Methods and compositions for stimulating immune activity

Abstract

The present invention relates to compositions and methods or the use of those compositions for amplifying immune activity to treat various diseases or conditions, particularly cancer. The method of treating a condition comprises administering to a subject an antigen-binding protein comprising (i) a first antigen-binding domain that binds to a tumor-specific antigen and (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell, thereby treating the condition. Preferably, the method further comprises a bridging molecule comprising (i) a targeting moiety that binds to a cell surface molecule on a target cell and (ii) a tumor-specific antigen epitope moiety that is bound by the first antigen-binding domain of the antigen-binding protein.

Description

[Technical field]

[0001]

[0001] The present invention relates to compositions and methods for stimulating immune activity or the use of those compositions to treat various diseases or conditions, particularly cancer. Related Applications

[0002] This application claims priority from Australian Provisional Application No. 2021902832, the entire contents of which are incorporated herein by reference. [Background technology]

[0002]

[0003] One of the most promising advances is a new class of treatments called active cellular immunotherapy (ACI). Cancer immunotherapy can be passive or active. Passive therapy is based on the adoptive transfer of immunomodulatory agents, including cytokines, tumor-specific antibodies or immune cells. These substances or cells are then administered to the patient to initiate the antitumor action. Generally, these treatments do not generate immune memory and therefore require chronic infusion-based treatments. On the other hand, active immunotherapy stimulates the patient's immune system with the aim of promoting antigen-specific antitumor effects using the body's own immune cells. Moreover, active immunotherapy aims to create a sustained antitumor response that can provide protection from minimal residual disease and tumor recurrence. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0004] Thus, despite advances in cancer therapy and infectious disease immunotherapy / vaccine technologies, there is an urgent need for new or improved effective immunotherapeutic approaches to the treatment of such diseases. [Means for solving the problem]

[0004]

[0005] The reference to prior art herein is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood to be relevant, considered to be relevant, and / or could reasonably be expected to be taken in combination with other prior art by a person of ordinary skill in the art.

[0005]

[0006] In one aspect, the present invention provides a method for treating a condition, comprising: (i) a first antigen-binding domain that binds to a tumor-specific antigen; (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and An antigen-binding protein comprising to the subject, thereby treating the condition.

[0006]

[0007] In another aspect, the present invention provides a method for producing a composition comprising: (i) a first antigen-binding domain that binds to a tumor-specific antigen; (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and An antigen-binding protein comprising Preferably, the composition further comprises a pharma- ceutically acceptable carrier, diluent or excipient.

[0007]

[0008] In another aspect, the present invention provides a method for producing a composition comprising: (i) a first antigen-binding domain that binds to a tumor-specific antigen; (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and An antigen-binding protein comprising and preferably the kit further comprises written instructions for using the antigen binding protein in the methods described herein.

[0008]

[0009] In one aspect, the present invention provides a method for treating a condition, comprising: (a)(i) a first antigen-binding domain that binds to a tumor-specific antigen; and (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and an antigen-binding protein comprising (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific antigen epitope portion that is bound by the first antigen-binding domain of the antigen-binding protein; a bridging molecule comprising to the subject, thereby treating the condition in the subject.

[0009]

[0010] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (a)(i) a first antigen-binding domain that binds to a tumor-specific antigen; and (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and an antigen-binding protein comprising (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific antigen epitope portion that is bound by the first antigen-binding domain of the antigen-binding protein; a bridging molecule comprising The present invention provides a two-component therapeutic agent comprising:

[0010]

[0011] In another aspect, the present invention provides a method for producing a composition comprising: (a)(i) a first antigen-binding domain that binds to a tumor-specific antigen; and (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and an antigen-binding protein comprising (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific antigen epitope portion that is bound by the first antigen-binding domain of the antigen-binding protein; a bridging molecule comprising Preferably, the composition further comprises a pharma- ceutically acceptable carrier, diluent or excipient.

[0011]

[0012] In another aspect, the present invention provides a method for producing a composition comprising: (a)(i) a first antigen-binding domain that binds to a tumor-specific antigen; and (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and an antigen-binding protein comprising (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific antigen epitope portion that is bound by the first antigen-binding domain of the antigen-binding protein; a bridging molecule comprising and preferably the kit further comprises written instructions for using the antigen binding protein in the methods described herein.

[0012]

[0013] In another aspect, the present invention provides a method for producing a composition comprising: (i) a first antigen-binding domain that binds to a tumor-specific antigen; (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and The present invention provides an antigen-binding protein comprising:

[0013]

[0014] In any aspect, tumor-specific antigen is the antigen expressed on solid or liquid tumor.In one embodiment, tumor-specific antigen is any one of dysfunctional P2X7 receptor, EGFRvIII or CLDN6.In any aspect, the first antigen-binding domain binds or specifically binds to dysfunctional P2X7, EGFRvIII or CLDN6.Preferably, tumor-specific antigen is dysfunctional P2X7.

[0014]

[0015] Thus, in a preferred embodiment, (i) a first antigen-binding domain that binds to a dysfunctional P2X7 receptor; (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and An antigen binding protein comprising:

[0015]

[0016] In any embodiment, the antigen binding protein (further defined herein as "orchestration molecule") comprising the first and second antigen binding domains may be at least a bivalent molecule, or may be a multivalent molecule, such as a tetravalent molecule. For example, the antigen binding protein may comprise a single binding domain for binding to a tumor-specific antigen (preferably dysfunctional P2X7) and a single binding domain for binding to a cell surface molecule on an immune cell, making the molecule a bivalent molecule. A non-limiting example of such a molecule may be a fusion protein comprising an scFv for binding to each of the first and second antigens, or a fusion protein comprising a monomeric IgG and an scFv. Alternatively, the tetravalent molecule may comprise an antigen binding protein in the form of a dimeric IgG molecule for binding to a cell surface molecule on an immune cell, and a scFv fused (e.g., via the C-terminus of each heavy chain) for binding to a tumor-specific antigen (e.g., dysfunctional P2X7 receptor). Furthermore, IgG-derived binding proteins with or without CH2 and / or CH3 domains are contemplated. Furthermore, the Fc binding domains of the CH2 and CH3 domains (if included) can be modified to attenuate or increase FcRN binding. Non-limiting examples of suitable structures for various OR molecules are also provided in the examples and figures of this specification. It is within the scope of those skilled in the art to be able to design and obtain suitable OR molecules based on these exemplary structures.

[0016]

[0017] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein as described herein.Preferably, the nucleic acid comprises a first nucleotide sequence encoding a first antigen-binding domain and a second nucleotide sequence encoding a second antigen-binding domain.In any aspect, the nucleic acid can be DNA or RNA.

[0017]

[0018] In another aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein as described herein and a nucleotide sequence encoding a bridge molecule as described herein. Preferably, the nucleic acid comprises a first nucleotide sequence encoding a first antigen binding domain and a second nucleotide sequence encoding a second antigen binding domain.

[0018]

[0019] In any aspect, the invention further comprises an immune cell or a precursor thereof expressing a receptor comprising an antigen recognition domain and a signaling domain. Preferably, the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface. For example, in any method of the invention, the method further comprises administering an immune cell or a precursor thereof expressing a receptor comprising an antigen recognition domain and a signaling domain. Preferably, the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface. Also, in another aspect, the invention provides a triple therapy comprising the dual therapy described herein, further comprising an immune cell or a precursor thereof expressing a receptor comprising an antigen recognition domain and a signaling domain. Preferably, the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface. In any embodiment, the immune cell is a T cell expressing a chimeric antigen receptor (CAR), i.e., a CAR-T cell.

[0019]

[0020] In any embodiment, the first antigen-binding domain binds to the pitope associated with the adenosine triphosphate (ATP) binding site of dysfunctional P2X7 receptor.In some embodiments, dysfunctional P2X7 receptor has reduced ability to bind ATP at ATP binding site compared with the ATP binding ability of functional P2X7 receptor (e.g., receptor with wild-type sequence and ATP-binding receptor conformation or fold).In some embodiments, dysfunctional P2X7 receptor cannot bind ATP at ATP binding site.

[0020]

[0021] In any embodiment, dysfunctional P2X7 receptor has conformational change that causes receptor to malfunction.In some embodiments, conformational change is the amino acid change from trans conformation to cis conformation.In some embodiments, the amino acid that changes from trans conformation to cis conformation is the proline at amino acid position 210 of dysfunctional P2X7 receptor.

[0021]

[0022] In any embodiment, the first antigen-binding domain binds to an epitope comprising a proline at amino acid position 210 of a dysfunctional P2X7 receptor. In some embodiments, the first antigen-binding site binds to an epitope comprising one or more amino acid residues from a glycine at amino acid position 200 to a cysteine ​​at amino acid position 216 (inclusive) of a dysfunctional P2X7 receptor.

[0022]

[0023] The first antigen-binding domain present can be any suitable molecule that can interact with and specifically bind to dysfunctional P2X7 receptor.However, in some embodiments, the first antigen-binding domain comprises amino acid sequence homology with the amino acid sequence of the antibody or its fragment that binds to dysfunctional P2X7 receptor.In some embodiments, the first antigen-binding domain comprises amino acid sequence homology with the amino acid sequence of the fragment-antigen binding (Fab) part of the antibody that binds to dysfunctional P2X7 receptor.In some embodiments, the antibody is a humanized antibody.

[0023]

[0024] In any embodiment, the first antigen-binding domain comprises amino acid sequence homology with the amino acid sequence of the single chain variable fragment (scFv) or multivalent scFv that binds to dysfunctional P2X7 receptor.In some embodiments, the multivalent scFv is a bivalent or trivalent scFv.

[0024]

[0025] In any embodiment, the first antigen binding domain comprises amino acid sequence homology to a single antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor.

[0026] In any embodiment, the first antigen-binding domain comprises a binding polypeptide that comprises amino acid sequence homology with one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide comprises one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L In a preferred embodiment, the binding polypeptide comprises amino acid sequence homology with the CDR1, 2 and 3 domains of the V chain of an antibody. H and / or V L The amino acid sequence of the CDR of the chain, or the V of the antibody H and / or V LThe antibody or fragment thereof may comprise any of the amino acid sequences described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,700,621, 8,820,632, 8,900,641, 8,100,652, 8,100,662, 8,100,672, 8,200,511, 8,200,521, 8,200,532, 8,300,672, 8,400,682, 8,500,692, 8,600,692, 8,700,692, 8,820,672, 8,100,672, 8,200,532, 8,300,672, 8,400,672, 8,50 ...200,532, 8, Nos. 9,425, 9,663,584, or 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010 / 0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, and 9,425, 9,663,584, and ... Nos. 10,597,451 or 19,944,701, PCT / AU2008 / 001365 (or corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385, PCT / AU2009 / 000869 (or corresponding U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716, PCT / AU2009 / 000869) , PCT / AU2010 / 001070 (corresponding to WO / 2011 / 020155, U.S. Patent No. 9,127,059, U.S. Patent No. 9,688,771, or U.S. Patent No. 10,053,508), and PCT / AU2010 / 001741 (corresponding to WO / 2011 / 075789 or U.S. Patent No. 8,835,609).Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 as described in PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09 as described in PCT / AU2007 / 001541 (or the corresponding U.S. Patent Application Publication No. 2010 / 0036101), and that produced by hybridoma AB253 deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101.

[0025]

[0027] In any embodiment, the cell surface molecule on the immune cell is present on the surface of a lymphoid or myeloid cell. The lymphocyte can be an innate lymphoid cell, an invariant NK cell, an NK cell, or a subtype of T lymphocyte (e.g., a cytotoxic T cell, an immunoregulatory T cell, a γδ T cell, or an NKT cell), or a subset of B lymphocytes. The myeloid cell can be a subtype of monocyte, macrophage, dendritic cell, or granulocyte. The cell surface molecule on the immune cell can be any molecule present on the immune cell that can be bound by or detected by the antigen binding domain. Preferably, the cell surface molecule is present only on the immune cell, and not on a non-immune cell. Preferably, the cell surface molecule is a receptor that directly or indirectly causes the activation of the immune cell. Typically, the activation of the immune cell leads to an increase in the ability to reduce the survival of cancer cells.

[0026]

[0028] In any aspect, the second antigen-binding domain binds or specifically binds to a cell surface molecule on an immune cell as described herein. In one embodiment, the second antigen-binding domain binds or specifically binds to a cell surface molecule on a T cell; optionally, the cell surface molecule is a T cell receptor, or a molecule associated with the T cell receptor, such as the TCR-alpha or beta chain, or a chain of the CD3 T cell receptor complex, such as the epsilon chain. In another embodiment, the cell surface molecule is a costimulatory receptor, such as CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137) and ICOS. In another embodiment, the cell surface molecule can be an Fc receptor or a part thereof, such as FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b). In other examples, the second antigen-binding domain binds or specifically binds to a cell surface molecule on an innate immune effector cell, preferably an innate immune effector cell. Examples of cell surface molecules expressed on innate immune effector cells include CD16 (also known as FcγRIIIa), NKp46, NKG2D, NKp44, and DNAM-1, and others.

[0027]

[0029] The second antigen-binding domain can be any molecule that binds to a cell surface molecule on an immune cell. For example, the second antigen-binding domain can include or be a portion of an antibody or an antigen-binding fragment thereof. Alternatively, the second antigen-binding domain can be an Fc region or a portion thereof that can bind to an Fc receptor, such as FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b). In any aspect or embodiment, the second antigen-binding domain can be an antibody Fc region, or a polypeptide that includes an Fc receptor binding domain.

[0028]

[0030] In any aspect, the Fc region of the antibody is an Fc region of an IgG, more preferably an IgG1, more preferably a human IgG1. In some embodiments, the IgG Fc region is a mouse IgG1.

[0029]

[0031] In any embodiment, one or more of the two or more polypeptides in a dimer of a chimeric or fusion protein, or one or both of the receptor binding domains, can be fused at the C-terminus to an Fc region, or one or both of the two or more polypeptides in a dimer of a chimeric or fusion protein, or one or both of the receptor binding domains can be fused at the C-terminus to an Fc region via a linker.

[0030]

[0032] Preferably, the Fc region comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of the heavy chain. In one embodiment, the heavy chain fragments are linked via disulfide bonds. Alternatively, neither the heavy chain fragments nor the Fc region are disulfide linked or linked in any way.

[0031]

[0033] In any embodiment, the bridging molecule can be a polypeptide or a polypeptide conjugated to a bridging molecule, such as a molecule with the function of a DNA aptamer. The polypeptide can be expressed by immune cells or their precursors. Alternatively, the therapeutic agent, composition or kit can include a polypeptide or a nucleic acid encoding the polypeptide.

[0032]

[0034] The bridging molecule may be a polypeptide, such as a fusion protein or a chimeric protein. In alternative embodiments, the bridging molecule may comprise a polypeptide or peptide linked via a linking molecule.

[0033]

[0035] In any embodiment, the cell surface molecule to which the targeting moiety binds or specifically binds may include an antigen, preferably an antigen described herein.

[0036] The cell surface molecule may be selected from a protein, a lipid moiety, a glycoprotein, a glycolipid, a carbohydrate, a polysaccharide, a nucleic acid, an MHC-binding peptide, or a combination thereof.

[0034]

[0037] Cell surface molecules can include parts of bacteria, viruses, and other microorganisms, such as envelopes, capsules, cell walls, flagella, pili, and toxins. Cell surface molecules can be expressed by target cells.

[0035]

[0038] The cell surface molecule may not be expressed by the target cell. By way of non-limiting example, the cell surface molecule may be a ligand expressed by a cell other than the target cell that binds to the target cell or the cell surface molecule of the target cell. Also by way of non-limiting example, the cell surface molecule may be a toxin, an exogenous molecule, or a viral protein that binds to the cell surface or cell surface receptor of the target cell.

[0036]

[0039] Target cell can be cancer cell or cell that can present peptide derived from infectious agent on MHC class receptor.Target cell can express or not express tumor-specific antigen, for example, dysfunctional P2X7 receptor.

[0037]

[0040] In any embodiment, the target cell can be any cell that expresses a dysfunctional P2X7 receptor, for example a cancer cell.

[0041] In any embodiment, two or more kinds of bridging molecules can be administered to a subject, and each bridging molecule comprises a targeting moiety that binds to a different cell surface molecule on a target cell.For example, in the context of a method for treating cancer, each bridging molecule administered can comprise a different targeting moiety and thus bind to a different tumor-associated antigen present on a cancer cell.Such an embodiment facilitates the redirection (including simultaneous) of a single class of CAR T cells to multiple antigens present on a tumor antigen, thus providing a multifaceted approach to killing cancer cells.

[0038]

[0042] Thus, in any embodiment, the method of treating cancer comprises administering two or more bridging molecules, each bridging molecule comprising a targeting moiety for binding to a different cell surface antigen on a target cell.

[0039]

[0043] In further embodiments, the bridging molecules may bind to different epitopes on the same cell surface antigen expressed by the cancer cells. Thus, in further embodiments, the methods of the invention include administering two or more bridging molecules, each bridging molecule comprising a targeting moiety for binding to a different epitope on the same cell surface antigen on the target cell.

[0040]

[0044] Furthermore, the present invention provides a method in which bridging molecules for redirecting immune cells to different cancer antigens can be synchronously administered to a subject in need thereof, allowing fine tuning of the therapeutic approach, such that immune cells can be directed to bind to cancer cells via different antigens at different times during the course of a patient's treatment regimen.

[0041]

[0045] In further embodiments, a single bridging molecule may contain more than one targeting moiety, such that a single molecule contains targeting moieties for more than one cell surface molecule on a target cell.

[0046] Furthermore, a single bridging molecule may contain more than one targeting moiety, such that a single molecule contains targeting moieties to the same cell surface molecule on a target cell, but the targeting moieties bind to different epitopes on the cell surface molecule.

[0042]

[0047] In any embodiment, the targeting moiety that binds to a cell surface molecule on the target cell comprises or consists of a peptide or an antibody or antibody fragment. Alternatively, the targeting moiety may comprise a ligand or binding partner for a protein or receptor present on the target cell surface.

[0043]

[0048] The targeting moiety may further comprise a soluble T cell receptor (TcR) or a single chain T cell receptor binding motif or a T cell receptor-like mAb. In such an embodiment, the targeting moiety is particularly suitable for binding peptides derived from intracellularly processed proteins from infectious agents that are presented on the cell surface via MHC (HLA) I and II molecules. The targeting moiety may also be suitable for binding peptides presented by MHC molecules, where the peptides comprise mutations associated with cancer, such as cancer testis antigens (WT1, NY-ESO-1, PRAME family (e.g., PRA100, PRA142, PRA300, PRA425 and others), MAGE family (e.g., MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A12 and others), CT83, SSX2, GAGE, BAGE, PAGE) or other cancer-specific mutations.

[0044]

[0049] In any aspect or embodiment, the targeting portion of the bridging molecule does not bind to the same antigen or epitope as that of the antigen recognition of the receptor.For example, the targeting portion of the bridging molecule does not bind to the dysfunctional P2X7 receptor, E200, E300, or E200 / E300 composite epitope, or any other epitope present on the dysfunctional P2X7 receptor described herein.

[0045]

[0050] The targeting moiety can be a targeting antibody or antibody fragment. The targeting antibody or antibody fragment can be an immunoglobulin (Ig). The immunoglobulin can be selected from IgG, IgA, IgD, IgE, IgM, fragments thereof or variants thereof. The immunoglobulin can be IgG. The IgG can be IgG1. The IgG can be any IgG subclass.

[0046]

[0051] In any embodiment, the bridge molecule of the present invention can comprise more than one targeting moiety.For example, in certain non-limiting embodiments, the bridge molecule can comprise two different antibodies, or fragments thereof.Antibodies can bind to different epitopes of the same cell surface molecule on target cells.Alternatively, antibodies can bind to epitopes of different cell surface molecules on target cells.

[0047]

[0052] In any of the embodiments of the aspects relating to methods of treatment herein, the antigen binding protein and / or bridging molecule may be delivered to the subject via injection or may be expressed by immune cells (e.g., those expressing a chimeric antigen receptor). The antigen binding protein and / or bridging molecule may be a polypeptide and encoded in an inducible or constitutive expression construct contained in the immune cell.

[0048]

[0053] In any embodiment, the tumor-specific antigen epitope portion comprises or consists of an epitope derived from a tumor-specific antigen.Typically, the tumor-specific antigen is any one of dysfunctional P2X7, EGFRvIII or CLDN6.In any embodiment, the tumor-specific antigen epitope portion can be bound by the first antigen-binding domain of the antigen-binding protein described herein.The tumor-specific antigen epitope portion can be any one described herein.

[0049]

[0054] In any aspect or embodiment, tumor-specific antigen epitope portion can be dysfunctional P2X7 receptor epitope portion.Dysfunctional P2X7 receptor epitope portion can be provided in the form of P2X7 receptor or the fragment of P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP.Such receptor cannot extend the opening of non-selective calcium channel to apoptotic pore.

[0050]

[0055] In any embodiment, the dysfunctional P2X7 receptor epitope portion comprises or consists of a fragment of dysfunctional P2X7 receptor. Exemplary fragments include GHNYTTRNILPGLNITC (SEQ ID NO: 2; also referred to herein as "E200 epitope") and variants thereof (exemplary variants are provided in SEQ ID NOs: 3-10 and 15-30, 168, 361-396, 437 and 438); KYYKENNVEKRTLIKVF (SEQ ID NOs: 12 and 13; also referred to herein as "E300" epitope); or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14; also referred to herein as "E200 / E300" or "composite" epitope).

[0051]

[0056] In any embodiment, the dysfunctional P2X7 receptor epitope portion is bound by an antibody that binds to the dysfunctional P2X7 receptor, but not to an antibody that binds to a functional P2X7 receptor.

