amount of antigen binding protein, but rather the invention encompasses any amount of antigen binding protein sufficient to achieve the stated result in a subject.

[0189] kit

[0202] The invention further includes kits comprising one or more of the following: (i) an antigen-binding protein of the invention or an expression construct encoding same; (ii) the cell of the present invention, (iii) a conjugate of the present invention, or (iii) A pharmaceutical composition of the present invention.

[0190]

[0203] In the case of a kit for detecting a dysfunctional P2X7 receptor, the kit may for example further comprise a detection means linked to the antigen binding protein of the invention.

[0204] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharma- ceutically acceptable carrier.

[0191]

[0205] Optionally, the kits of the invention are packaged with instructions for use in any of the examples of the methods described herein. EXAMPLES

[0192] Example 1: Identification of the light chain paired with the BIL03 heavy chain

[0206] Single-chain human variable heavy chain regions are poorly expressed, often aggregate after expression, and are difficult to handle and produce in clinically relevant quantities.

[0193]

[0207] The inventors sought to identify a suitable light chain to pair with the single domain heavy chain protein BIL03 (defined herein in Table 1).

[0208] More than 10 different light chain pairings were tested, including the variable light chain defined as GB1 in Table 1 herein.

[0194]

[0209] We found that a specific light chain pairing with WT B1 (defined in Table 1) resulted in stable expression and reduced aggregation. Surprisingly, this light chain-heavy chain pairing also significantly improved binding affinity to the target antigen, as shown in the table below.

[0195]

[0210]

[0196] [Table 2]

[0197]

[0211] Binding affinity was assessed for two different antigens derived from dysfunctional P2X7 receptors: E200 peptide (GHNYTTRNILPGLNITC) and Ext peptide 17 (GHNYTTRNILPGLNITSTFHKTSGSGK).

[0198]

[0212] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features set forth or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

Claims

1. Dysfunctional P2X ７ An antigen-binding protein that binds to a receptor, FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, and FR1a - CDR1a - FR2a - CDR2a - FR3a - CDR3a - FR4a wherein FR1, FR2, FR3 and FR4 are each framework regions, CDR1, CDR2 and CDR3 are each complementarity - determining regions, FR1a, FR2a, FR3a and FR4a are each framework regions, CDR1a, CDR2a and CDR3a are each complementarity - determining regions), and an antigen - binding protein, wherein the sequence of any of the framework regions or complementarity - determining regions is described herein.

2. The antigen - binding protein according to claim 1, wherein any one of the complementarity - determining regions has the amino acid sequence set forth in Table 1.

3. The antigen - binding protein according to claim 1 or 2, which competitively inhibits the binding of an antibody comprising a variable heavy chain (VH) containing the sequence shown in SEQ ID NO: 4 and a variable light chain (VL) containing the sequence shown in SEQ ID NO:

12.

4. The antigen - binding protein according to claim 1 or 2, wherein CDR1, CDR2 and CDR3 are sequences derived from the variable heavy chain (VH) of an antibody, CDR1a, CDR2a and CDR3a are sequences derived from the variable light chain (VL) of an antibody, or CDR1, CDR2 and CDR3 are sequences derived from VL, and CDR1a, CDR2a and CDR3a are sequences derived from VH.

5. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 - linker - FR1a - CDR1a - FR2a - CDR2a - FR3a - CDR3a - FR4a The antigen - binding protein according to claim 1 or 2, which comprises.

6. The antigen - binding protein according to claim 5, wherein the linker is a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine residues.

7. The antigen - binding protein according to claim 1 or 2, which comprises, consists essentially of, or consists of the amino acid sequences of SEQ ID NOs: 4 and 12 (in the order from the N - terminus to the C - terminus or from the C - terminus to the N - terminus), and preferably, in the order from the N - terminus to the C - terminus, comprises the sequences of SEQ ID NO: 12 and SEQ ID NO:

4.