[0052]

[0057] In any embodiment, the bridging molecule can comprise two or more kinds of dysfunctional P2X7 receptor epitope parts.Two or more kinds of dysfunctional P2X7 receptor epitope parts can comprise or consist of the same sequence or different sequences.For example, in any embodiment, the bridging molecule can comprise a dysfunctional P2X7 receptor epitope part in the form of E200 epitope and a further dysfunctional P2X7 receptor epitope part in the form of E300 epitope.Alternatively, in any embodiment, the bridging molecule can comprise a dysfunctional P2X7 receptor epitope part in the form of E200 epitope and a further dysfunctional P2X7 receptor epitope part in the form of composite epitope.Furthermore, in any embodiment, the bridging molecule can comprise a first dysfunctional P2X7 receptor epitope part in the form of E200 epitope and a further dysfunctional P2X7 receptor epitope part in the form of E200 epitope.

[0053]

[0058] In a further aspect of the present invention, an antigen-binding protein is provided that comprises an antigen-binding domain that binds to P2X7 receptors that are impaired in response to ATP such that they cannot form apoptotic pores under physiological conditions (i.e., dysfunctional or non-functional P2X7 receptors as defined herein).Preferably, the antigen-binding protein does not bind to the P2X7 receptors that function normally in response to ATP.

[0054]

[0059] Preferably, the antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and an antigen-binding domain comprising FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; The sequences of any of the complementarity determining regions are as set forth herein in Table 2.

[0055]

[0060] Preferably, the framework regions also have amino acid sequences as set forth in Table 2, including amino acid variations at specific residues that can be determined by aligning the various framework regions from each antibody.

[0056]

[0061] Further, the present invention provides an antigen binding protein that binds or specifically binds to a dysfunctional P2X7 receptor, the antigen binding protein comprising: A variable heavy chain (VH) comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and an antigen-binding domain comprising FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; The antigen binding domain comprises any CDR1-3 of the VH chain as defined in any one of SEQ ID NOs: 400 or 402. Preferably, the CDRs of an antigen binding protein can be determined using the Kabat Chothia or IMGT domain gap numbering systems, or the Martin system, more preferably using the Kabat system.

[0057]

[0062] In one aspect, the present invention provides an antigen binding protein comprising, consisting of, or consisting essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 400, 402 or 411.

[0058]

[0063] In another aspect, the present invention provides an antigen binding protein comprising an antigen binding domain of an antibody, wherein the antigen binding domain binds or specifically binds to a dysfunctional P2X7 receptor, the antigen binding domain comprising: (i) a VH comprising a complementarity determining region (CDR) 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 set forth in SEQ ID NO: 397, a CDR2 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 set forth in SEQ ID NO: 398, and a CDR3 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 set forth in any one of SEQ ID NOs: 399 or 401; (ii) a VH comprising a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in any one of SEQ ID NOs: 400 or 402; (iii) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 397, a CDR2 comprising the sequence set forth in any one of SEQ ID NOs: 398, and a CDR3 comprising the sequence set forth in any one of SEQ ID NOs: 399 or 401; (iv) a VH comprising a sequence as set forth in any one of SEQ ID NOs: 400 or 402; Includes at least one of the following:

[0059]

[0064] In any embodiment the antigen binding domain further comprises: (i) a VH comprising 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 set forth in any one of SEQ ID NOs: 403 or 404, a 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 set forth in SEQ ID NO: 405, a 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 set forth in SEQ ID NO: 406, and a 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 set forth in SEQ ID NO: 407; or (ii) a VH comprising FR1 comprising the sequence set forth in any one of SEQ ID NOs: 403 or 404, FR2 comprising the sequence set forth in SEQ ID NO: 405, FR3 comprising the sequence set forth in SEQ ID NO: 406, and FR4 comprising the sequence set forth in SEQ ID NO: 407; Includes at least one of the following:

[0060]

[0065] The present invention also provides an antigen binding protein that binds or specifically binds to a dysfunctional P2X7 receptor, which antigen binding protein competitively inhibits binding of an antigen binding protein comprising, consisting of, or consisting essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 400 or 402.

[0061]

[0066] In another aspect, the present invention also provides an antigen binding protein comprising an antigen binding domain of an antibody, wherein the antigen binding domain binds or specifically binds to a dysfunctional P2X7 receptor, the antigen binding domain comprising: (i) a VH comprising a complementarity determining region (CDR) 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 set forth in SEQ ID NO: 408, 416, 423 or 430, a CDR2 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 set forth in SEQ ID NO: 409, 417, 424 or 431, and a CDR3 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 set forth in any one of SEQ ID NOs: 410, 418, 424 or 432; (ii) a VH comprising a sequence that is at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 411; (iii) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 408, 416, 423 or 430, a CDR2 comprising the sequence set forth in any one of SEQ ID NO: 409, 417, 424 or 431, and a CDR3 comprising the sequence set forth in any one of SEQ ID NO: 410, 418, 424 or 432; (iv) VH comprising the sequence set forth in SEQ ID NO: 411 Includes at least one of the following:

[0062]

[0067] The antigen binding domain may further comprise: (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%, or at least 99% identical to the sequence set forth in any one of SEQ ID NOs: 412, 419, 426, or 433; a VH comprising a 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 set forth in SEQ ID NO:414, 421, 428 or 435, and a 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 set forth in SEQ ID NO:415, 422, 429 or 436; or (ii) a VH comprising FR1 comprising a sequence as set forth in any one of SEQ ID NOs: 412, 419, 426, or 433, FR2 comprising a sequence as set forth in SEQ ID NOs: 413, 420, 427, or 434, FR3 comprising a sequence as set forth in SEQ ID NOs: 414, 421, 428, or 435, and FR4 comprising a sequence as set forth in SEQ ID NOs: 415, 422, 429, or 436; may further include at least one of:

[0063]

[0068] In any of the above two aspects, the antigen binding protein may further comprise FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a, where FR1a, FR2a, FR3a, and FR4a are each framework regions, and CDR1a, CDR2a, and CDR3a are each complementarity determining regions. In certain embodiments, FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a corresponds to the sequence of a variable light chain (VL).

[0064]

[0069] In certain embodiments, the antigen binding protein comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a; or FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a-FR1-CDR1-FR2-CDR2-FR3a-CDR3a-FR4a-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0065]

[0070] As defined herein, the linker may be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues. In a preferred embodiment, the linker is composed of one or more amino acid residues.

[0066]

[0071] In any embodiment, the antigen binding protein comprises a variable light chain (VL) comprising any of the CDRs of the sequences defined in any one of SEQ ID NOs: 309, 310, 311, 312, 319, 330, 331, 332 or 333. In any embodiment, the antigen binding protein comprises a variable light chain (VL) comprising any of the CDR1, CDR2 and CDR3 of the sequences defined in any one of SEQ ID NOs: 309, 310, 311, 312, 319, 330, 331, 332 or 333. Preferably, the CDRs can be determined using the Kabat, Chothia, or IMGT domain gap numbering systems, or the Martin system, more preferably using the Kabat system.

[0067]

[0072] In any embodiment, the antigen binding protein comprises a variable light chain (VL) comprising a sequence defined in any one of SEQ ID NOs: 309, 310, 311, 312, 319, 330, 331, 332 or 333, or a sequence which is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical thereto.

[0068]

[0073] In certain preferred embodiments, the present invention provides an antigen binding protein comprising, consisting essentially of, or consisting of the amino acid sequence of (in order from N to C-terminus or C to N-terminus): i) any of SEQ ID NOs: 400, 402 or 411; and ii) any of SEQ ID NOs: 309, 310, 311, 312, 319, 330, 331, 332 or 333.

[0069]

[0074] 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:

[0070]

[0075] Further, as described herein, the antigen binding protein may be (i) Diabody; (ii) triabody; (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 a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; The compound may be in the form:

[0071]

[0076] In certain embodiments, the antigen-binding protein of the present invention is a protein that does not contain a constant region from an immunoglobulin. For example, the antigen-binding protein can be an scFv, a dimeric scFv, an Fv fragment, a single domain antibody (dAb), a diabody, or a fusion protein or conjugate comprising the same.

[0072]

[0077] In certain embodiments, the antigen binding protein is in the form of a fusion protein as described herein in the context of an "orchestral molecule" (e.g., comprising one antigen binding domain for binding to the nfP2X7 receptor and connected or fused to a second antigen binding domain for binding to an antigen on an immune cell, preferably an immune effector cell, including an innate immune effector cell).

[0073]

[0078] Such antigen-binding proteins may also be referred to as antigen-binding domains of antibodies.

[0079] In certain embodiments, the complementarity determining region sequences (CDRs) of the antigen binding proteins of the invention may be defined according to the IMGT numbering system, the Kabat, Martin or Chothia systems.

[0074]

[0080] In this specification, reference to a protein or antibody that "binds" to a dysfunctional P2X7 receptor (nfP2X7 receptor) literally refers to the protein or antibody that "binds specifically to" or "specifically binds to" the nfP2X7 receptor.

[0075]

[0081] Preferably, the antigen-binding protein described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding protein is an antibody, for example, a monoclonal antibody. The antigen-binding protein may be in the form of a recombinant antibody or a 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 include chemical modifications such as conjugation to an active agent or a radioactive label, or an agent to improve solubility, or other modifications described herein.

[0076]

[0082] As used herein, an antigen binding protein can be a variable domain.

[0083] Any embodiment of the invention, and any antigen binding protein described herein, further comprises an Fc region that is engineered to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the reduced ability to induce ADCC is brought about by mutation, deletion or modification of amino acids in the Fc region that interact with Fc receptors.

[0077]

[0084] The 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.

[0078]

[0085] 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, including antigen binding proteins having sequences described herein or comprising the CDR and / or FR sequences described herein.

[0079]

[0086] The antigen-binding proteins described herein may comprise a human constant region, such as an IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region, or a mixture thereof. In the case of an antibody or protein comprising a VH and a VL, the VH may be linked to a heavy chain constant region, and the VL may be linked to a light chain constant region.

[0080]

[0087] One example is an antigen binding protein described herein that comprises a constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. One example is an IgG4 constant region that has 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)).

[0081]

[0088] In one example, an antigen binding protein described herein, or a composition of antigen binding proteins described herein, comprises a heavy chain constant region, including a stabilized heavy chain constant region, that comprises a mixture of sequences with or without a C-terminal lysine residue, either completely, partially or completely.

[0082]

[0089] In one example, the antigen binding protein comprises a VH disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as discussed above), and the VL is linked or fused to a kappa light chain constant region.

[0083]

[0090] In any aspect of the invention, the antibody is a naked antibody, specifically, the antibody is in unconjugated form and has not been adapted to form a conjugate.

[0091] 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 conjugated to a label or a cytotoxic agent.

[0084]

[0092] In embodiments of the invention relating to multiple polypeptide chains forming an antigen binding protein, the 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.

[0085]

[0093] In another example, the expression construct is a bicistronic expression construct, e.g., the following operably linked components in 5' to 3' order:

[0094] (i) Promoter

[0095] (ii) a nucleic acid encoding a first polypeptide;

[0096] (iii) an internal ribosome entry site; and

[0097] (iv) a nucleic acid encoding a second polypeptide. Including,

[0098] The first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0086]

[0099] The present invention also contemplates an expression construct encoding a first polypeptide comprising a VH and another expression construct encoding a second polypeptide comprising a VL. For example, the present invention also provides a composition comprising:

[0087]

[0100] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

[0101] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0088]

[0102] The invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified, or recombinant. One example is a cell comprising an expression construct of the invention, or

[0103] (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and

[0104] (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter. Including,

[0105] The first and second polypeptides associate to form the antigen binding protein of the invention.

[0089]

[0106] Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0107] 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.

[0090]

[0108] The invention provides vectors comprising the nucleic acids described herein.

[0109] The invention provides cells comprising a vector or nucleic acid described herein.

[0110] 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.

[0091]

[0111] The present invention provides a diagnostic composition comprising an antigen binding protein, or an antigen binding protein comprising the CDR and / or FR sequences described herein, or 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 described herein, a diluent and optionally a label.

[0092]

[0112] The invention provides kits or articles of manufacture 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.

[0093]

[0113] The present invention provides for the use of sequences according to one or more of CDR1, CDR2, FR1, FR2, FR3 and FR4 described herein to produce antigen binding proteins for binding to the nfP2X7 receptor.

[0094]

[0114] 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 having increased affinity for the nfP2X7 receptor.

[0095]

[0115] The present invention provides a library of nucleic acid molecules produced from mutations of the antigen binding proteins or CDR and / or FR sequences described herein, wherein at least one nucleic acid molecule in the library encodes an antigen binding protein for binding to the nfP2X7 receptor.

[0096]

[0116] The present invention provides a method for producing an antigen binding protein for binding to the nfP2X7 receptor described herein, comprising expressing a nucleic acid described herein in a cell or animal described herein.

[0097]

[0117] The functional characteristics of the antigen-binding proteins of the invention apply mutatis mutandis to the antibodies of the invention.

[0118] The antigen binding proteins described herein may be purified, substantially purified, isolated, and / or recombinant.

[0098]

[0119] The antigen binding protein of the invention may be part of the supernatant harvested from the culture medium in which a hybridoma expressing the antigen binding protein of the invention has been grown.

[0120] The present invention provides a method for preventing or treating the condition or disease associated with the expression of nfP2X7 in an individual, which comprises providing the 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, conjugate or pharmaceutical composition described herein to the individual who needs to treat the condition or disease.The disease or condition associated with the expression of nfP2X7 is preferably cancer.

[0099]

[0121] In another aspect, the present invention also provides a method of treating or preventing cancer in a subject, comprising administering to the subject an antigen binding protein of the present invention, thereby treating or preventing cancer in a 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.

[0100]

[0122] 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.

[0123] In another aspect, the present invention provides an antigen binding protein or a pharmaceutical composition comprising an antigen binding protein of the present invention for use in the treatment or prevention of cancer in a subject.

[0101]

[0124] As used herein, unless the context otherwise requires, the term "comprise" and variations of the term, such as "comprises," "comprises," and "comprised," are not intended to exclude additional additives, components, integers, or steps.

[0102]

[0125] 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 with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0103] [Figure 1]

[0126] Figure 1: Three different scenarios are shown as potential modes of action using examples of antigen binding proteins (also referred to herein as orchestration molecules (ORs)) and bridging molecules (also referred to herein as BRiDGE molecules). Scenario I shows direct recruitment of immune effector cells to cancer cells via OR molecules. Scenario II shows indirect recruitment of immune effector cells to cancer cells via BRiDGE molecules. Because BRiDGE molecules co-express targeting nfP2X7 E200-derived epitopes on the BRiDGE molecules, OR molecules may detect cancer cells despite the fact that they may only express nfP2X7 at very low levels or may not express nfP2X7 at all. Scenario III shows the most likely combined mode of action, where both direct and indirect targeting may occur. [Figure 2-1]

[0127] Figure 2: Exemplary OR molecules with a first antigen binding domain targeting a dysfunctional P2X7 receptor and a second antigen binding domain targeting CD3. OR19_6 is a CD19 / CD3 control based on blinatumomab. (a) OR1, OR2-3, OR13-3, OR14, OR15, OR17, and a CD33 bridging molecule. (b) Table of additional exemplary OR molecule formats. [Figure 2-2] FIG. 2: An exemplary OR molecule having a first antigen-binding domain that targets a dysfunctional P2X7 receptor and a second antigen-binding domain that targets CD3. [Figure 2-3] FIG. 2: An exemplary OR molecule having a first antigen-binding domain that targets a dysfunctional P2X7 receptor and a second antigen-binding domain that targets CD3. [Figure 2-4]

[0127]

[0127] Figure 2: An exemplary OR molecule having a first antigen-binding domain that targets a dysfunctional P2X7 receptor and a second antigen-binding domain that targets CD3. [Figure 3-1]

[0128] Figure 3: Binding of orchestration molecule variants on MOLM-13 (AML) wild type cell line. Indirect staining of engineered proteins on MOLM-13 cells via anti-HIS staining. CD33 BRiDGE (B031) was used as a control fusion protein. There is clear binding to MOLM-13 cells compared to the isotype control, which is anti-HIS antibody alone. Histogram overlay of HIS-FITC positivity on MOLM-13. Grey: isotype control (left), red: binding of HIS ab (right shift). HIS-ab was used according to the manufacturer at 1 μl per test and target cells were incubated with 50 μl of supernatant from Lenti-X™ 293T cells for 10 min at room temperature. Supernatants were collected 48 h after transfection. Transfection with CD33 Fab as positive control. (a) OR1, (b) OR2-3, (c) OR13-3, (d) OR14, (e) OR15, (f) OR17, and (g) CD33 cross-linking molecule. [Figure 3-2]FIG. 3: Binding of orchestration molecule variants on MOLM-13 (AML) wild-type cell line. [Figure 3-3] FIG. 3: Binding of orchestration molecule variants on MOLM-13 (AML) wild-type cell line. [Diagram 3-4] FIG. 3: Binding of orchestration molecule variants on MOLM-13 (AML) wild-type cell line. [Figure 4-1]

[0129] Figure 4: Flow cytometric detection of the binding capacity of OR molecules on T cells. Histogram overlay of HIS-FITC positivity on T cells. Grey: isotype control (left), red: binding of HIS antibody (right shift). HIS antibody was used according to the manufacturer at 1 μl per test and target cells were incubated with 50 μl of supernatant from Lenti-X™ 293T cells for 10 min at room temperature. Supernatants were harvested 48 h after transfection. T cells were isolated and cultured for 7 days in TexMacs medium with IL-7 / IL-15. (a) OR13-3, (b) OR14, (c) OR16, (d) OR17, and (e) OR19_6. [Figure 4-2] FIG. 4: Flow cytometric detection of the binding capacity of OR molecules on T cells. [Figure 4-3] FIG. 4: Flow cytometric detection of the binding capacity of OR molecules on T cells. [Diagram 5]

[0130] Figure 5: Bridging molecules in Fab format with a single E200 epitope linked directly ((a) and (b)) or via a linker ((c) and (d)) to the VH bind to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, where the E200 epitope is available for binding to an antibody (BIL03_2-2-1-AF647). The HIS tag is detected by a FITC antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show antibody binding to a dysfunctional P2X7 receptor epitope. [Figure 6]

[0131] Figure 6: Bridge molecules in scFv format with a single E200 epitope linked directly ((a) and (b)) or via a linker ((c) and (d)) to the VH bind to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, with the E200 epitope available for binding to the antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show antibody binding to the dysfunctional P2X7 receptor epitope. [Figure 7]

[0132] Figure 7: Bridging molecules in Fab format with a single E200 epitope linked directly ((a) and (b)) or via a linker ((c) and (d)) to the VL bind to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, with the E200 epitope available for binding to the antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show antibody binding to the dysfunctional P2X7 receptor epitope. [Figure 8]

[0133] Figure 8: Bridge molecules in scFv format with a single E200 epitope linked directly ((a) and (b)) or via a linker ((c) and (d)) to the VL bind to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, with the E200 epitope available for binding to the antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show antibody binding to the dysfunctional P2X7 receptor epitope. [Figure 9-1]

[0134] Figure 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. (a), (c), (e), (g), (i), (k), (m), (o), (q), (s), (u), (w) and (y) show anti-HIS antibody binding and (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitope. [Figure 9-2]FIG. 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. [Figure 9-3] FIG. 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. [Figure 9-4] FIG. 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. [Figure 9-5] FIG. 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. [Figure 9-6] FIG. 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. [Figure 9-7] FIG. 9: Binding of cross-linking molecules to various antigens CD37, CD79B, ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20. [Figure 10]

[0135] Figure 10: "Painting" of JeKo-1 cells with CD19-targeted Fab cross-linking molecules in the indicated formats as detected by flow cytometry. Cells were incubated with Fab cross-linking molecules at the indicated concentrations. CD33-targeted Fab cross-linking molecules served as negative controls at 10 ng / mL and 1000 ng / mL of JeKo-1. CD19-targeted Fab cross-linking molecules were used at 1 ng / mL, 10 ng / mL, 100 ng / mL and 1000 ng / mL. [Figure 11]

[0136] Figure 11: "Painting" of MOLM-13 cells with CD33-targeted Fab cross-linking molecules in the indicated formats as detected by flow cytometry. Cells were incubated with Fab cross-linking molecules at the indicated concentrations. CD19-targeted Fab cross-linking molecules served as negative controls at 10 ng / mL and 1000 ng / mL of JeKo-1. CD33-targeted Fab cross-linking molecules were used at 1 ng / mL, 10 ng / mL, 100 ng / mL and 1000 ng / mL. [Figure 12]

[0137] Figure 12: The gating strategy is shown, starting with a cell gate, a singlet gate to exclude doublets, and a live / dead discrimination gate using only live cells. T cells were identified from MOLM-13 via CD3 APCs, which were generated to constitutively express eGFP. Only the T cell population was analyzed for CD25 APC Vio770 and CD69 VioGreen expression. [Figure 13]

[0138] Figure 13: T cells were incubated with MOLM-13 cells under different conditions. In the left panel, neither OR nor BRiDGE molecules were added and basal T cell activation, referred to as CD25 and CD69 double positive cells, was 3.65%. In the middle panel, OR17 was added and specific activation increased to 8.05%, whereas the combination of OR17 and B031 increases specific activation to 31%. [Figure 14-1]