8. The protein comprises an antigen-binding domain that binds or specifically binds to the nfP2X ７ receptor, and the antigen-binding domain is (i)A VH comprising a Complementary Determining Region (CDR) 1 that includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 1, 29, 36 or 43, a CDR2 that includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 that includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 3, 31, 38 or 45, (ii)A VH that includes a sequence that is at least about 95% or 96% or 97% or 98% or 99% identical to the sequence shown in SEQ ID NO: 4, (iii)A VL comprising a CDR1 that includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 9, 50, 57 or 64, a CDR2 that includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 10, 51, 58 or 65, and a CDR3 that includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 11, 52, 59 or 66, (iv)A VL that includes a sequence that is at least about 95% identical to the sequence shown in SEQ ID NO: 12, (v)A VH comprising a CDR1 that includes the sequence shown in SEQ ID NO: 1, 29, 36 or 43, a CDR2 that includes the sequence shown in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 that includes the sequence shown in SEQ ID NO: 3, 31, 38 or 45, (vi)A VH that includes the sequence shown in SEQ ID NO: 4, (vii)A VL comprising a CDR1 that includes the sequence shown in SEQ ID NO: 9, 50, 57 or 64, a CDR2 that includes the sequence shown in SEQ ID NO: 10, 51, 58 or 65, and a CDR3 that includes the sequence shown in SEQ ID NO: 11, 52, 59 or 66, (viii)A VL that includes the sequence shown in SEQ ID NO: 12, (ix) A VH comprising a CDR1 comprising the sequence shown in SEQ ID NO: 1, 29, 36 or 43, a CDR2 comprising the sequence shown in SEQ ID NO: 2, 30, 37 or 44, and a CDR3 comprising the sequence shown in SEQ ID NO: 3, 31, 38 or 45, and a VL comprising a CDR1 comprising the sequence shown in SEQ ID NO: 9, 50, 57 or 64, a CDR2 comprising the sequence shown in SEQ ID NO: 10, 51, 58 or 65, and a CDR3 comprising the sequence shown in SEQ ID NO: 11, 52, 59 or 66, or (x) A VH comprising the sequence shown in SEQ ID NO: 4 and a VL comprising the sequence shown in SEQ ID NO: 12 The antigen-binding protein according to claim 1 or 2, comprising at least one of the above. **Claim 9** The antigen-binding domain is (i) A framework region (FR) 1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 5, 32, 39, or 46, an FR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 6, 33, 40 or 47, an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 7, 34, 41 or 48, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 8, 35, 42 or 49, a VH (ii) An FR1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 15, 55, 62 or 69, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 16, 56, 63 or 70, wherein the VL contains these sequences. (iii) An FR1 comprising the sequence shown in SEQ ID NO: 5, 32, 39, or 46, an FR2 comprising the sequence shown in SEQ ID NO: 6, 33, 40 or 47, an FR3 comprising the sequence shown in SEQ ID NO: 7, 34, 41 or 48, and an FR4 comprising the sequence shown in SEQ ID NO: 8, 35, 42 or 49, wherein the VH contains these sequences. (iv) An FR1 comprising the sequence shown in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising the sequence shown in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising the sequence shown in SEQ ID NO: 15, 55, 62 or 69, and an FR4 comprising the sequence shown in SEQ ID NO: 16, 56, 63 or 70, wherein the VL contains these sequences, or (v) An FR1 comprising the sequence shown in SEQ ID NO: 5, 32, 39, or 46, an FR2 comprising the sequence shown in SEQ ID NO: 6, 33, 40 or 47, an FR3 comprising the sequence shown in SEQ ID NO: 7, 34, 41 or 48, and an FR4 comprising the sequence shown in SEQ ID NO: 8, 35, 42 or 49, wherein the VH contains these sequences, and an FR1 comprising the sequence shown in SEQ ID NO: 13, 53, 60 or 67, an FR2 comprising the sequence shown in SEQ ID NO: 14, 54, 61 or 68, an FR3 comprising the sequence shown in SEQ ID NO: 15, 55, 62 or 69, and an FR4 comprising the sequence shown in SEQ ID NO: 16, 56, 63 or 70, wherein the VL contains these sequences The antigen-binding protein according to claim 8, further comprising at least one of the above.

10. (i) a single-chain Fv fragment (scFv), (ii) a dimeric scFv (di-scFv), or (iii) one of (i) or (ii) linked to a constant region of an antibody, Fc or heavy-chain constant domain (CH)2 and / or CH3 The antigen-binding protein according to claim 1 or 2, which is in the form of.

11. The protein is (i) a diabody, (ii) a triabody, (iii) a tetrabody, (iv) a Fab, (v) a Fab', (v) an F(ab')2, (vi) an Fv or a fragment thereof, (vii) a bispecific antibody or other form of multispecific antibody (including BiTE), (viii) one of (i) to (vii) linked to a constant region of an antibody, Fc or heavy-chain constant domain (CH)2 and / or CH3, or (ix) a linear antibody The antigen-binding protein according to claim 1 or 2, which is in the form of.

12. The antigen-binding protein according to claim 1 or 2, wherein the protein is a recombinant antibody or a modified antibody.