[0139] Figure 14: Luciferase-based cytotoxicity assay of T cells versus MOLM-13. Cytolysis was measured by bioluminescence activity of MOLM-13 wild-type cell line transduced to constitutively express firefly luciferase and eGFP. OR molecules alone led to a significant reduction of cancer cells, especially after 24 hours, which was further improved by the presence of bridging molecules. (a) % viability of MOLM-13 cells after 20 hours of incubation with T cells in the presence of different concentrations of OR molecules, without or with bridging molecules. (b) Overview of OR15, with and without CD33 bridging molecule (B031). (c) Overview of OR17, with and without CD33 bridging molecule (B031). (d) Overview of different OR molecules, with and without CD33 bridging molecule (B031). [Figure 14-2] FIG. 14: Luciferase-based cytotoxicity assay of T cells versus MOLM-13. [Figure 14-3] FIG. 14: Luciferase-based cytotoxicity assay of T cells versus MOLM-13. [Figure 15]

[0140] Figure 15: Kinetics of luciferase-based cytotoxicity assay of T cells from healthy donors versus JeKo-1 cells without bridging molecules. Cytolysis was measured by bioluminescence activity of the Jeko-1 cell line transduced to constitutively express firefly luciferase and eGFP. OR molecules alone result in a significant reduction of cancer cells, especially after 23 hours. Relatedly, OR17 showed comparable potency to OR19_6 (corresponding to a very similar protein like blinatumomab, a CD3xCD19 bispecific T-cell engager [BiTE]) and OR13+3, which was significantly more potent than the other OR molecules tested. N=1 healthy donor and 6 technical replicates. [Figure 16]

[0141] FIG. 16: The bridging molecule B19_8_Fab significantly increased the potency of many OR molecules compared to the data in FIG. 15. [Figure 17]

[0142] Figure 17: The bridging molecule B19_8_IgG1 significantly increased the potency of many OR molecules compared to the data in Figure 15. [Figure 18]

[0143] Figure 18: Cross-linking molecular barriers. [Figure 19]

[0144] Figure 19: Viability of JeKo-1 cells at an effector to target ratio of 10:1, without and with OR molecules after 24 hours of incubation. No bridging molecules are present. T cells from 2 healthy volunteer donors and 6 technical replicates. [Figure 20]

[0145] Figure 20: Viability of JeKo-1 cells at an effector cell to target ratio of 10:1, without and with OR molecules after 24 hours of incubation. B19_8_Fab cross-linking molecule is present at 100 ng / mL. CD19-targeted BRiDGE derived from Tafasitamab. T cells from 2 healthy volunteer donors and 6 technical replicates. [Figure 21]

[0146] Figure 21: Viability of JeKo-1 cells at an effector cell to target ratio of 10:1, without and with OR molecules after 24 hours of incubation. B19_8_IgG1 bridging molecule is present at 100 ng / mL. CD19-targeted BRiDGE derived from Tafasitamab. T cells from 2 healthy volunteer donors and 6 technical replicates. [Figure 22]

[0147] FIG. 22: Further bridge molecule variants with linkers or extended E200 epitope portions. [Figure 23]

[0148] Figure 23: Viability of JeKo-1 cells at an effector cell to target ratio of 10:1, without and with OR17 after 24 hours of incubation. B19_10_Fab cross-linking molecule shown at various concentrations. CD19-targeted BRiDGE derived from Tafasitamab. T cells from one healthy volunteer donor and 6 technical replicates. [Figure 24]

[0149] Figure 24: Viability of JeKo-1 cells at an effector cell to target ratio of 10:1, without and with OR17 after 24 hours of incubation. B19_10_IgG1 bridging molecule shown at various concentrations. CD19-targeted BRiDGE derived from Tafasitamab. T cells from one healthy volunteer donor and 6 technical replicates. [Diagram 25]

[0150] Figure 25: Viability of JeKo-1 cells at an effector cell to target ratio of 10:1, without and with OR17 after 24 hours of incubation. B19_11_Fab cross-linking molecule is shown at various concentrations. CD19-targeted BRiDGE derived from Tafasitamab. T cells from one healthy volunteer donor and 6 technical replicates. [Figure 26]

[0151] Figure 26: Viability of JeKo-1 cells at an effector cell to target ratio of 10:1, without and with OR17 after 24 hours of incubation. B19_11_IgG1 bridging molecule shown at various concentrations. CD19-targeted BRiDGE derived from Tafasitamab. T cells from one healthy volunteer donor and 6 technical replicates. [Figure 27]

[0152] FIG. 27: Comparison of different BRiDGE variants at the same concentration of 100 ng / mL in Fab format. [Figure 28]

[0153] FIG. 28: Comparison of different BRiDGE variants at the same concentration of 100 ng / mL in IgG1 format. [Figure 29]

[0154] Figure 29: Viability of MOLM-13 cells at an effector cell to target ratio of 10:1 without and with OR molecules after 30 hours of incubation. B027 bridging molecule targeting CD38 is present. T cells from healthy volunteer donors and 5 technical replicates. [Diagram 30]

[0155] Figure 30: A different representation of the data from Figure 29. [Diagram 31]

[0156] Figure 31: Activation of T cells measured by CD25+CD69+ cells with variable + / - OR17 and different bridge molecules in Fab and IgG1 format and tag variants OR19_7 (control without E200), B19_8, B19_10 and B19_11. 2 healthy donors. Effector / target ratio 10:1. Bridge molecule in Fab / IgG1 format, final concentration 100ng / mL, incubation time 24h. [Diagram 32]

[0157] Figure 32: Cytokine secretion of GMCSF under different conditions in T cells targeting JeKo-1 with different BRiDGE molecular variants in Fab format with variable + / - OR17 and control BRiDGE B19_7, and B19_8, B19_10 and B19_11. Effector / target ratio 10:1, 24 hour incubation. [Diagram 33]

[0158] Figure 33: Cytokine secretion of IL2 under different conditions in T cells targeting JeKo-1 with different BRiDGE molecular variants in Fab format with variable + / - OR17 and control BRiDGE B19_7, and B19_8, B19_10 and B19_11. Effector / target ratio 10:1, 24 hour incubation. [Diagram 34]

[0159] Figure 34: Cytokine secretion of TNFα under different conditions in T cells targeting JeKo-1 with different BRiDGE molecular variants in Fab format with variable + / - OR17 and B19_7 as control BRiDGE, and B19_8, B19_10 and B19_11. Effector / target ratio 10:1, 24 hour incubation. [Diagram 35]

[0160] Figure 35: Cytokine secretion of IFN gamma under different conditions in T cells targeting JeKo-1 with different BRiDGE molecular variants in Fab format with variable + / - OR17 and B19_7 as control BRiDGE, and B19_8, B19_10 and B19_11. Effector / target ratio 10:1, 24 hour incubation. [Figure 36-1]

[0161] Figure 36: Structure of a bispecific and tetravalent molecule (orchestration molecule) for engaging innate immune cells and cancer cells via the nfP2X7 receptor. [Figure 36-2] FIG. 36: Structure of a bispecific and tetravalent molecule (orchestration molecule) for engaging innate immune cells and cancer cells via the nfP2X7 receptor. [Figure 36-3] FIG. 36: Structure of a bispecific and tetravalent molecule (orchestration molecule) for engaging innate immune cells and cancer cells via the nfP2X7 receptor. [Figure 36-4] FIG. 36: Structure of a bispecific and tetravalent molecule (orchestration molecule) for engaging innate immune cells and cancer cells via the nfP2X7 receptor. [Figure 36-5] FIG. 36: Structure of a bispecific and tetravalent molecule (orchestration molecule) for engaging innate immune cells and cancer cells via the nfP2X7 receptor. [Figure 37-1]

[0162] Figure 37: Binding of orchestration molecules that engage innate immune cells to NK cells. [Figure 37-2] FIG. 37: Binding of orchestration molecules that engage innate immune cells to NK cells. [Figure 38]

[0163] Figure 38: Cell killing of JeKo cancer cells by PBMCs in the presence of orchestration molecules + / - BRiDGE molecules. [Figure 39]

[0164] Figure 39: Cell killing of MOLM-13 cells by PBMCs in the presence of orchestration molecules + / - BRiDGE molecules. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] [Table 1-1]

[0105] [Table 1-2]

[0106] [Table 1-3]

[0107] [Table 1-4]

[0108] [Table 1-5]

[0109] [Table 1-6]

[0110] [Table 1-7]

[0111] [Table 1-8]

[0112] [Table 1-9]

[0113] [Table 1-10]

[0114]

Table 1-11

[0115]

Table 1-12

[0116]

Table 1-13

[0117]

Table 1-14

[0118]

Table 1-15

[0119]

Table 1-16

[0120]

Table 1-17

[0121]

Table 1-18

[0122]

Table 1-19

[0123]

Table 1-20

[0124]

Table 1-21

[0125]

Table 1-22

[0126]

Table 1-23

[0127]

Table 1-24

[0128]

Table 1-25

[0129]

Table 1-26

[0130]

Table 1-27

[0131]

Table 1-28

[0132]

Table 1-29

[0133]

Table 1-30

[0134]

Table 1-31

[0135]

Table 1-32

[0136]

Table 1-33

[0137]

Table 1-34

[0138]

Table 1-35

[0139]

Table 1-36

[0140]

Table 1-37

[0141]

Table 1-38

[0142]

Table 1-39

[0143]

Table 1-40

[0144]

Table 1-41

[0145]

Table 1-42

[0146]

Table 1-43

[0147]

Table 1-44

[0148]

Table 1-45

[0149]

Table 1-46

[0150]

Table 1-47

[0151]

Table 1-48

[0152]

Table 1-49

[0153]

Table 1-50

[0154]

Table 1-51

[0155]

Table 1-52

[0156]

Table 1-53

[0157]

Table 1-54

[0158]

Table 1-55

[0159]

Table 1-56

[0160]

Table 1-57

[0161]

Table 1-58

[0162]

Table 1-59

[0163]

Table 1-60

[0164]

Table 1-61

[0165]

Table 1-62

[0166]

Table 1-63

[0167]

Table 1-64

[0168]

Table 1-65

[0169]

Table 1-66

[0170]

Table 1-67

[0171]

Table 1-68

[0172]

Table 1-69

[0173]

Table 1-70

[0174]

Table 1-71

[0175]

Table 1-72

[0176]

Table 1-73

[0177]

Table 1-74

[0178]

Table 1-75

[0179] [Table 1-76]

[0180] [Table 2-1]

[0181] [Table 2-2]

[0182] [Table 2-3]

[0183] Detailed Description of the Preferred Embodiments

[0165] 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 these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0184]

[0166] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0185]

[0167] It is understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0186]

[0168] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0169] The present invention is directed to addressing one or more of the shortcomings of the prior art and is based on the inventors' recognition that cancer-specific expression of tumor-specific antigens, such as dysfunctional P2X7 receptors, can be exploited by: a) redirecting adaptive or innate immune cells, such as T cells, NK cells, macrophages, monocytes and granulocytes, to tumor-specific antigens, such as dysfunctional P2X7 receptor; redirecting immune cells to cancerous cells by modified proteins, such as antibodies, such as IgG1 antibodies, with enhanced Fc receptor binding ability, for example, SDIE modification.In addition, bispecific or trispecific fusion proteins are also considered as multifunctional fusion proteins, and recruit immune cells through immune receptors, such as CD3 on T cells, CD16 on NK cells, or CD32 or CD64 on macrophages / monocytes and granulocytes.The format of fusion can vary from full-size antibodies to bispecific T cell engagers and their variations.

[0187] b) Providing a tunable, "switchable" approach to targeted cell killing in a variety of settings that minimizes on-target and off-tumor effects; c) minimizing aberrant immune responses against adaptor molecules, and d) Amplifying the immune response by bringing target cells and immune cells into close proximity and activating the immune cells to act on the target cells.

[0188]

[0170] The present invention provides a novel treatment modality that includes a first component and a second component. The first component is the administration (or expression) of an antigen-binding protein (also referred to herein as "orchestration" (OR) molecule), such as a bispecific fusion protein that binds to a tumor-specific antigen (preferably the nfP2X7 receptor, more preferably the nfP2X7E200 epitope), and an antigen on immune cells, preferably the antigen is an activating receptor, thereby recruiting immune cells to tumor cells. Examples of suitable immune antigens for binding by the OR molecule are further provided herein. The use of the OR molecule itself has a strong anti-tumor effect mediated by engaged immune cells, such as T cells via a bispecific T cell recruiting fusion protein, or NK cells via a bispecific NK cell recruiting fusion protein. As used herein, the antigen-binding protein defined herein may also be referred to as an OR molecule.

[0189] The second component is the administration (or expression) of a bridging molecule that can redirect immune cells in the context of OR molecules. The second component, the bridging molecule, can bind to other cell surface molecules, such as tumor-associated antigens, for example, intracellularly processed proteins that are presented as peptides of various lengths via CD19, CD20, CD33, MHC I and II, or any other mechanism of accessible surface antigen exposure. The design of the bridging molecule incorporates tumor-specific epitope moieties, such as nfP2X7-derived peptide antigens (herein referred to as dysfunctional P2X7 receptor epitope moieties). The enrichment of the bridging molecule on cancer cells results in an increase in nfP2X7 target antigens on all cells targeted by the bridging molecule, for example, a CD19 Fab-based bridging molecule can bind to CD19-positive cancer cells and physiological CD19-expressing B lineage-derived cells. The OR molecule can then be enriched in cancer cells by specifically binding to cancer cells expressing nfP2X7, as well as the dysfunctional P2X7 receptor epitope portion (e.g., nfP2X7E200-derived peptide antigen) that is part of the bridging molecule, in the following steps.

[0190]

[0173] In general, the bridging molecule can redirect naturally occurring immune cells, such as T cells, NK cells, macrophages, monocytes, etc., to cancer cells via targeting tumor-associated antigens, and can simultaneously redirect cells expressing CARs, if present, for the purpose of recruiting the natural immune system. Through the combination of OR molecules, CAR-expressing cells and bridging molecules, all different types of effector cells are redirected to specifically target cancer cells and target tumor-associated and / or tumor-specific antigens. The orchestration of the natural current immune system and the artificial transgenic CAR-expressing cell subset significantly amplifies the anti-cancer response, resulting in improved cancer control.

[0191] The cell surface molecule targeted by the bridging molecule may be associated with a cancer / tumor (such as a tumor-associated antigen expressed on the surface of a cancer cell). In further embodiments, the targeted cell surface molecule may be associated with an infection or associated with any other disease (including autoimmune disease). In such embodiments, the molecule may be a cell surface antigen associated with a disease or may include a peptide / HLA complex presented on a cell. In one particular example, the molecule may include CD19 in malignancies of the B-cell lineage. The molecule may include a targeting peptide for a cancer-specific protein (genetic abnormalities such as cancer testis antigens and others specific to cancer patients). The molecule may be a peptide / HLA complex including peptides from infectious agents, such as viruses, bacteria, protozoa, virions, prions, and fungi. The molecule may be a peptide / HLA complex including peptides associated with autoimmune disease (e.g., Sm peptides associated with lupus). Additionally, the targeted antigen may be a processed sugar molecule (GD2) or a lipid.

[0192]

[0175] Furthermore, the present invention offers several advantages over existing antibody or bispecific fusion protein based therapies because the primary targeting is tumor specific (the first antigen binding domain of the antigen binding protein or ORN molecule binds to a tumor specific antigen) and not a tumor associated antigen. Targeting of the tumor associated antigen only works by introducing a bridging molecule.

[0193]

[0176] While today's antibody therapies are limited to using antibodies that directly target tumor-associated antigens or redirect non-functional antibodies against tumor-associated antigens, the orchestration technology works by a dual principle: in addition to providing cancer-specific targeting as a maintenance therapy without direct toxicity to healthy tissues, it also offers the ability to enhance the effector function of OR molecule function by introducing bridging molecules that target alternative target antigens. The nature and origin of potential target antigens are outlined above.

[0194] In another example, the adapter molecule contains a small peptide tag to redirect standard scFv-based adapter CARs. However, concerns have been raised again regarding the immunogenicity of small peptide tags contained in the adapter molecule, especially when the tag contains non-human sequences or sequences derived from human nuclear proteins.

[0195]

[0178] In contrast to prior art approaches, the present invention exploits both the specificity of the OR molecule that binds to dysfunctional P2X7 and the unique properties of the epitope derived from the dysfunctional P2X7 receptor.

[0196]

[0179] The present invention offers advantages over existing mono-antigen directed antibody and fusion protein based therapies in that: · Utilize functional OR molecules (i.e., no need to use switching molecules to activate existing immune cells); · Dysfunctional P2X7 receptors are only exposed on the surface of cancer cells, thus utilizing cancer-specific immune cell recruitment and not binding to healthy cells; Contains a non-immunogenic, naturally occurring human epitope on the cross-linking molecule in the form of an epitope derived from the dysfunctional P2X7 receptor.

[0197]

[0180] Thus, the present invention provides a new concept and approach in the use of OR molecules together with adaptor / bridging molecules for cancer-specific and cancer-associated targeting.

[0198]

[0181] More specifically, the specificity of OR molecules (also referred to herein as nfP2X7CAR) that bind to dysfunctional nfP2X7 receptors is due to the fact that dysfunctional P2X7 is only exposed on the surface of transformed cells. Furthermore, by including an epitope derived from nfP2X7 in the bridging molecule, OR molecule-mediated recognition can be broadened to include any target antigen of interest via the corresponding bridging molecule. Although nfP2X7-targeting OR molecules only recognize dysfunctional P2X7 receptors, such as E200 (or E300 or E200-300 complex) epitopes, the use of bridging molecules facilitates unlimited targeting by any accessible recognition site expressed on the cell surface. For example, nfP2X7-recruited OR molecules can be further directed (or redirected) to bind to CD19-positive cells by using a bridging molecule that includes a targeting moiety for CD19 binding on the cell surface and an E200 epitope moiety derived from nfP2X7.

[0199] P2X7 is a human receptor protein that is commonly expressed in human tissues, especially in immune and neuronal cells. No autoimmune responses against P2X7 receptors have been reported or registered. Exemplary targeted epitopes such as E200, E300 and E200 / E300 are not genetically defined, but are only due to conformational changes in the tertiary structure of P2X7. Therefore, they are non-immunogenic peptide sequences that are non-altered parts of the P2X7 sequence. Only under artificial conditions using adjuvants and conjugates can immune responses against targets be generated.

[0200] The advantage of non-immunogenic recognition sites in the bridge molecule, such as the peptide sequences E200 or E300, or the composite peptide E200-300, facilitates the long-term application of bridge molecules with various specificities without the induction of neutralizing antibodies and / or T cell-mediated rejection of cells, which represents a significant advantage over the bridge / adapter molecule designs described in the prior art.

[0201]

[0184] Furthermore, nfP2X7CAR specifically targets only cancer tissues, and therefore the approach of the present invention presents minimal risk of "on-target, off-tumor" effects and damage to healthy tissues through off-target binding of OR molecules that are cancer specific. Thus, the present invention exploits the specificity of nfP2X7-targeting OR molecules in two ways: first, it relies on the fact that nfP2X7OR molecules only target cells that express nfP2X7 (cancer cells only), and second, it relies on the fact that immune cells via OR molecules can be redirected to other tumor-associated antigens and / or specific target antigens in a switchable and adjustable manner using bridge molecules engineered to express peptide epitope portions derived from nfP2X7E200.

[0202]

[0185] Thus, the use of the bridging molecules of the present invention allows for the redirection of immune cells recruited by OR molecules for targeting of any surface-expressed target antigen. A particular advantage of this approach is that targeting is limited to the period in vivo when the bridge molecule therapeutic agent persists in circulation. This means that any toxicity resulting from the "on target, off tumor" expression of the target antigen in healthy tissues is minimized. This is because once the bridge molecule is cleared from the body, the cells to which the nfP2X7-targeting OR molecule has been recruited can only target nfP2X7 in a tumor-specific manner again. Furthermore, administration of the OR molecule can be started, stopped, and restarted at any time. In other words, targeting of target antigens other than nfP2X7 is bridge molecule dependent, so that targeting can be controlled by application of the bridge molecule. This makes it easy to perform a simple approach to "switch on" and "switch off" targeting of cancer cells via antigens that have been bound by the targeting portion of the bridge molecule, so that the immune cells to which the OR molecule has been redirected are transiently directed to cancer cells via antigens other than nfP2X7. Furthermore, the length of time that the OR molecule-redirected immune cells are redirected to other cancer antigens can be regulated by the time that the bridging molecule is administered to the patient in need thereof. Once application of the OR molecule is terminated, cancer-specific targeting of the OR molecule-engaged immune cells is likewise terminated according to the pharmacokinetics and pharmacodynamics of this particular OR molecule or the OR molecule applied to the patient.

[0203]

[0187] Thus, it should be clear that the present invention finds application in a variety of settings. For example, in the context of oncological treatment, the present invention allows the use of a single class of immune effector cell recruiting OR molecule (i.e., for binding to dysfunctional P2X7 receptors present on cancer cells) or in combination with a bridging molecule to target multiple antigens present on cancer cells. More specifically, using a bridging molecule that includes a targeting moiety for binding CD19, for example, the OR molecule recruited immune cells can be targeted to cancer cells at both tumor-specific antigens (e.g., dysfunctional P2X7 receptors) and the tumor-associated antigen CD19. This maximizes the chances that the cancer cells will be recognized and eliminated, since they have been targeted at multiple sites. Furthermore, it should be clear that the use of multiple bridging molecules, or bridging molecules that include more than one targeting moiety, facilitates the "painting" of the cancer cell surface by CAR T cells. In other words, the present invention provides the use of a variety of different bridge molecules, each of which contains an epitope for binding an OR molecule or a panel of OR molecules that has the ability to recruit various immune effector cells specifically to cancer, both directly via nfP2X7 targeting and by redirection via additionally introduced BRiDGE molecules. Thus, the OR molecule contains the ability to engage immune effector cells to be directed or redirected to bind to cancer cells via multiple cancer antigens at the same time (e.g., nfP2X7, CD19, CD20, CD22, etc.). In this way, cancer cells are targeted and bound by immune effector cells recruited by OR molecules via multiple sites, resulting in increased anti-cancer effector function and in the latest example, elimination of cancer cells.

[0204] This approach is also particularly useful in the case of cancers that express low levels of dysfunctional P2X7 receptors, such as Burkitt's lymphoma or various subcategories of solid tumors arising from epithelial, mesenchymal, neural or embryonic origin. Another example of a low-expressing cancer cell type can be triple-negative breast cancer (e.g., MDA-MB-231 cell line). Other examples include solid tumor tissues tested in tissue arrays from PDX models, some of which show lower receptor expression than other cancers. Such examples include, but are not limited to, neuroblastoma, colon cancer, lung cancer, breast cancer or brain cancer.

[0205] In a further example, the present invention finds application in the context of preventing or minimizing the severity of infection by pathogens, preferably intracellular pathogens. Although not limited to oncological situations, this may be particularly useful in the treatment of patients who have undergone cancer treatment and are immunocompromised (and therefore susceptible to infection by opportunistic or other pathogens). Thus, patients who have undergone (or continue to undergo) treatment with OR molecules that bind to dysfunctional P2X7 receptors can simultaneously be administered bridging molecules that facilitate the redirection of immune effector cells from infectious agents to cells that present peptides on MHC molecules on the cell surface. In other words, the present invention provides a platform for the simultaneous or sequential treatment of cancer and infectious diseases as well as autoimmune diseases.

[0206]

[0190] The basic principle and engagement of effector cells expressing nfPX7CAR via peptide-tagged bridge molecules derived from nfP2X7E200 and different formats of bridge molecules are illustrated and outlined in Figures 1-3.

[0207]

[0191] With nfP2X7R OR molecules in the absence of a bridging molecule, the recruited effector cells exhibit cancer-specific targeting (Figure 1: Scenario I). To broaden the applicability of nfP2X7 to functionally negative cancers (very low or negative for nfP2X7), the immune effector cells recruited by OR molecules can be redirected to cancer cells via bridging molecules that target cancer-associated antigens, such as CD33, or cancer-specific antigens via TcR-like mAbs. The specificity of the bridging molecules means that, without limitation, any surface-expressed target antigen or presented antigen can engage effector cells expressing nfP2X7CAR in the same mode of action, in the context of MHC peptide presentation (class I and II) via TcR-like mAbs or ligands (Figure 1: Scenario II).

[0208] In most cases, the dual function of OR molecules to recruit immune effector cells is utilized (Figure 1: Scenario III). This is a combination of scenarios I and II, meaning that OR molecules directly recruit and engage immune effector cells to cancer cells via nfP2X7 expressed on cancer cells, and further recruit them to cancer cells via bridging molecules targeting cancer-associated antigens, such as CD33, or cancer-specific antigens via TcR-like mAbs.

[0209] definition

[0194] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0210]

[0195] For purposes of interpreting this specification, the following definitions shall generally apply and, where appropriate, terms used in the singular shall also include the plural and vice versa.

[0196] "Purinergic receptor" generally refers to a receptor that uses purine (eg, ATP) as a ligand.

[0211]

[0197] "P2X7 receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, at least one monomer having the amino acid sequence substantially as set forth in SEQ ID NO:1 below.

[0212] SEQ ID NO: 1 MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWMDPQSKGIQ TGRCVVYEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNFRYAK YYKENNVEKRTLIKVFGIRFDILVFGTGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLAL HDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY

[0199] Insofar as the P2X7 receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X7 receptor" encompasses naturally occurring variants of the P2X7 receptor, for example, P2X7 monomers are isoforms (e.g., forms consisting of extracellular domain sequences or truncated forms thereof), naturally occurring variant forms (e.g., alternatively spliced ​​forms) and naturally occurring allelic variants, including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of the monomers that form the P2X7 receptor. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the P2X7 receptor may have a modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO:1 may be substituted, deleted, or residues may be inserted.

[0213]

[0200] "Functional P2X7 receptor" generally refers to a form of P2X7 receptor that has three intact binding sites or grooves for binding ATP. When bound to ATP, functional receptor forms a non-selective sodium / calcium channel that transforms into a pore-like structure, allowing calcium ions and molecules up to 900 Da to enter the cytoplasm, one of the consequences of which may be the induction of programmed cell death. In normal homeostasis, the expression of functional P2X7 receptor is generally restricted to cells undergoing programmed cell death, such as thymocytes, dendritic cells, lymphocytes, macrophages and monocytes. Also, there may be some expression of functional P2X7 receptor on erythrocytes and other cell types.

[0214] A "dysfunctional P2X7 receptor" (also called "non-functional" or (nf)P2X7) is a P2X7 receptor with impaired response to ATP such that it is unable to form an apoptotic pore under physiological conditions. Dysfunctional P2X7 receptor or (nfP2X7 receptor) generally refers to a form of P2X7 receptor that has a different conformation from functional P2X7, whereby the receptor is unable to form an apoptotic pore but can operate as a non-selective channel by maintaining a single functional ATP binding site located between adjacent monomers. One example occurs when one or more monomers have cis isomerization at Pro210 (according to SEQ ID NO:1). Isomerization can result from any molecular event that leads to misfolding of the monomer, including, for example, mutation of the monomer primary sequence or abnormal post-translational processing. One consequence of isomerization is that the receptor is unable to bind ATP at one, more specifically two, of the ATP binding sites on the trimer, and as a result is unable to extend the opening of the channel. In this situation, the receptor cannot form pores, which limits the degree to which calcium ions can enter the cytoplasm.Dysfunctional P2X7 receptor is expressed in a wide range of epithelial and hematopoietic cancers.As used herein, the term "dysfunctional P2X7 receptor" can be used interchangeably with the term "non-functional P2X7 receptor" or "nfP2X7 receptor".

[0215]

[0202] 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.

[0216]

[0203] "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC, and variants thereof (e.g., SEQ ID NOs: 2-11, 15-30, 168, 361-396, 437, and 438).

[0217]

[0204] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK, and variants thereof (SEQ ID NOs: 12 and 13).

[0205] "Composite epitope" generally refers to an epitope formed from the adjoining of the E200 and E300 epitopes or portions of these epitopes. An example of a composite epitope that includes the E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14).

[0218]

[0206] An "antibody" or "immunoglobulin" or "Ig" is a gamma globulin protein found in blood or other body fluids of vertebrates that functions in the immune system to bind to antigens, thus identifying and / or neutralizing foreign substances.

[0219]

[0207] Antibodies are generally heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to a H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfides BRiDGE.

[0220]

[0208] The heavy and light chains define specific Ig domains. More specifically, each heavy chain contains a variable domain (V H ), followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) followed by a constant domain (CL) at the other end. L is V H Aligned with C L is aligned with the first constant domain of the heavy chain (CH1).

[0221]

[0209] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in 3 / 4 sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chains of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of the constant domain.

[0222]

[0210] The constant domain comprises the Fc portion, which includes the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody, such as ADCC, are determined by the sequence of the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

[0223]

[0211] V H and V L The pairing of these together forms a "variable region" or "variable domain" which comprises the amino terminal domain of either the heavy or light chain of an antibody. The variable domain of the heavy chain is called the "V H The variable domain of the light chain may be referred to as "V L The V domain contains the "antigen binding site" which influences antigen binding and defines the specificity of a particular antibody for a particular antigen. The V region spans about 110 amino acid residues and consists of relatively invariant stretches called framework regions (FRs) (generally about 4 amino acids) of 15-30 amino acids separated by short regions of extreme variability called "hypervariable regions" (generally about 3 amino acids), which are generally 9-12 amino acids long each. The FRs adopt a predominantly β-sheet structure, and the hypervariable regions form loops that connect the β-sheet structures and in some cases form part of the β-sheet structures.

[0224] "Hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions; H Three of them (H1, H2, H3) and V L Includes three of them (L1, L2, L3).

[0225] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens by means of an antigen-binding domain contained within the Fv. The term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant 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).

[0226] Antibodies generally contain a constant domain, which can be arranged into a constant region or constant fragment or crystallizable fragment (Fc). Exemplary forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies contain 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 domain(s) linked to further constant domain(s) 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, two heavy chains and heavy and light chains are held together by interchain disulfide bonds, and non-covalent interactions. The number of interchain disulfide bonds varies among different types of antibodies. Each chain contains an N-terminal variable region (V H or V L ; each of which is about 110 amino acids in length) and one or more constant domains at the C-terminus. L) is the first constant domain of the heavy chain (C H1 The light chain variable region aligns with the heavy chain variable region and forms a disulfide bond. The antibody heavy chain has two or more additional C H Domain (e.g., C H2 , C H3 etc.), C H1 and C. H2 It may include a hinge region between the constant domains. 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 (e.g., human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, similarly humanized, chimerized, CDR-grafted, or deimmunized.

[0227]

[0215] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain with an 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.

[0228]

[0216] As used herein, "variable region" refers to a portion of the light and / or heavy chain of an antibody 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) and three CDRs. H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0229] As used herein, the term "complementarity determining region" (synonymous CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region whose presence is the primary contributor to specific antigen binding. Each variable region domain (V H or V L ) 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 V H CDR 1 corresponds to V H CDR 2 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 extended 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 systems, or the numbering systems 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.

[0230] In one example, CDRs are defined according to the Kabat numbering system. In some cases, 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, demonstrated that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0231] "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.

[0232]

[0220] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0221] An "antigen-binding domain" generally refers to a molecule that comprises at least the hypervariable and framework regions necessary to confer antigen-binding function to a V domain. The antigen-binding protein or domain may be in the form of an antibody or antibody fragment, such as a mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD-Fv, Fv, F(ab')2 or scFv.

[0233] An "intact" or "whole" antibody comprises an antigen-binding site, as well as a C L and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0234]

[0223] "Whole antibody fragments containing the variable domain" include SD-mAbs, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0235]

[0224] A "Fab fragment" is a fragment of the variable region domain of the H chain (V H ) and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0236] "Fab' fragments" differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0237]

[0226] The "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments which have divalent antigen-binding activity and are still capable of cross-linking antigen.

[0227] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.

[0238] In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. The folding of these two domains gives rise to six hypervariable loops (three loops each from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0239]

[0229] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and refers to VFs connected to form a single polypeptide chain. H and V L An scFv polypeptide is an antibody fragment that contains an antibody domain. H Domain and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for antigen binding.

[0240]

[0230] A "single variable domain" is typically one half of an Fv (containing only the three CDRs specific for an antigen) that has the ability to recognize and bind to an antigen, although with lower affinity than the entire binding site.

[0241]

[0231] "Diabody" refers to an antibody fragment that has two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to a heavy chain variable domain (V H Small antibody fragments include V domains such that interchain, but not intrachain, pairing of the V domains is achieved, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. H Domain and V L They are prepared by constructing an sFv fragment (see previous paragraph) with a short linker (about 5-10 residues) between the domains.

[0242] Diabodies can be bivalent or bispecific. Bispecific diabodies are diabodies that combine the V of two antibodies. H Domain and V L They are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Triabodies and tetrabodies are also commonly known in the art.

[0243]

[0233] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its existing environment. Contaminating components are substances that would interfere with therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0244]

[0234] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering antigen to transgenic animals that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled.

[0245] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0246]

[0236] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies can be prepared by the hybridoma method. "Monoclonal antibodies" can also be isolated from phage antibody libraries using molecular engineering techniques.

[0247] The term "anti-P2X7 receptor antibody" or "antibody that binds to P2X7 receptor" refers to an antibody that can bind to P2X7 receptor with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting P2X7 receptor, typically non-functional P2X7 receptor or cancer-related P2X7 receptor.Preferably, the degree of binding of P2X7 receptor antibody to unrelated proteins is less than about 10% of the binding of the antibody to P2X7 receptor, as measured, for example, by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Biacor or flow cytometry.In certain embodiments, the antibody that binds to P2X7 receptor has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. Anti-nf2X7 receptor antibodies are generally antibodies that have some or all of these serological characteristics and bind to dysfunctional P2X7 receptors but not to functional P2X7 receptors.

[0248]

[0238] An "affinity matured" antibody is an antibody with one or more modifications in one or more hypervariable regions that result in improved affinity of the antibody for antigen compared to a parent antibody without those modification(s). Preferred affinity matured antibodies have nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art.

[0249]

[0239] A "blocking antibody" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0250]

[0240] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0241] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0251]

[0242] As used herein, the term "antigen" is intended to include a substance that binds to or induces the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, such as glycosylated proteins or glycolipids. As used herein, the term "antigen" refers to a molecular entity that can be expressed on a target cell and recognized by the adaptive immune system, including, but not limited to, an antibody or TCR, or a transgenic TCR, a CAR, an scFv or multimer thereof, an Fab-fragment or multimer thereof, an antibody or multimer thereof, a single-chain antibody or multimer thereof, or any other molecule that can bind to the structure with high affinity.

[0252]

[0243] "Epitope" generally refers to that portion of an antigen that is bound by the antigen-binding site of an antibody. An epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen-binding site bind to a sequence of amino acids as in the primary protein structure. In certain embodiments, the epitope is a "conformational epitope", i.e., one that binds to residues when the hypervariable loops of the CDRs are present in a tertiary or quaternary protein structure.

[0253] 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 Fv comprising both VH and VL. The antigen-binding domain need not be in the context of a complete antibody, for example it may be isolated (e.g., a domain antibody) or may be in another form as described herein, such as, for example, an scFv.

[0254]

[0245] As used herein, the term "target cell" refers to the cell that expresses a dysfunctional P2X7 receptor (e.g., nfP2X7 receptor) or the cell surface molecule that the targeting portion of the bridging molecule binds to. Target cell can be a cancer cell or any other diseased cell.

[0255]

[0246] The term "disorder" or "condition" refers to a functional abnormality or impairment in a subject, such as cancer, an autoimmune disease, or a viral, bacterial, parasitic, or other infection.

[0256] For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can also exist in a non-native environment, such as, for example, a host cell.

[0257]

[0248] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey, or human. Preferably, the subject is a human. The subject may be a subject (patient) suffering from a disorder such as cancer, although the subject may also be a healthy subject.

[0258]

[0249] The term "autologous" as used herein refers to any material from the same subject into which it is subsequently reintroduced.

[0250] The term "allogenic" as used herein refers to any material derived from a different subject of the same species as the subject into which the material is reintroduced.

[0259]

[0251] The term "therapeutically effective amount" or "therapeutically effective population" refers, for example, to an amount of a cell population that provides a therapeutic benefit in a subject. The terms "bind", "specifically bind", or "specific for", for example, in the bridging molecules disclosed herein, or in the context of a targeting moiety used in a CAR in reference to an antigen-binding domain, mean that it recognizes and binds to a specific antigen, but does not substantially recognize or bind to other molecules in a sample. An antigen-binding domain or targeting moiety that specifically binds to an antigen from one species may also bind to an antigen from another species. This cross-species reactivity is typical of many antibodies, and therefore does not violate the definition that an antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (allelic variants, splice variants, isoforms, etc.) or homologous variants of this antigen from the same gene family. This cross-reactivity is typical of many antibodies, and therefore does not violate the definition that an antigen-binding domain is specific.

[0260]

[0253] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. These terms mean that the cells contain and / or express foreign genes or nucleic acid sequences that in turn modify the genotype or phenotype of the cells or their progeny. In particular, these terms refer to the fact that cells, preferentially immune cells, can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins that are not naturally expressed in these cells. For example, immune cells are engineered to express artificial constructs such as chimeric antigen receptors on their cell surface. For example, CAR sequences can be delivered into cells using adenovirus, adeno-associated virus (AAV)-based, retrovirus or lentivirus vectors, or any other pseudotyped variations thereof, or any other gene delivery mechanism such as electroporation or lipofection with CRISPR / Cas9, transposons (e.g., Sleeping-Beauty), or variations thereof. Gene delivery can be in the form of mRNA (transient) or DNA (transient or permanent).

[0261]

[0254] The term "immune cell" or "immune effector cell" refers to a cell that is part of the immune system, either adaptive (i.e., cellular or humoral) immunity or innate immune system, and exerts a specific effector function, such as alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes or macrophages, or any hematopoietic progenitor cell, such as pluripotent stem cells and early progenitor cell subsets that can mature or differentiate into somatic cells. The cells can be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (such as iPSCs and other artificial cell types). The immune cells can be artificial cell subsets including induced pluripotent stem cells and cells matured therefrom. Preferred immune cells are cells with cytotoxic effector function, such as alpha-beta T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells or gamma-delta T cells. "Effector function" refers to a special function of a cell, for example, in T cells, the effector function can be cytolytic activity or helper cell activity, including secretion of cytokines.

[0262]

[0255] As used herein, the term "treating" a disorder means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0263]

[0256] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell. Antigen-binding proteins

[0257] The present invention relates to (i) a first antigen-binding domain that binds to a tumor-specific antigen; (ii) a second antigen-binding domain that binds to a cell surface molecule on an immune cell; and The antigen-binding protein comprises

[0264] In any aspect, the tumor-specific antigen is an antigen expressed on a solid tumor. In one embodiment, the tumor-specific antigen is any one of a dysfunctional P2X7 receptor, EGFRvIII, or CLDN6.

[0265]

[0259] In any aspect, the antigen binding protein can be any binding molecule, such as a full size antibody or fragment thereof, or any antibody or fragment thereof described herein, an immunocytokine (an antibody linked to a cytokine or fragment thereof), a ligand (a protein-related peptide, processed molecule, cytokine, hormone), a soluble T cell receptor (TcR), a single chain (sc) TcR, a single chain T cell receptor binding motif, a T cell receptor-like mAb, or a D domain (e.g., a de novo designed α-helical bundle derived D domain, α3D).

[0266]

[0260] In any embodiment, the first antigen binding domain binds to or specifically binds to a dysfunctional P2X7 receptor, EGFRvIII or CLDN6. In any embodiment, the first antigen-binding domain binds to an epitope associated with the adenosine triphosphate (ATP) binding site of the dysfunctional P2X7 receptor.In some embodiments, the dysfunctional P2X7 receptor has a reduced ability to bind ATP at the ATP binding site compared to the ATP binding ability of a functional P2X7 receptor (e.g., a receptor having a wild-type sequence and having the conformation or fold of an ATP-binding receptor).In some embodiments, the dysfunctional P2X7 receptor cannot bind ATP at the ATP binding site.

[0267] In any embodiment, the dysfunctional P2X7 receptor has a conformational change that causes the receptor to malfunction. In some embodiments, the conformational change is an amino acid change from trans conformation to cis conformation. In some embodiments, the amino acid that is changed from trans conformation to cis conformation is a proline at amino acid position 210 of the dysfunctional P2X7 receptor.

[0268] In any embodiment, the first antigen binding domain binds to an epitope that includes a proline at amino acid position 210 of a dysfunctional P2X7 receptor. In some embodiments, the first antigen binding site binds to an epitope that includes one or more amino acid residues from a glycine at amino acid position 200 to a cysteine ​​at amino acid position 216 (inclusive) of a dysfunctional P2X7 receptor.

[0269] The first antigen-binding domain present can be any suitable molecule that can interact with and specifically bind to the dysfunctional P2X7 receptor. However, in some embodiments, the first antigen-binding domain comprises an amino acid sequence homology with the amino acid sequence of an antibody or a fragment thereof that binds to the dysfunctional P2X7 receptor. In some embodiments, the first antigen-binding domain comprises an amino acid sequence homology with the amino acid sequence of the fragment-antigen-binding (Fab) portion of an antibody that binds to the dysfunctional P2X7 receptor. In some embodiments, the antibody is a humanized antibody.

[0270] In any embodiment, the first antigen-binding domain comprises an amino acid sequence homologous to the amino acid sequence of a single chain variable fragment (scFv) or a multivalent scFv that binds to a dysfunctional P2X7 receptor. In some embodiments, the multivalent scFv is a bivalent or trivalent scFv.

[0271] In any embodiment, the first antigen binding domain comprises amino acid sequence homology to a single antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor. In any embodiment, the first antigen-binding domain comprises a binding polypeptide that comprises amino acid sequence homology with one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide comprises one or more complementarity determining regions (CDRs) of an antibody that binds to a dysfunctional P2X7 receptor. H and / or V L In a preferred embodiment, the binding polypeptide comprises amino acid sequence homology with the CDR1, 2 and 3 domains of the V chain of an antibody.H and / or V L The amino acid sequence of the CDR of the chain, or the V of the antibody H and / or V LThe antibody or fragment thereof may comprise any of the amino acid sequences described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,700,621, 8,820,632, 8,900,641, 8,100,652, 8,100,662, 8,100,672, 8,200,511, 8,200,521, 8,200,532, 8,300,672, 8,400,682, 8,500,692, 8,600,692, 8,700,692, 8,820,672, 8,100,672, 8,200,532, 8,300,672, 8,400,672, 8,50 ...200,532, 8, Nos. 9,425, 9,663,584, or 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010 / 0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, and 9,425, 9,663,584, and ... Nos. 10,597,451 or 19,944,701, PCT / AU2008 / 001365 (or corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385, PCT / AU2009 / 000869 (or corresponding U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716, PCT / AU2009 / 000869) , PCT / AU2010 / 001070 (corresponding to WO / 2011 / 020155, U.S. Patent No. 9,127,059, U.S. Patent No. 9,688,771, or U.S. Patent No. 10,053,508), and PCT / AU2010 / 001741 (corresponding to WO / 2011 / 075789 or U.S. Patent No. 8,835,609).Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 as described in PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09 as described in PCT / AU2007 / 001541 (or the corresponding U.S. Patent Application Publication No. 2010 / 0036101), and that produced by hybridoma AB253 deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101.