13. The antigen-binding protein according to claim 1 or 2, wherein the protein is a naked antibody.

14. The antigen-binding protein according to claim 1 or 2, wherein the protein is an antibody comprising an Fc region modified so as to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), preferably, the reduction in the ability to induce ADCC is conferred by a mutation, deletion, or modification of an amino acid of the Fc region that interacts with an Fc receptor.

15. The antigen-binding protein according to claim 1 or 2, wherein the amino acid sequence forming one or more of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 is a human sequence.

16. The antigen-binding protein according to claim 1 or 2, wherein the protein comprises a human constant region of an immunoglobulin.

17. The antigen-binding protein according to claim 1 or 2, wherein the protein may comprise a mixture of sequences that completely or partially contain or do not contain a C-terminal lysine residue and may comprise a stabilized heavy-chain constant region, and comprises a heavy-chain constant region.

18. A conjugate comprising the antigen-binding protein according to claim 1 or 2 conjugated to a label comprising a radioactive label, a cytotoxic agent, or another chemically active agent.

19. A fusion protein comprising the antigen-binding protein according to claim 1 or 2. **Claim 20** A nucleic acid encoding an antigen-binding protein according to claim 1 or 2, or a fusion protein comprising the antigen-binding protein according to claim 1 or 2, optionally comprising any one of SEQ ID NOs: 71 to 86, preferably the nucleotide sequence shown in SEQ ID NO: 74 or 82, or a sequence at least 80% identical thereto. **Claim 21** An expression construct comprising the nucleic acid according to claim 20. **Claim 22** A cell comprising the nucleic acid according to claim 20 or an expression construct comprising the nucleic acid according to claim 20. **Claim 23** A conjugate comprising an antigen-binding protein according to claim 1 or 2 conjugated to a label comprising a radioactive label, a cytotoxic agent, or another chemically active agent, a fusion protein comprising the antigen-binding protein according to claim 1 or 2, a nucleic acid encoding an antigen-binding protein according to claim 1 or 2 or a fusion protein comprising the antigen-binding protein according to claim 1 or 2, or a cell comprising the nucleic acid or an expression construct comprising the nucleic acid; and a pharmaceutically acceptable carrier, diluent, or excipient comprising a pharmaceutical composition. **Claim 24** A conjugate comprising an antigen-binding protein according to claim 1 or 2 conjugated to a label comprising a radioactive label, a cytotoxic agent, or another chemically active agent, a fusion protein comprising the antigen-binding protein according to claim 1 or 2, a nucleic acid encoding an antigen-binding protein according to claim 1 or 2 or a fusion protein comprising the antigen-binding protein according to claim 1 or 2, or a cell comprising the nucleic acid or an expression construct comprising the nucleic acid; a diluent; and optionally a label comprising a diagnostic composition. **Claim 25** A kit or product comprising a conjugate comprising an antigen-binding protein according to claim 1 or 2 conjugated to a label comprising a radioactive label, a cytotoxic agent, or another chemically active agent, a fusion protein comprising the antigen-binding protein according to claim 1 or 2, a nucleic acid encoding an antigen-binding protein according to claim 1 or 2 or a fusion protein comprising the antigen-binding protein according to claim 1 or 2, or a cell comprising the nucleic acid or an expression construct comprising the nucleic acid.

26. Use of a conjugate comprising an antigen-binding protein according to claim 1 or 2 conjugated to a label comprising an antigen-binding protein according to claim 1 or 2, a radiolabel, a cytotoxic agent, or another chemically active agent, a fusion protein comprising an antigen-binding protein according to claim 1 or 2, a cell comprising a nucleic acid encoding an antigen-binding protein according to claim 1 or 2 or a fusion protein comprising an antigen-binding protein according to claim 1 or 2, or a cell comprising an expression construct comprising said nucleic acid, in the manufacture of a medicament for the treatment or prevention of cancer in a subject.

27. A pharmaceutical composition for use in the treatment or prevention of cancer, comprising a conjugate comprising an antigen-binding protein according to claim 1 or 2 conjugated to a label comprising an antigen-binding protein according to claim 1 or 2, a radiolabel, a cytotoxic agent, or another chemically active agent, a fusion protein comprising an antigen-binding protein according to claim 1 or 2, a cell comprising a nucleic acid encoding an antigen-binding protein according to claim 1 or 2 or a fusion protein comprising an antigen-binding protein according to claim 1 or 2, or a cell comprising an expression construct comprising said nucleic acid.