[0272] In any embodiment, the cell surface molecule on the immune cell is present on the surface of a lymphoid cell or a myeloid cell. The lymphoid cell can be a natural killer cell or a lymphocyte. The lymphocyte can be a T lymphocyte (e.g., a cytotoxic T cell, a gd T cell, or a NKT cell) or a B lymphocyte. The myeloid cell can be a monocyte, such as a macrophage. The cell surface molecule on the immune cell can be any molecule present on the immune cell that can be bound or detected by an antigen binding domain. Preferably, the cell surface molecule is present only on the immune cell and not on a non-immune cell. Preferably, the cell surface molecule is a receptor that directly or indirectly causes activation of the immune cell. Typically, activation of the immune cell results in an increased ability to reduce the viability of cancer cells.

[0273] In any aspect, the second antigen-binding domain binds or specifically binds to a cell surface molecule on an immune cell as described herein. In one embodiment, the cell surface molecule is a T cell receptor, or a molecule associated with a T cell receptor, such as a TCR-alpha or beta chain, or CD3. In another embodiment, the cell surface molecule is a costimulatory receptor, such as CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS. In another embodiment, the cell surface molecule can be an Fc receptor, or a portion thereof, such as FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b).

[0274] The second antigen-binding domain can be any molecule that binds to a cell surface molecule on an immune cell. For example, the second antigen-binding domain can include or be a portion of an antibody or an antigen-binding fragment thereof. Alternatively, the second antigen-binding domain can be an Fc region or a portion thereof that can bind to an Fc receptor, such as FcRI or FcRIIIa. In any aspect or embodiment, the second antigen-binding domain can be an Fc region of an antibody, or a polypeptide that includes an Fc receptor binding domain.

[0275]

[0271] In any embodiment, the Fc region of the antibody is an IgG, more preferably an IgG1, more preferably a human IgG1 Fc region.

[0272] Preferably, the Fc region comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of said heavy chains. In one embodiment, the heavy chain fragments are linked via disulfide linkages. In another embodiment, the fragments are not linked. In another embodiment, the Fc region comprises one or more modifications that inhibit or prevent homo- or heterodimerization, e.g., that prevent assembly or dimerization at the hinge region.

[0276]

[0273] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region contains two heavy chain fragments that contain the CH2 and CH3 domains of an antibody. In the context of the present invention, the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of said heavy chain. The two heavy chain fragments may be held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ.

[0277] The term "Fc region" also includes native sequence Fc regions and variant Fc regions. The Fc region may include the carboxyl terminus of the heavy chain. The antibody produced by the host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, the antibody produced by the host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain or it may include a truncated variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the Fc region of the antibody may be contemplated. Amino acid sequence variants of the Fc region of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions and / or substitutions of residues within, the amino acid sequence of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., induction or support of an anti-inflammatory response.

[0278]

[0275] The Fc region of an antibody can be the Fc region of any of the classes of antibodies, such as IgA, IgD, IgE, IgG, and IgM. The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, as used in the context of the present invention, an antibody can be an IgG Fc region. For example, an antibody Fc region can be an IgG1, IgG2, IgG2b, IgG3, or IgG4 Fc region. In some embodiments, the fusion protein of the present invention comprises an IgG of the antibody Fc region. In the context of the present invention, an antibody Fc region is an IgG, preferably an IgG1 Fc region.

[0279]

[0276] The Fc region is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response against a given antigen.

[0280]

[0277] An Fc receptor binding domain is any protein or polypeptide that binds to an Fc receptor on a cell surface. An Fc receptor binding domain can be the antigen binding domain of an antibody. An Fc receptor binding domain also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0281]

[0278] The Fc region may contain one or more mutations or modifications that increase affinity for binding to an Fc receptor.

[0279] The Fc region may contain one or more mutations or modifications that reduce affinity for binding to an Fc receptor. For example, the Fc region may have one or more mutations or modifications that weaken binding to an Fc receptor.

[0282] In any aspect or embodiment, the antigen binding protein may have a first antigen binding domain for binding to a tumor-specific antigen and a second antigen binding domain for binding to one of CD3 or CD16. Preferably, the antigen binding protein comprises a first antigen binding domain for binding to a dysfunctional P2X7 receptor and a second antigen binding domain for binding to CD3 or CD16.

[0283] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs:313 and 314, SEQ ID NOs:313 and 315, SEQ ID NOs:313 and 316, SEQ ID NOs:313 and 317, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0284] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 318 and 319, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0285] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:320, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0286] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:321, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0287] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:322, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0288] In any embodiment, the antigen binding protein for binding to a dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:324, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0289] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:325, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0290] In any embodiment, the antigen binding protein for binding to a dysfunctional P2X7 receptor and CD16 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:326, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0291] In any embodiment, the antigen binding protein for binding to a dysfunctional P2X7 receptor and CD16 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:327, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0292] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs:328 and 329, SEQ ID NOs:328 and 330, SEQ ID NOs:328 and 331, SEQ ID NOs:328 and 332, or SEQ ID NOs:328 and 333, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0293] In any embodiment, the antigen binding protein for binding to dysfunctional P2X7 receptor and CD16 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs:334 and 335, SEQ ID NOs:334 and 336, SEQ ID NOs:334 and 337, SEQ ID NOs:334 and 338, or SEQ ID NOs:334 and 339, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0294]

[0292] In any embodiment, an antigen binding protein for binding to CLDN6 and CD3 can comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 340 and 341, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0295]

[0293] In any embodiment, an antigen binding protein for binding to CLDN6 and CD3 can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 342, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0296]

[0294] In any embodiment, an antigen binding protein for binding to CLDN6 and CD16 can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 343, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0297] In any embodiment, an antigen binding protein for binding to EGFRvIII and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 344 and 345, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0298]

[0296] In any embodiment, an antigen binding protein for binding to EGFRvIII and CD3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO:346 or SEQ ID NO:347, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0299] In any embodiment, the antigen binding protein (or OR molecule) can comprise or consist of an amino acid sequence identified in the sequence information table above, e.g., any one or more of SEQ ID NOs:307-347, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% at least 98%, or at least 99% identical thereto.

[0300] In any aspect, the antigen binding proteins (or OR molecules) described herein do not have a HIS tag. Also contemplated are OR molecules that include an amino acid sequence identified in the sequence information table above, e.g., any one or more of SEQ ID NOs: 307-347, but where no HIS tag is identified in the sequence, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Furthermore, in one embodiment, the OR molecule may include a tag other than a HIS tag, or may include an amino acid sequence identified in the sequence information table above, e.g., any one or more of SEQ ID NOs: 307-347, but where no HIS tag is identified in the sequence, but where a different tag is identified.

[0301] Chimeric antigen receptors (CARs) The term cancer-specific CAR (T cell) targeting refers to the use of CAR T cells to bind to target antigens that are presented on the cell surface of tumor cells but are not typically found on the surface of healthy cells. In other words, normal cells under normal circumstances can be characterized by the absence of target antigens on the extracellular membrane (hence the presence of antigens on the cell surface cannot be detected). However, such cells can express mRNAs that code for the antigens at the intracellular level. Because CAR T cells only recognize surface-expressed antigens, intracellular expression of targeting proteins does not result in CAR engagement.

[0302] The targeted epitopes E200 and E300 of P2X7 receptor are not exposed in the form of the receptor found in healthy tissue, and therefore these epitopes can be considered to be cancer-specific. In other words, E200 and E300 epitopes are only exposed and available for binding when P2X7 receptor has an altered non-functional conformation, such as occurs in the context of cancer (in this case, the receptor is called nfP2X7 receptor). Another example of a targeted epitope that is cancer-specific can be derived from splice variant EGFRvIII. Yet another example is the antigen CLDN6, which is mostly restricted to embryonic and fetal life, has very limited expression in healthy cells after early postnatal stages, and can be considered to be highly restricted and relatively overexpressed in cancer. The present invention contemplates binding to any such tumor-specific antigens, including nfP2X7, EGFRvIII and CLDN6 for cancer-specific targeting, and engages CAR T cells to cancer-associated antigens via the bridging molecules described herein.

[0303] Generally, a CAR may comprise an extracellular domain (extracellular portion) that includes an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain may be linked to the transmembrane domain by a linker. The extracellular domain may also include a signal peptide. The extracellular portion of the CAR of the present invention comprises a tumor-specific antigen binding domain. For example, the tumor-specific antigen may be any of those described herein, including nfP2X7, EGFRvIII, or CLDN6.

[0304] The tumor-specific antigen-binding domain can be an nfP2X7 binding domain that recognizes the E200 (or E300 or E200-300 complex) epitope, as disclosed herein. Specifically, the CAR disclosed herein has an extracellular nfP2X7E200 binding domain as the antigen-binding domain. Alternatively, the tumor-specific antigen-binding domain can be an EGFRvIII binding domain that recognizes an epitope resulting from the fusion of the amino acid sequence starting with LEEKK at positions 25-29, followed by the insertion of G, and then the amino acid sequence NYVVTDH at 298-304, where the entire epitope is a 13-mer composed of the sequence LEEKKGNYVVTDH (SEQ ID NO: 267). Alternatively, the tumor-specific antigen-binding domain may be a CLDN6 binding domain that recognizes an epitope in the second extracellular domain of CLDN6 [UniProtKB-P56747 (CLDN6_HUMAN)] via the amino acid sequence of SEQ ID NO: 273, 274 or 275.

[0305] Typically, the antigen recognition domain comprises a binding polypeptide that comprises amino acid sequence homology with one or more complementarity determining regions (CDRs) of an antibody that binds to a tumor-specific antigen (such as a dysfunctional P2X7 receptor, EGFRvIII or CLDN6). In any embodiment, the binding polypeptide comprises one or more complementarity determining regions (CDRs) of an antibody that binds to a tumor-specific antigen (such as a dysfunctional P2X7 receptor, EGFRvIII or CLDN6). H and / or V L It contains amino acid sequence homology with the CDR1, 2 and 3 domains of the chain.

[0306]

[0304] In a particularly preferred embodiment of the present invention, the antigen recognition domain of the CAR binds to an epitope of the tumor-specific antigen nfP2X7. In such embodiments, the binding polypeptide is a polypeptide as disclosed in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, US Patent No. 6,226,41 ... No. 9,663,584, or U.S. Patent No. 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, and 9,94 No. 4,701 or U.S. Pat. No. 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716). No. 9,127,059, U.S. Patent No. 9,688,771, or U.S. Patent No. 10,053,508), and PCT / AU2010 / 001741 (or any one of the corresponding WO 2011 / 075789 or U.S. Patent No. 8,835,609). H and / or V LPreferably, the antibody comprises the amino acid sequence of the CDR of the chain of 2-2-1 described in PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or the CDR amino acid sequence of BPM09 described in WO2013185010A1 or WO2019056023, which is produced by hybridoma AB253 described in PCT / AU2007 / 001541 (or the corresponding U.S. Patent Application Publication No. 2010-0036101) and deposited at the European Collection of Cultures (ECACC) under accession number 06080101.

[0307] CAR binding polypeptides may be prepared using methods described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 8,710,426, 8,820,428, 8,930,429, 8,102,430, 8,112,431, 8,122,432, 8,136,433, 8,140,434, 8,152,435, 8,162,436, 8,172,437, 8,182,438, 8,192,439, 8,232,436, 8,242,437, 8,252,438, 8,262,439, 8,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 8,262,436, 8,136,436, 8,252,436, 8,152,436, 8,26 ... Nos. 9,663,584, or 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010-0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, and 9, Nos. 944,701 or 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716) ), PCT / AU2010 / 001070 (corresponding to WO / 2011 / 020155, U.S. Pat. No. 9,127,059, U.S. Pat. No. 9,688,771, or U.S. Pat. No. 10,053,508), and PCT / AU2010 / 001741 (corresponding to WO / 2011 / 075789 or U.S. Pat. No. 8,835,609). H and / or V LPreferably, the antibody comprises the CDR amino acid sequence of 2-2-1 described in PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09 described in PCT / AU2007 / 001541 (or the corresponding U.S. Patent Application Publication No. 2010-0036101), and is produced by hybridoma A253 (WO 20135010 or WO 2019056023) deposited at the European Collection of Cultures (ECACC) under accession number 060101.

[0308] CAR binding polypeptides may be prepared using methods described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Patent Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,581, 9,709,425, 9,871, 9,9 ... No. 4, or U.S. Patent No. 10,450,380), PCT / AU2007 / 001540 (or corresponding U.S. Patent No. 8,067,550), PCT / AU2007 / 001541 (or corresponding U.S. Patent Application Publication No. 2010 / 0036101), PCT / AU2008 / 001364 (or corresponding U.S. Patent Nos. 8,440,186, 9,181,320, 9,944,701, or U.S. Patent No. No. 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Pat. Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Pat. Nos. 8,597,643, 9,328,155 or 10,238,716), PCT / AU2010 No. 9,127,059, U.S. Pat. No. 9,688,771, or U.S. Pat. No. 10,053,508), and PCT / AU2010 / 001741 (corresponding to WO / 2011 / 075789 or U.S. Pat. No. 8,835,609).Preferably, the antibody comprises the CDR amino acid sequence of 2-2-1 as described in PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patent Nos. 9,127,059, 9,688,771, or 10,053,508), or BPM09 as described in PCT / AU2007 / 001541 (or the corresponding U.S. Patent Application Publication No. 2010 / 0036101), and those produced by hybridoma AB253 deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101, those described in WO201385010 or WO2019056023.

[0309]

[0308] "Signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a particular organelle (eg, the endoplasmic reticulum) and / or to the cell surface.

[0310]

[0309] Generally, "antigen binding domain" refers to the region of a CAR that specifically binds to an antigen (and thereby can target a cell containing the antigen). The CAR of the present invention may contain one or more antigen binding domains. Generally, the targeting region on a CAR is extracellular. The antigen binding domain may comprise an antibody or an antibody binding fragment thereof. The antigen binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody or a diabody. Any molecule that specifically binds to a given antigen can be used as an antigen binding domain, such as an affibody or a ligand binding domain from a naturally occurring receptor. Often the antigen binding domain is an scFv. Usually, in an scFv, the variable regions of immunoglobulin heavy and light chains are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3-linker" and variations thereof, although one skilled in the art will appreciate that a variety of linker sequences and formats may be used.

[0311] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR is used. For example, if it is planned to be used therapeutically in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise a human or humanized antibody or an antigen-binding fragment thereof. Human or humanized antibodies or antigen-binding fragments thereof can be produced by various methods well known in the art. The CAR disclosed herein has an extracellular linker / tag epitope binding domain as an antigen-binding domain, allowing it to indirectly bind to an antigen expressed on a target cell via a target cell binding molecule disclosed herein.

[0312]

[0311] As used herein, "spacer" or "hinge" refers to a hydrophilic region between the antigen-binding domain and the transmembrane domain. The CAR of the present invention may include an extracellular spacer domain, but it is also possible to remove such a spacer. The spacer may include, for example, an Fc fragment of an antibody or a fragment thereof, a hinge region of an antibody or a fragment thereof, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A prominent example of a spacer is the CD8 alpha hinge.

[0313] The transmembrane domain of the CAR can be derived from any desired natural or synthetic source of such a domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8 alpha or CD28. If the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides, the CAR can have two (or more) transmembrane domains. The division of the key signaling and antigen recognition modules allows small molecule-dependent, titratable and reversible control of CAR cell expression due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR (Wu et al, 2015, Science 350: 293-303).

[0314] The cytoplasmic domain (or intracellular signaling domain) of a CAR is involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. By "effector function" is meant a special function of a cell, e.g., in T cells, the effector function can be cytolytic activity or helper cell activity, including secretion of cytokines. An intracellular signaling domain refers to a portion of a protein that transmits an effector function signal and directs the cell expressing the CAR to perform a special function. An intracellular signaling domain can include any complete, mutated or truncated portion of the intracellular signaling domain of a given protein sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0315]

[0314] The function of the intracellular domain may be pro-inflammatory and / or immunomodulatory, or a combination of such.

[0315] Prominent examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signaling following antigen receptor engagement.

[0316]

[0316] In general, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences, firstly, those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and secondly, those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can include one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0317]

[0317] Primary cytoplasmic signaling sequences that act in a stimulatory manner are likely to contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0318] Examples of ITAMs containing primary cytoplasmic signaling sequences that are frequently used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is the sequence derived from CD3 zeta.

[0318]

[0319] The cytoplasmic domain of the CAR can be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, and are necessary for efficient lymphocyte response to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

[0319]

[0320] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order, with or without a linker. A short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A prominent linker is a glycine-serine doublet.

[0320]

[0321] As an example, the cytoplasmic domain may include a signaling domain of CD3-zeta and a signaling domain of CD28. In another example, the cytoplasmic domain may include a signaling domain of CD3-zeta and a signaling domain of CD27. In a further example, the cytoplasmic domain may include a signaling domain of CD3-zeta, a signaling domain of CD28, and a signaling domain of CD27.

[0321]

[0322] As mentioned above, the extracellular portion or transmembrane or cytoplasmic domain of the CAR may also contain a heterodimerization domain for the purpose of splitting the key signaling and antigen recognition modules of the CAR.

[0322]

[0323] Non-limiting examples of CARs that may be used in accordance with the present invention are set forth in SEQ ID NOs: 165-167, 266 or 272. Examples of the structures of various CAR molecules are also provided herein in FIG.

[0323]

[0324] CARs for use according to the present invention, i.e., CARs comprising an nfP2X7E200 binding domain, can be designed to comprise any portion or part of the above domains described herein in any order and / or combination, resulting in a functional CAR.

[0324]

[0325] The CAR disclosed herein, or a polypeptide(s) derived therefrom, a nucleic acid molecule(s) encoding said CAR, or a recombinant expression vector cell, or a cell population expressing said CAR, can be isolated and / or purified. The term "isolated" means altered or removed from a natural state. For example, an isolated cell population refers to the enrichment of such cells and separation from other cells that normally associate with said isolated cells in their naturally occurring state. An isolated cell population refers to a population of substantially purified cells that is a more homogeneous cell population than found in nature. Preferably, the enriched cell population comprises at least about 90% of a selected cell type. In certain embodiments, the cell population comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of a selected cell type.

[0325]

[0326] The affinity with which the dysfunctional P2X7 receptor binding domain of the CAR binds to the nfP2X7 recognition site E200 of the bridging molecule can vary, but generally the binding affinity can be in the range of 100 μm, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, and even more preferably at least about 100 pM.

[0326]

[0327] CAR T cells targeted to EGFRvIII can be used to treat solid cancers. EGFRvIII is a frequent splice variant of EGFR skipping exons 2-7. EGFRvIII is tumor specific and does not occur in healthy cells because EGFR is tightly regulated in normal cells. EGFRvIII is commonly expressed in glioblastomas, but also in breast and head and neck cancers. EGFRvIII-CAR T in this context can have the sequence (SEQ ID NO: 266) and can be targeted to an epitope resulting from the fusion of an amino acid sequence starting from LEEKK at positions 25-29, followed by a G insertion followed by the amino acid sequence NYVVTDH at positions 298-304, with the entire epitope comprising or consisting of the sequence LEEKKGNYVVTDH (SEQ ID NO: 267). The complete EGFR sequence is found in UniProtKB-P00533 (EGFR_HUMAN) and the complete protein counts 1210 amino acids in isoform 1.

[0327]

[0328] EGFRvIII targeting CAR T cells can be used to treat glioblastoma in the conventional way of targeting EGFRvIII on cancer cells, but can also be redirected to other cancer-related target antigens via the bridging molecule described herein, when the EGFRvIII epitope portion is incorporated into the sequence of the bridging molecule.Then, EGFRvIII CAR T cells can be used in the same way as outlined for the nfP2X7CAR targeting approach described herein.The peptide tag can be the 13-mer peptide LEEKKGNYVVTDH of SEQ ID NO:267, or its shortened or extended natural or artificial variants.

[0328]

[0329] The amino acid sequences of EGFRvIII CAR compatible bridge molecules targeted to CD33 and Her2 are set forth in Table 1 as SEQ ID NOs: 268 and 269, and SEQ ID NOs: 270 and 271, respectively.

[0329]

[0330] CLDN6-targeted CAR T cells can be used to treat solid cancers, such as ovarian cancer. The CLDN6-CAR T in this context may have the sequence (SEQ ID NO: 272) and may be targeted to the second extracellular domain of CLDN6 [UniProtKB-P56747(CLD6_HUMAN] cells directly via the amino acid sequence [ECD2,>sp|P56747|138-160WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 273)], but may also be redirected to other cancer-associated target antigens, e.g., CD33 or Her2, via the bridging molecules described herein, if a CLDN6 epitope portion is incorporated into the sequence of the bridging molecule. The CLDN6 CAR T cells can then be used in the same way as outlined for the nfP2X7CAR targeting approach described herein. The peptide tag may be the 23-mer peptide WTAHAIIRDFYNPLVAEAQKREL, or a shortened or extended natural or artificial variant thereof, e.g., SEQ ID NO: 274 or 275.

[0330] crosslinking molecule

[0331] It is understood that the bridging molecule can be in any form, provided that it comprises a) a targeting portion for binding to target cell, and b) a tumor-specific antigen epitope portion.Preferably, the tumor-specific antigen epitope portion is a dysfunctional P2X7 receptor epitope portion, an EGFRvIII epitope portion or a CLDN6 epitope portion.

[0331]

[0332] As used herein, reference to a bridge molecule may also be by using the term "BRiDGE."

[0333] Typically, the targeting moiety is in the form of a fusion protein in which the targeting moiety is linked directly or via a linker to a tumor-specific antigen epitope moiety, preferably a dysfunctional P2X7 receptor epitope moiety.

[0332]

[0334] Any suitable linker can be used. The linker can comprise a polypeptide, a peptide, or a chemical group.

[0335] The linker can be a peptide with a length of up to 20, 30, 40 or 50 amino acids. The term "linked to" or "fused to" refers to a covalent bond formed between two moieties, such as a peptide bond. Thus, in the context of the present invention, the linker can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acids. For example, the bridging molecule provided herein can include a linker between the targeting moiety and the tumor-specific antigen epitope moiety, preferably a dysfunctional P2X7 receptor epitope moiety. Such a linker has the advantage that it can increase the likelihood that different polypeptides of the fusion protein fold independently and behave predictably.

[0333]

[0336] Those skilled in the art are familiar with the design and use of various peptide linkers, including various amino acids and various lengths, that are suitable for use as linkers according to the present invention.Linkers can include various combinations of repeated amino acid sequences.Linkers can be flexible linkers (e.g., those that include repeats of glycine and serine residues), rigid linkers (e.g., those that include glutamic acid and lysine residues, adjacent alanine repeats), and / or cleavable linkers (e.g., sequences that are more susceptible to protease cleavage).

[0334]

[0337] The peptide linker may be any one or more repeats of Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS), or variations thereof. In one embodiment, the linker may comprise or consist of the sequence GGGGSGGGGSGGGGS, i.e., (G4S)3.

[0335]

[0338] In one embodiment, the peptide linker can comprise the amino acid sequence GGGGGS (a linker of 6 amino acids in length) or longer. The linker can be a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n, where n is any number from 1 to 15 or more. For example, the linker can be (GS)3 (i.e., GSGSGS) or longer (GS) 11 It is understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more.

[0336]

[0339] The peptide linker can consist of a series of repeats of Thr-Pro (TP) with one or more additional amino acids N- and C-terminal to the repeat sequence. For example, the linker can comprise or consist of the sequence GTPTPTPTPTGEF (also known as the TP5 linker). In further embodiments, the linker can be a short and / or alpha-helical rigid linker (e.g., A(EAAAK)3A, PAPAP, or a dipeptide such as LE or CC).

[0337]

[0340] Linkers can also be composed of glycine-serine based linkers and linkers derived from immunoglobulin hinge regions. Examples of such linker combinations are provided in Table 1 (e.g., G4S+hinged+G4S+E200 sequence derived from IgG). It is also understood that the targeting moiety and tumor specific antigen epitope moiety, preferably dysfunctional P2X7 receptor epitope moiety, can be linked via their C- or N-terminal regions. For example, the E200 epitope sequence can be linked via its C-terminus to the N-terminal region of the targeting moiety (including either the heavy or light chain of the targeting moiety). Similarly, the E200 epitope sequence can be linked via its N-terminus to the C-terminal region of the targeting moiety (including either the heavy or light chain of the targeting moiety). It is within the scope of those skilled in the art that as described herein, suitable cross-linking molecules can be designed and generated for use according to the present invention.

[0338] Targeting moiety of the bridging molecule

[0341] The targeting portion of the bridge molecule may bind to a cell surface molecule on a target cell. The cell surface molecule may include an antigen. The cell surface molecule may be selected from a protein, a lipid moiety, a glycoprotein, a glycolipid, a carbohydrate, a polysaccharide, a nucleic acid, an MHC-binding peptide, or a combination thereof. The cell surface molecule may include parts of bacteria, viruses, and other microorganisms (e.g., capsules, cell walls, flagella, pili, and toxins). The cell surface molecule may be expressed by a target cell. The cell surface molecule may not be expressed by a target cell. By way of non-limiting example, the cell surface molecule may be a ligand expressed by a cell other than the target cell that binds to the target cell or the cell surface molecule of the target cell. Also by way of non-limiting example, the cell surface molecule may be a toxin, an exogenous molecule, or a viral protein that binds to the cell surface or cell surface receptor of the target cell.

[0339]

[0342] The bridge molecule may interact with multiple target cells. The target cell may be an infected cell. The target cell may be a pathogenically infected cell. The target cell may be a diseased cell. The target cell may be a genetically modified cell. The target cell may not be a host cell. The target cell may be derived from an invaded organism (e.g., yeast, nematode, bacteria, fungi). Further disclosed herein are bridge molecules that interact with molecules on non-cellular targets. The non-cellular target may be a virus or a part thereof. The non-cellular target may be a fragment of a cell. The non-cellular target may be an extracellular matrix component or a protein.

[0340]

[0343] The target cells can be derived from tissues. The tissues can be selected from brain, esophagus, breast, digestive tract, intestine, colon, lung, glia, ovary, uterus, testis, prostate, gastrointestinal tract, bladder, liver, spleen, thymus, bone, fat and skin. The target cells can be derived from one or more endocrine glands. Alternatively, or in addition, the target cells can be derived from one or more endocrine glands. The endocrine glands can be lymph glands, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonads or pineal gland.

[0341]

[0344] The target cell may be selected from a stem cell, a pluripotent cell, a hematopoietic stem cell or a progenitor cell. The target cell may be a circulating cell. The target cell may be an immune cell.

[0345] The target cells can be cancer stem cells. The target cells can be cancer cells. The cancer cells can be derived from tissues. The tissues can be selected from, by way of non-limiting example, brain, esophagus, breast, colon, lung, glia, ovary, uterus, testis, prostate, gastrointestinal tract, bladder, liver, thyroid and skin. The cancer cells can be derived from bone. The cancer cells can be derived from blood. The cancer cells can be derived from B cells, T cells, monocytes, platelets, leukocytes, neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, or endothelial cell progenitor cells. The cancer cells can be derived from CD19 positive B lymphocytes. The cancer cells can be derived from stem cells. The cancer cells can be derived from pluripotent cells. The cancer cells can be derived from one or more endocrine glands. The endocrine glands can be lymphatic glands, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonads, or pineal gland.

[0342]

[0346] The cell surface molecule of the target cell can be a receptor. The receptor can be an extracellular receptor. The receptor can be a cell surface receptor. As a non-limiting example, the receptor can bind to a hormone, a neurotransmitter, a cytokine, a growth factor, or a cell recognition molecule. The receptor can be a transmembrane receptor. The receptor can be an enzyme-linked receptor. The receptor can be a G-protein coupled receptor (GPCR). The receptor can be a growth factor receptor. As a non-limiting example, the growth factor receptor can be selected from epidermal growth factor receptor, fibroblast growth factor receptor, platelet derived growth factor receptor, neuronal growth factor receptor, transforming growth factor receptor, bone morphogenetic protein growth factor receptor, hepatocyte growth factor receptor, vascular endothelial growth factor receptor, stem cell factor receptor, insulin growth factor receptor, somatomedin receptor, erythropoietin receptor, and homologs and fragments thereof. The receptor can be a hormone receptor. The receptor can be an insulin receptor. As non-limiting examples, the receptor may be selected from eicosanoid receptors, prostaglandin receptors, estrogen receptors, follicle-stimulating hormone receptors, progesterone receptors, growth hormone receptors, gonadotropin-releasing hormone receptors, homologs thereof, and fragments thereof. The receptor may be an adrenergic receptor. The receptor may be an integrin. The receptor may be an Eph receptor. The receptor may be a luteinizing hormone receptor. The cell surface molecule may be at least about 50% homologous to the luteinizing hormone receptor. The receptor may be an immune receptor. As non-limiting examples, the immune receptor may be selected from pattern recognition receptors, Toll-like receptors, NOD-like receptors, killer activating receptors, killer inhibitor receptors, Fc receptors, B-cell receptors, complement receptors, chemokine receptors, and cytokine receptors. As non-limiting examples, the cytokine receptor may be selected from interleukin receptors, interferon receptors, transforming growth factor receptors, tumor necrosis factor receptors, colony-stimulating factor receptors, homologs thereof, and fragments thereof. The receptor may be a receptor kinase. The receptor kinase may be a tyrosine kinase receptor. The receptor kinase may be a serine kinase receptor. The receptor kinase may be a threonine kinase receptor.As non-limiting examples, the receptor kinase can activate a signaling protein selected from Ras, Raf, PI3K, protein kinase A, protein kinase B, protein kinase C, AKT, AMPK, phospholipase, homologs thereof, and fragments thereof. The receptor kinase can activate the MAPK / ERK signaling pathway. The receptor kinase can activate Jak, Stat, or Smad.

[0343]

[0347] The cell surface molecule can be a non-receptor cell surface protein. The cell surface molecule can be a cluster of differentiation proteins. As a non-limiting example, the cell surface molecule can be selected from CD3, CD4, CD8, CD11a, CD11b, CD13, CD14, CD15, CD16, CD22, CD24, CD25, CD30, CD31, CD33, CD34, CD38, CD45, CD56, CD61, CD91, CD114, CD117, CD182, CD200, fragments thereof, and homologs thereof.

[0344]

[0348] The cell surface molecule of the target cell may be a non-peptide molecule. The cell surface molecule may comprise a lipid. The cell surface molecule may comprise a lipid moiety or lipid group. The lipid moiety may comprise a sterol. The lipid moiety may comprise a fatty acid. The antigen may comprise a glycolipid. The cell surface molecule may comprise a carbohydrate.

[0345]

[0349] The cell surface molecule of the target cell can be an antigen. The antigen can be at least a portion of a surface antigen or cell surface marker on the cell. The antigen can be a receptor or co-receptor on the cell. The antigen can refer to a molecule or molecular fragment that can undergo binding of the major histocompatibility complex (MHC) and be presented to a T cell receptor. The term "antigen" can also refer to an immunogen. An immunogen can trigger an adaptive immune response when injected into a subject alone. An immunogen can induce an immune response by itself. An antigen can be a superantigen, a T-dependent antigen, or a T-independent antigen. The antigen can be an exogenous antigen. An exogenous antigen is typically an antigen that enters the body from outside the body, for example, by inhalation, ingestion, or injection. Some antigens start out as exogenous antigens and can later become endogenous (e.g., intracellular viruses). The antigen can be an endogenous antigen. An endogenous antigen can be an antigen that is produced within a cell as a result of normal cellular metabolism or due to a pathogenic infection (e.g., viral, bacterial, fungal, parasitic). The antigen may be an autoantigen. An autoantigen may be a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a particular autoimmune disease. These antigens are not targets of the immune system under normal conditions, but due to genetic and / or environmental factors, normal immune tolerance to such antigens should not be present in these patients. The antigen may be present or overexpressed due to a condition or disease. The condition or disease may be cancer or leukemia. The condition may be an inflammatory disease or condition. The condition or disease may be a metabolic disease. The condition may be a genetic disorder.

[0346]

[0350] The present invention can also find application for treating specific B or T cell lineage-related autoimmune diseases, for example, by using anti-idiotypic antibodies or their fragments or their ligands to target B cell receptors and / or T cell receptors. Such diseases include myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), hyperacute rejection, acute rejection, chronic rejection or mixed rejection of solid organ transplants, rejection of bone marrow or stem cell transplants, graft-versus-host disease. The present invention can also find application in immune regulation, more broadly, for example, targeting one or more of the following proteins / receptors on immune cells: PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, and KIR to promote immune suppression.

[0347]

[0351] The cell surface molecules of the target cells may be antigens designated as tumor antigens. Tumor antigens or neoantigens may be antigens presented by MHC I or MHC II molecules on the surface of tumor cells. These antigens may be presented by tumor cells and not by normal antigens. In this case, they are called tumor-specific antigens (TSA) and generally result from tumor-specific mutations. More common are antigens presented by tumor cells and normal cells, which are called tumor-associated antigens (TAA). Cytotoxic T lymphocytes that recognize these antigens can destroy tumor cells before they proliferate or metastasize. Tumor antigens may also be on the surface of tumors, for example in the form of mutated receptors, in which case they can be recognized by B cells.

[0348]

[0352] The cell surface molecule of the target cell can be an antigen selected from the group consisting of any surface expressed antigen. Exemplary target antigens include, but are not limited to, CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRβ, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), ...44V6, CD47, CD184 (CXCR4), CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (L1CAM), CD200 (OX-2), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and EGFR. The cell surface molecule of the target cell may include any accessible epitope that can be recognized by a TCR, MHC I or II presented peptide, sugar, lipid, carbohydrate chain, or binding domain. The antigen may be any of those mentioned in Table 1 in conjunction with the bridging (BRiDGE) molecule.

[0349]

[0353] Suitable cancer antigens that may be conjugated to the targeting portion of the cross-linking molecule include, but are not limited to, mesothelin (MSLN), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, epithelial glycoprotein (EGFP), and / or EGFR-specific markers. 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α and β (FRα and β), ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2 / ERB2), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), mucin 16 (Muc -16), mucin 1 (Muc-1), NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), tyrosine protein kinase transmembrane receptor type 1 (ROR1), B7-H3 (CD276), B7-H6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX accessory molecule (DNAM-1), ephrin type A receptor 2 (EpHA2), fibroblast-associated protein (FAP), Gpl00 / HLA-A2, glypican 3 (GPC3), HA-1H, HERK-V, IL-11Ra, latent membrane protein 1 (LMP1), neural cell adhesion molecule (N-CAM / CD56) and TRAIL receptor (TRAIL R). It will be appreciated that these or other cancer antigens can be utilized for targeting by the bridging molecules in the present invention.

[0350]

[0354] The targeting moiety of the bridging molecule can be any binding molecule, such as a full size antibody or fragment thereof, any antibody or fragment thereof described herein, an immunocytokine (an antibody linked to a cytokine or fragment thereof), a ligand (a protein-related peptide, a sugar molecule, a processed molecule, a lipid, a cytokine, a hormone), a soluble T cell receptor (TcR), a single chain (sc) TcR, a single chain T cell receptor binding motif and a T cell receptor-like mAb, an aptamer (such as DNA or RNA), a peptide (e.g., an aptamer or a bicyclic peptide), a toxin, a lipid or a carbohydrate.

[0351]

[0355] The targeting portion of the bridging molecule may be a polypeptide and may be a targeting antibody or antibody fragment. The targeting antibody or antibody fragment may be an immunoglobulin (Ig). The immunoglobulin may be selected from IgG, IgA, IgD, IgE, IgM, fragments thereof or variants thereof. The immunoglobulin may be an IgG. The IgG may be an IgG1. The IgG may be an IgG2. The IgG may be an IgG3. The IgG may be an IgG4. The IgG may have one or more Fc mutations to modulate endogenous T cell FcR binding to the bridging molecule. The IgG may have one or more Fc mutations to remove Fc binding ability to FcR of FcR positive cells. The one or more Fc mutations may remove glycosylation sites. The one or more Fc mutations may be selected from E233P, L234V, L235A, delG236, A327G, A330S, P331S, N297Q and any combination thereof. The one or more Fc mutations may be in IgG1. The one or more Fc mutations in IgG1 may be L234A, L235A, or both. Alternatively, or in addition, the one or more Fc mutations in IgG1 may be L234A, L235E, or both. Alternatively, or in addition, the one or more Fc mutations in IgG1 may be N297A. Alternatively, or in addition, the one or more mutations may be in IgG2. The one or more Fc mutations in IgG2 may be V234A, V237A, or both.

[0352]

[0356] The targeting antibody or antibody fragment may be an Fc null immunoglobulin or a fragment thereof.

[0357] As used herein, the term "antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components present in a full-length antibody, and engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, and bispecific antibodies. Unless otherwise specified, statements and claims using the term "antibody" or "antibodies" can specifically include "antibody fragment" and "antibody fragments".

[0353]

[0358] The targeting antibody fragment may be a human, fully human, humanized, human engineered, non-human, and / or chimeric antibody. The non-human antibody may be humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. A chimeric antibody may refer to an antibody created through the joining of two or more antibody genes originally coded for separate antibodies. A chimeric antibody may contain at least one amino acid from a first antibody and at least one amino acid from a second antibody, where the first and second antibodies are different. At least a portion of the antibody or antibody fragment may be from a bovine species, a human species, or a murine species. At least a portion of the antibody or antibody fragment may be from a rat, a goat, a guinea pig, or a rabbit. At least a portion of the antibody or antibody fragment may be from a human. At least a portion of the antibody or antibody fragment may be from a cynomolgus monkey.

[0354]

[0359] Targeting antibody or antibody fragment can be based on or derived from antibody or antibody fragment from mammal, bird, fish, amphibian or reptile.Mammals include, but are not limited to, carnivores, rodents, elephants, marsupials, rabbits, bats, primates, seals, anteaters, whales, ungulates, and ungulates.Mammals can be humans, non-human primates, mice, sheep, cats, dogs, cows, horses, goats, or pigs.

[0355]

[0360] The targeting antibody or antibody fragment can recognize or bind to an antigen selected from, by way of non-limiting example, CD19, Her2, CLL-1, CD33, EGFRvIII, CD20, CD22, BCMA, or a fragment thereof. The antigen can include a wild-type antigen. The antigen can include one or more mutations.

[0356]

[0361] The targeting antibody or antibody fragment may be an anti-CD19 antibody or a fragment thereof. The targeting polypeptide may be an anti-CD22 antibody. The targeting polypeptide may be an anti-BCMA antibody or a fragment thereof. The targeting polypeptide may be an anti-EGFRvIII antibody or a fragment thereof. The targeting polypeptide may be an anti-Her2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD20 antibody or an antibody fragment. The targeting polypeptide may comprise rituximab. The targeting polypeptide may comprise an anti-EGFR antibody or an antibody fragment. The targeting polypeptide may comprise an anti-CEA antibody or an antibody fragment. The targeting polypeptide may comprise an anti-CLL-1 antibody or an antibody fragment. The targeting polypeptide may comprise an anti-CD33 antibody or an antibody fragment. The targeting polypeptide may comprise an anti-EpCAM antibody or a fragment thereof.

[0357]

[0362] Targeting antibody or antibody fragment can be selected from any commercially available antibody.Targeting antibody or antibody fragment can be selected from ado-trastuzumab emtansine, alemtuzumab, bevacizumab, brentuximab, vedotin, gemtuzumab, ozogamicin, ipilimumab, ibritumomab, tiuxetan, panitumumab, cetuximab, erbitux, rituximab, trastuzumab and their fragments.Targeting antibody or antibody fragment can be any of those mentioned in Table 1.

[0358]

[0363] The targeting moiety of the bridging molecule can target a peptide-MHC complex, and in such embodiments, the targeting moiety can be a soluble TcR molecule or a single chain TcR molecule.

[0359]

[0364] Non-limiting examples of sequences of various targeting antibodies, or antigen-binding fragments thereof, are provided herein in Table 1. Dysfunctional P2X7 receptor epitope region

[0365] The dysfunctional P2X7 receptor epitope part can be provided in the form of dysfunctional P2X7 receptor or the fragment of dysfunctional P2X7 receptor, which has at least one of three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP.Such receptor cannot extend the opening of non-selective calcium channel to apoptotic pore.

[0360]

[0366] A range of peptide fragments of dysfunctional P2X7 receptors are known and may be found in PCT / AU2002 / 000061 (corresponding International Publication No. WO 2002 / 057306, as well as U.S. Pat. Nos. 7,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 9,663,584, or 10,462, the entire contents of which are incorporated herein by reference in their entirety. 50,380), PCT / AU2008 / 001364 (corresponding International Publication No. WO 2009 / 033233, and U.S. Pat. Nos. 8,440,186, 9,181,320, 9,944,701, or 10,597,45), and PCT / AU2009 / 000869 (corresponding International Publication No. WO 2010 / 000041, and U.S. Pat. Nos. 8,597,643, 9,328,155, or 10,238,716). Exemplary peptides within these specifications that contain epitopes contemplated for use in the present invention are described below. PCT Application Published Peptide Sequence WO 2002 / 057306 GHNYTTRNILPGLNITC (SEQ ID NO:2) (also referred to herein as the "E200" epitope) WO 2009 / 033233 KYYKENNVEKRTLIKVF (SEQ ID NO: 12) (also referred to herein as the "E300" epitope) WO 2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14) (also referred to herein as the "E200 / E300" or "composite" epitope)

[0367] In any embodiment, the amino acid sequence of the dysfunctional P2X7 receptor epitope portion of any of the cross-linking molecules described herein is a sequence shown in any of SEQ ID NOs: 2-30, 168, 361-396, 437 and 438, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.Preferably, the dysfunctional P2X7 receptor epitope portion comprises at least the sequence of SEQ ID NO: 11.

[0361]

[0368] The dysfunctional P2X7 receptor epitope portion can have any functional group, such as a carboxyl group, active ester, acetamide or maleimide, that can be coupled to a targeting moiety disclosed herein, such as an antibody or fragment thereof, using an NH2 or SH group for coupling.

[0362] EGFRvIII epitope portion

[0369] In any embodiment, the amino acid sequence of the EGFRvIII epitope portion of any of the cross-linking molecules described herein is a sequence set forth in any of SEQ ID NOs:267, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical thereto. Preferably, the EGFRvIII epitope portion comprises at least the sequence of SEQ ID NO:267.

[0363] CLDN6 epitope portion

[0370] In any embodiment, the amino acid sequence of the CLDN6 epitope portion of any of the bridge molecules described herein is a sequence set forth in any of SEQ ID NOs: 273, 274 or 275, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical thereto. Preferably, the CLDN6 epitope portion comprises at least the sequence of SEQ ID NO: 273, 274 or 275.

[0364] Exemplary Cross-Linking Molecules

[0371] The present specification provides various non-limiting examples of tumor-specific antigen epitope moiety (eg, dysfunctional P2X7 receptor epitope moiety) / targeting moiety pairs.

[0365]

[0372] Exemplary bridging molecules are described in Table 1. For those bridging molecules described in Table 1 that contain an nfP2X7 epitope portion, the present specification includes those BRiDGEs, but with the nfP2X7 epitope portion replaced with an EGFRvIII or CLDN6 epitope portion.

[0366]

[0373] In examples where the bridging molecule comprises a targeting moiety for binding to CD19, the targeting moiety can comprise a combination of heavy and paired light chain variable chains set forth in SEQ ID NOs: 31 and 32; or 143 and 144 (heavy and light chains, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical thereto.

[0367]

[0374] In the above embodiment, the bridging molecule can comprise tumor-specific antigen epitope portion (e.g., dysfunctional P2X7 receptor epitope portion) conjugated to heavy chain, or tumor-specific antigen epitope portion (e.g., dysfunctional P2X7 receptor epitope portion) conjugated to light chain.Preferably, tumor-specific antigen epitope portion (e.g., dysfunctional P2X7 receptor epitope portion) is conjugated to light chain of target binding portion.

[0368]

[0375] In any embodiment in which the bridging molecule comprises a CD19-binding heavy / light chain pair, when the heavy chain comprises a dysfunctional P2X7 receptor epitope portion, the sequence of the variable sequence of the heavy and light chain pair is preferably selected from SEQ ID NOs: 33 and 32; 34 and 32, 37 and 32; 37 and 38 (respectively the heavy and light chain sequences listed), or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical thereto.

[0369]

[0376] In any embodiment in which the bridging molecule comprises or consists of a CD19-binding heavy chain / light chain pair, where the light chain comprises a dysfunctional P2X7 receptor epitope portion, the sequence of the variable sequences of the heavy and light chain pair is preferably SEQ ID NO: 31 and 35; 31 and 36; 39 and 31; 52 and 51; 143 and 145; 143 and 146; 143 and 147; 143 and 148; 143 and 149; 143 and 150; 143 and 151; 143 and 152; 143 and 153; 143 and 154; 143 and 155; 143 and 1561; 143 and 157; 143 and 158; 143 and 159; or 143 and 164 (heavy and light chain sequences as listed, respectively), or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In another embodiment, where the bridging molecule comprises a CD19-binding heavy / light chain pair, the light chain is any one of the light chains above and the heavy chain is selected from SEQ ID NO: 141 or 142.

[0370]

[0377] The targeting moiety can be in the form of an scFv comprising a heavy and a light chain.

[0378] In any embodiment, a CD19-binding scFv for use in a bridging molecule of the invention may have a sequence as set forth in SEQ ID NO: 40 or 41, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In the context of scFv, it is understood that the dysfunctional P2X7 receptor epitope portion can be conjugated to the light chain of the scFv, such as any of SEQ ID NOs: 42, 43, 46, 48, or the heavy chain of the scFv, such as any of SEQ ID NOs: 44, 45, 360, 47, 49, 50, or to a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0371]

[0379] In any embodiment, a bridging molecule for binding to CD20 can comprise or consist of a sequence set forth in SEQ ID NOs: 53 and 54, or SEQ ID NOs: 55 and 56 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0372]

[0380] In any embodiment, a bridging molecule for binding to CD22 can comprise or consist of SEQ ID NOs: 57 and 58, or SEQ ID NOs: 59 and 60 (the light and heavy chain sequences listed, respectively), or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0373]

[0381] In any embodiment, a bridging molecule for binding to CD79B may comprise or consist of a sequence as set forth in SEQ ID NOs: 61 and 62 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0374]

[0382] In any embodiment, a bridging molecule for binding to CD37 can comprise or consist of a sequence set forth in SEQ ID NOs: 63 and 64 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0375]

[0383] In any embodiment, a bridging molecule for binding to CD38 can comprise or consist of a sequence set forth in SEQ ID NOs: 65 and 66 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0376]

[0384] In any embodiment, a bridging molecule for binding to CD70 can comprise or consist of a sequence set forth in SEQ ID NOs: 67 and 68 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0377]

[0385] In any embodiment, a bridging molecule for binding to CD30 can comprise or consist of a sequence set forth in SEQ ID NOs: 39 and 70 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0378]

[0386] In any embodiment, a bridging molecule for binding to CD33 can comprise or consist of a sequence set forth in SEQ ID NOs: 71 and 72 or 73 and 74 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0379]

[0387] In any embodiment, a bridging molecule for binding to Her2 can comprise or consist of a sequence set forth in SEQ ID NOs: 75 and 75; or 77 and 78 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0380]

[0388] In any embodiment, a bridging molecule for binding to EGFR can comprise or consist of a sequence as set forth in SEQ ID NOs: 79 and 80 or 81 and 82 or 83 and 84 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0381]

[0389] In any embodiment, a bridging molecule for binding to CD276 can comprise or consist of a sequence set forth in SEQ ID NOs: 85 and 86 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0382]

[0390] In any embodiment, a bridging molecule for binding to GD2 can comprise or consist of a sequence set forth in SEQ ID NOs: 87 and 88 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0383]

[0391] In any embodiment, a bridging molecule for binding to BCMA may comprise or consist of a sequence as set forth in SEQ ID NOs: 89 and 90 (the light and heavy chain sequences listed respectively), or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0384]

[0392] In any embodiment, a bridging molecule for binding to CD371 can comprise or consist of a sequence set forth in SEQ ID NOs: 91 and 92 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0385]

[0393] In any embodiment, a bridging molecule for binding to CD135 can comprise or consist of a sequence set forth in SEQ ID NOs: 93 and 94 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0386]

[0394] In any embodiment, a bridging molecule for binding to CD123 can comprise or consist of a sequence set forth in SEQ ID NOs: 95 and 95 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0387]

[0395] In any embodiment, a bridging molecule for binding to CD105 can comprise or consist of a sequence set forth in SEQ ID NOs: 97 and 98 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0388]

[0396] In any embodiment, a bridging molecule for binding to ROR-1 can comprise or consist of a sequence set forth in SEQ ID NOs: 99 and 100 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0389]

[0397] In any embodiment, a linking molecule for binding to PD-L1 may comprise or consist of a sequence as set forth in SEQ ID NOs: 101 and 102 (the listed light and heavy chain sequences, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0390]

[0398] In any embodiment, a bridging molecule for binding to MET-R can comprise or consist of a sequence set forth in SEQ ID NOs: 103 and 104 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0391]

[0399] In any embodiment, a bridging molecule for binding to PDGFRalpha can comprise or consist of a sequence set forth in SEQ ID NOs: 105 and 106 or 107 and 108 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0392]

[0400] In any embodiment, a bridging molecule for binding to Her3 can comprise or consist of a sequence set forth in SEQ ID NOs: 109 and 110 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0393]

[0401] In any embodiment, a bridging molecule for binding to FR alpha can comprise or consist of the sequences set forth in SEQ ID NOs: 111 and 112 (the light and heavy chain sequences listed, respectively), or sequences that are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0394]

[0402] In any embodiment, a bridging molecule for binding to CGPC3 can comprise or consist of a sequence set forth in SEQ ID NOs: 113 and 114 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0395]

[0403] In any embodiment, a bridging molecule for binding to SLAMF7 can comprise or consist of a sequence set forth in SEQ ID NOs: 115 and 116 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0396]

[0404] In any embodiment, a bridging molecule for binding to TNFRSF10B can comprise or consist of a sequence set forth in SEQ ID NOs: 117 and 118 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0397]

[0405] In any embodiment, a bridging molecule for binding to GPNMB can comprise or consist of a sequence set forth in SEQ ID NOs: 119 and 120 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0398]

[0406] In any embodiment, a bridging molecule for binding to VEGFR2 can comprise or consist of a sequence set forth in SEQ ID NOs: 121 and 122 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0399]

[0407] In any embodiment, a bridging molecule for binding to α4β7 and / or αEβ7 can comprise or consist of a sequence set forth in SEQ ID NOs: 123 and 124; or 125 and 126 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0400]

[0408] In any embodiment, a bridging molecule for binding to CSPG4 can comprise or consist of a sequence set forth in SEQ ID NOs: 127 and 128 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0401]

[0409] In any embodiment, a bridging molecule for binding to CD80 can comprise or consist of a sequence set forth in SEQ ID NOs: 129 and 130 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0402]

[0410] In any embodiment, a bridging molecule for binding to CCR4 can comprise or consist of a sequence set forth in SEQ ID NOs: 131 and 132 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0403]

[0411] In any embodiment, a bridging molecule for binding to CD115 can comprise or consist of a sequence set forth in SEQ ID NOs: 133 and 134 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0404]

[0412] In any embodiment, a bridging molecule for binding to ENOX-2 can comprise or consist of a sequence set forth in SEQ ID NOs: 135 and 136 (respectively the listed light and heavy chain sequences), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0405]

[0413] In any embodiment, a bridging molecule for binding to CD56 can comprise or consist of a sequence set forth in SEQ ID NOs: 137 and 138 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0406]

[0414] In any embodiment, a bridging molecule for binding to huVH1-69 can comprise or consist of a sequence set forth in SEQ ID NOs: 139 and 140 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0407]

[0415] In any embodiment, a bridging molecule for binding to CD117 can comprise or consist of a sequence set forth in SEQ ID NOs: 169 and 170 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0408]

[0416] In any embodiment, a bridging molecule for binding to CD133 can comprise or consist of a sequence set forth in SEQ ID NOs: 171 and 172 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0409]

[0417] In any embodiment, a bridge molecule for binding to MUC1 can comprise or consist of a sequence set forth in SEQ ID NOs: 173 and 174 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0410]

[0418] In any embodiment, a bridging molecule for binding to mesothelin can comprise or consist of a sequence set forth in SEQ ID NOs: 175 and 176 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0411]

[0419] In any embodiment, a bridging molecule for binding to ROR2 can comprise or consist of a sequence set forth in SEQ ID NOs: 177 and 178 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0412]

[0420] In any embodiment, a bridging molecule for binding to IL13Ra2 can comprise or consist of a sequence as set forth in SEQ ID NOs: 179 and 180 (respectively the listed light and heavy chain sequences), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0413]

[0421] In any embodiment, a bridge molecule for binding to IL13Ra2 can comprise or consist of a sequence as set forth in SEQ ID NO: 181, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0414]

[0422] In any embodiment, a bridging molecule for binding to EPHA2 can comprise or consist of a sequence set forth in SEQ ID NOs: 182 and 183 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0415]

[0423] In any embodiment, a bridging molecule for binding to EGFRvIII can comprise or consist of a sequence set forth in SEQ ID NOs: 184 and 185 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0416]

[0424] In any embodiment, a linking molecule for binding to PSMA can comprise or consist of a sequence set forth in SEQ ID NOs: 186 and 187 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical thereto.

[0417]

[0425] In any embodiment, a bridging molecule for binding to CEA can comprise or consist of a sequence set forth in SEQ ID NOs: 188 and 189 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0418]

[0426] In any embodiment, a linking molecule for binding to PSCA can comprise or consist of a sequence set forth in SEQ ID NOs: 190 and 191 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0419]

[0427] In any embodiment, a bridging molecule for binding to Lewis Y can comprise or consist of a sequence set forth in SEQ ID NOs: 192 and 193 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0420]

[0428] In any embodiment, a bridging molecule for binding to CD171 LICAM can comprise or consist of a sequence as set forth in SEQ ID NOs: 194 and 195 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0421]

[0429] In any embodiment, a bridging molecule for binding to EpCAM can comprise or consist of a sequence set forth in SEQ ID NOs: 196 and 197 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0422]

[0430] In any embodiment, a bridge molecule for binding to ALK can comprise or consist of a sequence set forth in SEQ ID NOs: 198 and 199 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0423]

[0431] In any embodiment, a bridging molecule for binding to IGF-1R CD221 can comprise or consist of a sequence set forth in SEQ ID NOs: 200 and 201 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0424]

[0432] In any embodiment, a bridging molecule for binding to Nectin 4 can comprise or consist of a sequence set forth in SEQ ID NOs: 202 and 203 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0425]

[0433] In any embodiment, a bridging molecule for binding to a FAP can comprise or consist of a sequence set forth in SEQ ID NOs: 204 and 205 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0426]

[0434] In any embodiment, a bridging molecule for binding to AXL can comprise or consist of a sequence set forth in SEQ ID NOs: 206 and 207 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0427]

[0435] In any embodiment, a bridging molecule for binding to CD138 can comprise or consist of a sequence set forth in SEQ ID NOs: 208 and 209 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0428]

[0436] In any embodiment, a bridging molecule for binding to CLDN6 can comprise or consist of a sequence set forth in SEQ ID NOs: 210 and 211 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0429]

[0437] In any embodiment, a bridging molecule for binding to Her4 can comprise or consist of a sequence set forth in SEQ ID NOs: 212 and 213 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0430]

[0438] In any embodiment, a bridging molecule for binding to claudin 18.2 can comprise or consist of a sequence set forth in SEQ ID NOs: 214 and 215 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0431]

[0439] In any embodiment, a bridging molecule for binding to O-acetylated GD2 can comprise or consist of a sequence set forth in SEQ ID NOs: 216 and 217 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0432]

[0440] In any embodiment, a bridging molecule for binding to GD3 can comprise or consist of a sequence set forth in SEQ ID NOs:218 and 219 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0433]

[0441] In any embodiment, a bridging molecule for binding to GM2 can comprise or consist of a sequence set forth in SEQ ID NOs:220 and 221 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99% identical thereto.

[0434]

[0442] In any embodiment, a bridging molecule for binding to TM4SF1 can comprise or consist of a sequence set forth in SEQ ID NOs: 222 and 223 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0435]

[0443] In any embodiment, a bridging molecule for binding to CD147 can comprise or consist of a sequence set forth in SEQ ID NOs: 224 and 225 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0436]

[0444] In any embodiment, a cross-linking molecule for binding to CEACAM5 can comprise or consist of the sequences set forth in SEQ ID NOs: 226 and 227 (the light and heavy chain sequences listed, respectively), or sequences that are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0437]

[0445] In any embodiment, a bridging molecule for binding to VEGFR-1 can comprise or consist of a sequence set forth in SEQ ID NOs: 228 and 229 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0438]

[0446] In any embodiment, a bridging molecule for binding to podoplanin (PDPN) can comprise or consist of a sequence set forth in SEQ ID NOs: 230 and 231 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0439]

[0447] In any embodiment, a bridging molecule for binding to WT1 can comprise or consist of a sequence set forth in SEQ ID NOs: 232 and 233 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0440]

[0448] In any embodiment, a bridging molecule for binding to GPC2 can comprise or consist of a sequence as set forth in SEQ ID NOs: 234 and 235 (respectively the listed light and heavy chain sequences), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0441]

[0449] In any embodiment, a bridging molecule for binding to FGFR4 can comprise or consist of a sequence set forth in SEQ ID NOs: 236 and 237 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0442]

[0450] In any embodiment, a bridging molecule for binding to EphB4 can comprise or consist of a sequence set forth in SEQ ID NOs: 238 and 239 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0443]

[0451] In any embodiment, a bridging molecule for binding to STEAP-1 can comprise or consist of a sequence set forth in SEQ ID NOs: 240 and 241 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0444]

[0452] In any embodiment, a bridging molecule for binding to STEAP-2 can comprise or consist of a sequence set forth in SEQ ID NOs: 242 and 243 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0445]

[0453] In any embodiment, a bridging molecule for binding to IL11Ra may comprise or consist of a sequence as set forth in SEQ ID NOs: 244 and 245 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0446]

[0454] In any embodiment, a bridging molecule for binding to CD163 can comprise or consist of a sequence set forth in SEQ ID NOs: 246 and 247 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0447]

[0455] In any embodiment, a bridging molecule for binding to chlorotoxin can comprise or consist of a sequence set forth in SEQ ID NOs:248 and 249 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0448]

[0456] In any embodiment, a bridging molecule for binding to CD206 can comprise or consist of a sequence set forth in SEQ ID NO:250 (heavy chain sequence listed), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0449]

[0457] In any embodiment, a bridging molecule for binding to IL1RAP can comprise or consist of a sequence set forth in SEQ ID NOs: 251 and 252 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0450]

[0458] In any embodiment, a bridging molecule for binding to MICA can comprise or consist of a sequence set forth in SEQ ID NOs:253 and 254 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0451]

[0459] In any embodiment, a bridging molecule for binding to MAGE-A1 may comprise or consist of the sequence set forth in SEQ ID NO:255, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0452]

[0460] In any embodiment, a bridging molecule for binding to MAGE-A1 may comprise or consist of a sequence as set forth in SEQ ID NOs: 256 and 257, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0453]

[0461] In any embodiment, a bridging molecule for binding to MAGE-A1 may comprise or consist of a sequence as set forth in SEQ ID NOs: 258 and 259, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0454]

[0462] In any embodiment, a bridging molecule for binding to TRBC1 can comprise or consist of a sequence set forth in SEQ ID NOs: 260 and 261 (the light chain and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0455]

[0463] In any embodiment, a bridging molecule for binding to TRBC2 can comprise or consist of a sequence set forth in SEQ ID NOs: 262 and 263 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0456]

[0464] In any embodiment, a bridging molecule for binding to urokinase-type plasminogen activator receptor (uPAR) can comprise or consist of SEQ ID NOs: 264 and 265 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0457]

[0465] In any embodiment, a bridging molecule for binding to CD33 can comprise or consist of a sequence set forth in SEQ ID NOs: 268 and 269 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0458]

[0466] In any embodiment, a bridge molecule for binding to Her2 can comprise or consist of a sequence set forth in SEQ ID NOs: 276 and 277 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0459]

[0467] In any embodiment, a bridging molecule for binding to CD33 can comprise or consist of a sequence set forth in SEQ ID NOs: 278 and 279 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0460]

[0468] In any embodiment, a bridge molecule for binding to Her2 can comprise or consist of a sequence set forth in SEQ ID NOs: 270 and 271 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0461]

[0469] In any embodiment, a bridging molecule for binding to B7-H7 (HHLA2) can comprise or consist of a sequence set forth in SEQ ID NOs: 280 and 281 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0462]

[0470] In any embodiment, a bridging molecule for binding to CD34 can comprise or consist of a sequence set forth in SEQ ID NOs: 282 and 283 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0463]

[0471] In any embodiment, a bridging molecule for binding to CD7 can comprise or consist of a sequence set forth in SEQ ID NOs: 284 and 285 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0464]

[0472] In any embodiment, a bridging molecule for binding to CD7 can comprise or consist of a sequence set forth in SEQ ID NO:286 (heavy chain sequence), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0465]

[0473] In any embodiment, a bridging molecule for binding to GPRC5D can comprise or consist of a sequence set forth in SEQ ID NOs: 287 and 288 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0466]

[0474] In any embodiment, a bridging molecule for binding to TIM-3 can comprise or consist of a sequence set forth in SEQ ID NOs: 289 and 290 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0467]

[0475] In any embodiment, a bridging molecule for binding to CD191 (CCR1) can comprise or consist of a sequence set forth in SEQ ID NOs: 291 and 292 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0468]

[0476] In any embodiment, a bridging molecule for binding to CD66b (CEACAM8) can comprise or consist of a sequence set forth in SEQ ID NOs: 293 and 294 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0469]

[0477] In any embodiment, a bridging molecule for binding to CD11b (MAC-1) can comprise or consist of a sequence set forth in SEQ ID NOs: 295 and 296 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0470]

[0478] In any embodiment, a bridging molecule for binding to EMR2 (ADGRE2) can comprise or consist of a sequence set forth in SEQ ID NOs:297 and 298 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0471]

[0479] In any embodiment, a bridge molecule for binding to MUC16 can comprise or consist of a sequence set forth in SEQ ID NOs:299 and 300 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0472]

[0480] In any embodiment, a bridging molecule for binding to NYESO-1 HLA-A2 can comprise or consist of a sequence set forth in SEQ ID NOs: 301 and 302, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0473]

[0481] In any embodiment, a bridging molecule for binding to survivin HLA-A2 can comprise or consist of a sequence set forth in SEQ ID NOs: 303 and 304, or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0474]

[0482] In any embodiment, a bridging molecule for binding to BCMA may comprise or consist of a sequence as set forth in SEQ ID NO: 305, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0475]

[0483] In any embodiment, a bridging molecule for binding to BCMA may comprise or consist of a sequence as set forth in SEQ ID NO: 306, or a sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0476]

[0484] In any embodiment, a bridging molecule for binding to CD200 can comprise or consist of a sequence set forth in SEQ ID NOs: 349 and 348 (the light and heavy chain sequences listed, respectively), or a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0477]

[0485] In any aspect, the bridge molecule described herein does not have a HIS tag. Also contemplated are bridge molecules that include the amino acid sequence specified in the above sequence information table, but without the HIS tag specified in the sequence, or that include a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Furthermore, in one embodiment, the bridge molecule may include a tag other than a HIS tag, or may include an amino acid sequence specified in the above sequence information table, but with a different tag at the position of the HIS tag specified in the sequence.

[0478] nucleic acid

[0486] In a second aspect, the present invention provides a nucleic acid molecule encoding an antigen binding protein of the present invention, or a part thereof. The nucleic acid may further encode a bridge molecule as described herein.

[0479]

[0487] A nucleic acid molecule can include any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified, RNA or DNA. For example, a nucleic acid molecule can include single-stranded and / or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that include DNA and RNA that can be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, a nucleic acid molecule can include triple-stranded regions that include RNA or DNA, or both RNA and DNA. A nucleic acid molecule can also include one or more modified bases for stability, or DNA or RNA backbones that are modified for other reasons. Various modifications can be made to DNA and RNA, and thus the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0480]

[0488] In some embodiments of the second aspect of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 307-347. Preferably, the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein as described above. Preferably, the nucleic acid further comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 2-30, 168, 361-396, 437 or 438. Preferably, the nucleic acid comprises a nucleotide sequence encoding the heavy and light chain pair of the bridge molecule as described above.

[0481]

[0489] Furthermore, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding an antigen-binding protein molecule of the present invention, or a portion thereof. The nucleic acid construct may further comprise one or more of an origin of replication for one or more hosts; a selectable marker gene active in one or more hosts; and / or one or more transcription control sequences.

[0482]

[0490] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on the cell in which it is expressed, facilitating the identification and / or selection of cells transfected or transformed with a construct.

[0483]

[0491] A "selection marker gene" includes any nucleotide sequence that, when expressed by cells transformed with a construct, confers a phenotype on the cells that facilitates identification and / or selection of these transformed cells. A series of nucleotide sequences encoding suitable selection markers are known in the art (e.g., Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5). Exemplary nucleotide sequences encoding selection markers include the adenosine deaminase (ADA) gene; the cytosine deaminase (CDA) gene; the dihydrofolate reductase (DHFR) gene; the histidinol dehydrogenase (hisD) gene; the puromycin-N-acetyltransferase (PAC) gene; the thymidine kinase (TK) gene; the xanthine-guanine phosphoribosyltransferase (XGPRT) gene, or antibiotic resistance genes such as the ampicillin resistance gene, the puromycin resistance gene, the bleomycin resistance gene, the hygromycin resistance gene, the kanamycin resistance gene and the ampicillin resistance gene; fluorescent reporter genes, such as genes encoding green, red, yellow or blue fluorescent proteins; and luminescence-based reporter genes, such as the luciferase gene, among others, that allow for optical selection of cells using techniques such as fluorescence-activated cell sorting (FACS).

[0484]

[0492] Furthermore, it should be noted that the selectable marker gene can be a separate open reading frame in the construct or can be expressed as a fusion protein with another polypeptide (e.g., CAR).

[0485]

[0493] As mentioned above, the nucleic acid construct may also include one or more transcription control sequences. The term "transcription control sequence" should be understood to include any nucleic acid sequence that affects the transcription of an operably connected nucleic acid. Transcription control sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer or upstream activation sequence, and a transcription terminator. Typically, a transcription control sequence includes at least a promoter. The term "promoter" as used herein describes any nucleic acid that confers, activates, or enhances the expression of a nucleic acid in a cell.

[0486]

[0494] In some embodiments, at least one transcription control sequence is operably connected to the nucleic acid molecule of the second aspect of the present invention.For the purposes of this specification, a transcription control sequence is considered to be "operably connected" to a given nucleic acid molecule if the transcription control sequence can promote, inhibit or otherwise regulate the transcription of the nucleic acid molecule.Thus, in some embodiments, the nucleic acid molecule is under the control of a transcription control sequence, such as a constitutive promoter or an inducible promoter.

[0487]

[0495] A "nucleic acid construct" may be in any suitable form, such as in the form of a plasmid, phage, transposon, cosmid, chromosome, vector, and is capable of replicating when associated with appropriate control elements and transferring the genetic sequences contained within the construct between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, the nucleic acid construct is a vector. In some embodiments, the vector is a viral vector.

[0488]

[0496] A promoter can regulate the expression of an operably linked nucleic acid molecule constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs. Thus, a promoter can include, for example, a constitutive promoter or an inducible promoter. A "constitutive promoter" is a promoter that is active under most environmental and physiological conditions. An "inducible promoter" is a promoter that is active under a specific environmental or physiological condition. The present invention contemplates the use of any promoter that is active in the cell of interest. Thus, a wide range of promoters can be easily ascertained by one skilled in the art.

[0489]

[0497] Mammalian constitutive promoters can include, but are not limited to, Simian Virus 40 (SV40), Cytomegalovirus (CMV), P-actin, ubiquitin C (UBC), elongation factor-1 alpha (EF1A), phosphoglycerate kinase (PGK) and CMV early enhancer / chicken beta actin (CAGG).

[0490]

[0498] Inducible promoters can include, but are not limited to, chemically inducible promoters and physically inducible promoters.Chemically inducible promoters include promoters whose activity is regulated by compounds such as alcohol, antibiotics, steroids, metal ions or other compounds.Examples of chemically inducible promoters include, among others, tetracycline-regulated promoters (see, for example, U.S. Pat. No. 5,851,796 and U.S. Pat. No. 5,464,758); steroid-responsive promoters such as glucocorticoid receptor promoters (see, for example, U.S. Pat. No. 5,512,483), ecdysone receptor promoters (see, for example, U.S. Pat. No. 6,379,945); and metal-responsive promoters such as metallothionein promoters (see, for example, U.S. Pat. No. 4,940,661, U.S. Pat. No. 4,579,821 and U.S. Pat. No. 4,601,978).

[0491]

[0499] It will be appreciated that in the context of the present invention it may be desirable in certain circumstances for expression of the bridge molecule to be under the control of an inducible promoter, which allows expression of the nucleic acid encoding the bridge molecule to be switched on and off.

[0492]

[0500] In certain embodiments, and in the case of inducible expression construct, the immune cells expressing CAR can be genetically modified with a) the nucleic acid encoding antigen-binding receptor, and b) the inducible expression construct encoding antigen-binding protein and / or bridging molecule.When dysfunctional P2X7 receptor binds, immune cells induce the expression of the gene encoding antigen-binding protein and / or bridging molecule.In certain embodiments, the expression of such gene promotes and / or improves the treatment of cancer.

[0493]

[0501] As mentioned above, the control sequence may also include a terminator. The term "terminator" refers to the DNA sequence at the end of a transcription unit that signals the end of transcription. A terminator is generally a 3' non-translated DNA sequence that contains a polyadenylation signal, facilitating the addition of a polyadenylation sequence to the 3' end of a primary transcript. Similar to a promoter sequence, a terminator may be any terminator sequence that is operable in the cell, tissue or organ in which it is intended to be used. Suitable terminators are known to those skilled in the art.

[0494]

[0502] As will be appreciated, the nucleic acid constructs of the present invention can further include additional sequences, such as sequences that allow for enhanced expression, cytoplasmic or membrane transport, and location signals. Specific non-limiting examples include an internal ribosome entry site (IRES) or a cleavage site (e.g., P2A, T2A).

[0495]

[0503] The present invention extends to all genetic constructs essentially as described herein. These constructs may further comprise nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotic organisms and / or for the integration of the genetic construct or a part thereof into the genome of a eukaryotic cell.

[0496]

[0504] Methods for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the third aspect of the present invention, into eukaryotic cells are known in the art.As will be understood, the most suitable method for introducing a nucleic acid construct into a desired host cell depends on many factors, such as the size of the nucleic acid construct, the type of host cell, the desired efficiency of transfection / transduction, and the final desired or required survival rate of the transfected / transduced cells.Non-limiting examples of such methods include chemical transfection with chemicals such as cationic polymers, calcium phosphate, or structures such as liposomes and dendrimers; non-chemical methods such as electroporation, sonoporation, heat shock, or optical transfection; particle-based methods such as "gene gun" delivery, magnetofection, or impalefection or viral transduction.

[0497]

[0505] The nucleic acid construct is selected depending on the desired method of transfection / transduction. In some embodiments of the third aspect of the invention, the nucleic acid construct is a viral vector and the method of introducing the nucleic acid construct into the host cell is viral transduction. Methods are known in the art for inducing expression of CAR in PBMCs using viral transduction (Parker, LL. et al. Hum Gene Ther. 2000;11: 2377-87), and more generally using retroviral systems to transduce mammalian cells (Cepko, C. and Pear, W. Curr Protoc Mol Biol. 2001, unit 9.9). In other embodiments, the nucleic acid construct is a plasmid, cosmid, artificial chromosome, etc., and can be transfected into cells by any suitable method known in the art.

[0498] Modified cells

[0506] As described herein, in certain embodiments, the present invention includes a treatment method involving the use of cells expressing a chimeric antigen receptor (CAR) that includes an antigen recognition domain, for example, the antigen recognition domain recognizes a dysfunctional P2X7 receptor expressed on the cell surface. For example, the orchestration molecule of the present invention can be used to promote the killing of cancer (target) cells by recruiting cells expressing a chimeric antigen receptor. Such orchestration molecules typically include a first antigen binding domain for binding to a tumor-specific antigen (e.g., dysfunctional P2X7 receptor) and a second antigen binding domain for binding to any immune effector cell. In some cases, the immune effector cell can be a cell expressing a CAR (e.g., if the cell is a CAR T cell, the second binding domain can bind to CD3 or other antigens expressed by the CAR T cell).

[0499]

[0507] The cell can be an "engineered cell", "genetically modified cell", "immune cell" or "immune effector cell" as described herein. Furthermore, the cell can be differentiated into an immune cell. The cell that can be differentiated into an immune cell (e.g., a T cell expressing a dysfunctional P2X7CAR) can be a stem cell, a multilineage progenitor cell, or an induced pluripotent stem cell.

[0500]

[0508] In any embodiment, the cell may be a T cell, optionally wherein the T cell does not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0501]

[0509] In any embodiment, the cell may be an immune cell, and optionally the cell does not express accessory molecules that may be checkpoint, exhaustion or apoptosis-related signaling receptors, as well as ligands, such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R (e.g., by knocking out one of these genes at the genetic or functional level).

[0502]

[0510] In some embodiments, the genetically modified cells comprise two or more different CARs.

[0511] In some embodiments of the invention, the genetically modified cells comprise two or more nucleic acid molecules or nucleic acid constructs encoding two or more different CARs. In some embodiments of the invention, the genetically modified cells comprise two or more nucleic acid molecules or two or more nucleic acid constructs, each encoding a different CAR.

[0503]

[0512] As referred to herein, a "genetically modified cell" includes any cell that contains a non-native and / or introduced nucleic acid molecule or nucleic acid construct encompassed by the present invention. The introduced nucleic acid molecule or nucleic acid construct may be maintained within the cell as a discrete DNA molecule or may be integrated into the genomic DNA of the cell.

[0504]

[0513] The genomic DNA of a cell should be understood in its broadest context to include all endogenous DNA that constitutes the genetic complement of the cell. Thus, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, etc. Thus, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, etc. The "genomically integrated form" of a construct may be all or part of the construct. However, in some embodiments, the genomically integrated form of a construct comprises at least the nucleic acid molecule of the second aspect of the present invention.

[0505]

[0514] As used herein, the term "different CAR" or "different chimeric antigen receptor" refers to any two or more CARs that have either non-identical antigen recognition domains and / or non-identical signaling domains.In one example, "different CAR" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize dysfunctional P2X7 receptor), but have different signaling domains, such as one CAR that has a signaling domain with a part of an activating receptor, and the other CAR that has a signaling domain with a part of a costimulatory receptor.As will be understood, at least one of the two or more CARs in this embodiment has an antigen recognition domain that recognizes dysfunctional P2X7 receptor, and the other CAR(s) can take any suitable form and be directed against any suitable antigen.

[0506]

[0515] Thus, in some embodiments of the present invention, two or more different CARs have different signaling domains and can have the same or different antigen recognition domains.Specifically, the genetically modified cells of the present invention can include a first chimeric antigen receptor having a signaling domain that includes a portion derived from an activating receptor, and a second chimeric antigen receptor having a signaling domain that includes a portion derived from a costimulatory receptor.

[0507]

[0516] In some embodiments, the activating receptor (from which a portion of the signaling domain is derived) is a CD3 coreceptor complex or an Fc receptor.

[0517] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD27, CD28, CD-30, CD40, DAP10, OX40, 4-1BB (CD137), and ICOS.

[0508]

[0518] In some embodiments, the costimulatory receptor (from which a portion of the signaling domain is derived) is selected from the group consisting of CD28, OX40, or 4-1BB.

[0519] In some embodiments, the genetically modified cells are further modified to constitutively express a costimulatory receptor.

[0509]

[0520] As mentioned above, a cellular immune response is typically induced only when an activation signal (typically in response to an antigen) and a costimulatory signal are experienced simultaneously. Thus, by having a genetically modified cell according to some of the above embodiments, which includes two or more CARs that provide both an intracellular activation signal and an intracellular costimulatory signal in combination, it ensures that a sufficient immune response can be induced in response to the recognition of their cognate antigen by the CAR(s). Alternatively, the genetically modified cell can include only one CAR that has an antigen recognition domain that recognizes a dysfunctional P2X7 receptor and can constitutively express a costimulatory receptor, thereby increasing the possibility that costimulation is provided at the same time that the CAR is activated. Alternatively, the genetically modified cell can be further modified to constitutively express both the costimulatory receptor(s) and / or their ligand(s). In this way, the cell experiences continuous costimulation and only requires activation of the CAR, together with a signaling domain that includes a portion from the activating receptor, for immune activation of the cell.

[0510]

[0521] Thus, in some embodiments, the genetically modified cells expressing CAR are further modified to constitutively express costimulatory receptors.In further embodiments, the genetically modified cells are further modified to express the ligands of costimulatory receptors, thereby promoting the self-stimulation of cells.Examples of CAR-expressing T cells that also express both costimulatory receptors and their cognate ligands (to induce self-stimulation) are known in the art, and include those disclosed in Stephen MT. et al. Nat Med, 2007; 13: 1440-9, among others.

[0511]

[0522] The ability of the genetically modified cells containing CAR can be enhanced by further modifying the cells to secrete cytokines, preferably proinflammatory or growth-promoting cytokines. This cytokine secretion provides autocrine support for the cells expressing CAR and changes the local environment surrounding the cells expressing CAR, allowing other cells of the immune system to be recruited and activated. In conclusion, in some embodiments of the fourth or fifth aspect of the present invention, the genetically modified cells are further modified to secrete cytokines. This secretion can be constitutive or inducible by the recognition of the CAR's cognate antigen of its ligand.

[0512]

[0523] While any one or more cytokines can be selected depending on the desired immune response, preferred cytokines and / or chemokines include IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21, CCL19, CCL21 or combinations thereof.

[0513]

[0524] The immune cells may be any suitable immune cell, or a precursor thereof, or may be a homogenous or heterogenous population of cells. In some embodiments, the cells are leukocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ T cells, CD8+ T cells, natural killer cells, natural killer T cells, or γδ T cells.

[0514]

[0525] The immune cell may be a T cell, and optionally the T cell does not express TcRαβ, PD1, CD3 or CD96 (e.g., by knocking down or knocking out one of these genes at the genetic or functional level).

[0515]

[0526] The immune cells may not express accessory molecules that may be checkpoint, exhaustion or apoptosis-related signaling receptors, as well as ligands such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R (e.g., by knocking out or knocking down one of these genes at the genetic or functional level).

[0516] Methods of Treatment and Administration

[0527] As discussed further in this document, the present invention finds application in the treatment of a variety of conditions, preferably in the treatment of cancer.

[0517]

[0528] The present invention also contemplates various scenarios for the use of the antigen binding proteins described herein, preferably in conjunction with bridging molecules. Optionally, modified or engineered immune cells are also used.

[0518]

[0529] In one scenario, an individual in need of treatment is administered a single composition containing both the CAR T cells and the antigen binding protein, optionally including a bridging molecule.

[0530] In a further scenario, an individual in need of treatment is administered a population of CAR T cells, which contain an expression vector encoding an antigen binding protein, preferably a bridging molecule. The expression vector may facilitate constitutive or inducible expression of nucleic acid sequences encoding the antigen binding protein and / or the bridging molecule.

[0519]

[0531] Furthermore, an individual in need of treatment can be administered CAR T cells and later administered a composition comprising an antigen binding protein (and optionally a bridging molecule) (e.g., via infusion), or a nucleic acid sequence encoding an antigen binding protein, and optionally a bridging molecule. Such a scenario may be appropriate in a situation where an individual is first treated with CAR T cells for targeted treatment of cancer that is positive for dysfunctional P2X7 receptor, and the subsequent administration of antigen binding protein is intended to increase the potency of CAT T cells or further recruitment of endogenous immune cells. The bridging molecule is typically intended to redirect CAR or endogenous immune cells to alternative cancer antigens, or to peptides derived from infectious agents and presented on MHC I or II molecules of cells.

[0520]

[0532] Thus, the antigen binding protein (referred to herein as an orchestration molecule), preferably also a bridging molecule, can be administered before, simultaneously with, or after the subject is treated with the CAR T cells.

[0521]

[0533] When the antigen binding protein, preferably also the bridging molecule, and the CAR T cells are administered to a subject at the same time, they can be administered via the same route of administration (including a single composition) or alternatively via different routes of administration. For example, the CAR T cells can be administered by injection into the bloodstream of the subject, while the antigen binding protein (preferably also the bridging molecule) can be administered via another route of administration, such as intramuscular, intradermal, subcutaneous or intraperitoneal.

[0522]

[0534] The antigen binding proteins and / or bridging molecules may be produced or expressed in vivo by genetically engineered cells that naturally secrete the antigen binding proteins and / or bridging molecules, or upon stimulation via a stimulant, e.g., a small molecule. Alternatively, the cells may continuously secrete the antigen binding proteins and / or bridging molecules and cease their secretion upon application of a stimulant, e.g., a small molecule.

[0523]

[0535] Although this specification refers specifically to application in humans, it is clearly understood that the present invention is also useful for veterinary purposes. Thus, in all aspects, the present invention is useful for domestic animals such as cows, sheep, horses and poultry; companion animals such as cats and dogs; and zoo animals. Thus, the general term "subject" or "subject to be treated / being treated" is understood to include all animals, such as humans, apes, dogs, cats, horses and cows.

[0524]

[0536] The term "administered" refers to the administration of a therapeutically effective dose of the composition comprising individual cells to an individual. A "therapeutically effective amount" refers to a dose that produces the effect for which it is administered. The exact dose depends on the purpose of the treatment and can be ascertained by the skilled artisan using known techniques. As known in the art and described above, adjustments for systemic versus local delivery, age, weight, general health, sex, diet, time of administration, drug interactions and severity of the condition may be necessary and can be ascertained by routine experimentation by the skilled artisan.

[0525]

[0537] Subjects in need of treatment include those who already have a benign, precancerous, or non-metastatic tumor, as well as those in which the development or recurrence of cancer is to be prevented. The subject may have metastatic cells, such as metastatic cells present in the ascites and / or lymph nodes.

[0526]

[0538] The purpose or outcome of treatment may be to reduce the number of cancer cells; reduce primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more of the symptoms associated with the disorder.

[0527]

[0539] The efficacy of treatment can be measured by assessing survival, time to disease progression, response rate (RR), duration of response, and / or quality of life.

[0540] The method is particularly useful for extending the time to disease progression.

[0528]

[0541] The methods are particularly useful for extending human survival, including overall survival and progression-free survival.

[0542] The methods are particularly useful for providing a complete response to therapy, whereby all signs of the cancer disappear in response to treatment, which does not necessarily mean that the cancer has been cured.

[0529]

[0543] The methods are particularly useful for providing a partial response to therapy, whereby there is a decrease in the size of one or more tumors or lesions, or the extent of cancer in the body, in response to treatment.

[0530]

[0544] The objective or outcome of treatment may be any one or more of the following: -Reducing the number of cancer cells: - Reducing the size of the primary tumor; - inhibiting (i.e. slowing to some extent and preferably stopping) the invasion of cancer cells into peripheral organs; - inhibiting (i.e. slowing to some extent and preferably stopping) tumor metastasis; - Inhibit tumor growth to some extent; -Relieving to some extent one or more of the symptoms associated with the disorder.

[0531]

[0545] In one embodiment, the subject in need of treatment includes a subject with a benign, pre-cancerous, non-metastatic tumor.

[0546] In one embodiment, the cancer is precancerous or preneoplastic.

[0532]

[0547] In one embodiment, the cancer is secondary cancer or metastasis.Secondary cancer can be located in any organ or tissue, especially in the organ or tissue with relatively high hemodynamic pressure, such as lung, liver, kidney, pancreas, intestine and brain.Secondary cancer can be detected in ascites and / or lymph node.

[0533]

[0548] In one embodiment, the cancer may be substantially undetectable.

[0549] "Precancerous" or "preneoplastic" generally refers to a condition or growth that typically precedes or develops into cancer. A "precancerous" growth can have cells characterized by aberrant cell cycle regulation, proliferation, or differentiation, as can be determined by cell cycle markers.

[0534]

[0550] Cancers can be solid tumors or "liquid" tumors. In other words, cancers can grow in tissues (such as carcinomas, sarcomas, adenomas, etc.) or can be cancers that are present in body fluids such as blood or bone marrow (e.g., lymphomas and leukemias).

[0535]

[0551] In certain preferred embodiments, the cancer that requires treatment may be a cancer that is characterized by low-level expression of dysfunctional P2X7 receptor. Examples of such cancers include Burkitt's lymphoma. However, immunohistochemical analysis of the surface expression of dysfunctional P2X7 (nfP2X7) receptor in tumor biopsies of patients revealed that IHC scores range from 1+ to 3+. Thus, low expression samples can be found in a wide range of tumor types. Examples are found in various types of solid tumors, including but not limited to neuroblastoma, colon cancer, lung cancer, kidney cancer, skin cancer, breast cancer, brain cancer and prostate cancer. Such differences in expression levels in different tissues may be due to tumors being formed from cells that are in an early state of transformation (the tissue with the highest receptor expression may be the tissue with the highest proliferation rate).

[0536]

[0552] Other examples of cancers that can be treated according to the methods of the invention include blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, pancreatic islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, lung cancer, e.g., small cell lung cancer (SCKC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal carcinoma, hepatocellular carcinoma, Gastric or stomach cancer, such as gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancer.

[0537]

[0553] In a further example, the methods of treatment contemplated within the scope of the present invention include methods of treating or preventing infectious diseases. Thus, the bridging molecules of the present invention can be utilized to redirect CAR T cells to additional surface accessible antigens, e.g., where the antigen is a non-cancer associated pathogenic antigen presented on an MHC I or MHC II molecule as further described herein.

[0538]

[0554] The subject who needs to treat infectious disease may be at risk or diagnosed with disease.At risk subjects include immunocompromised patients.Thus, the method of the present invention also allows to prevent the onset of infectious disease in individuals undergoing treatment (e.g., for treating cancer), which makes them immunocompromised and therefore susceptible to infection.

[0539]

[0555] Examples of intracellular pathogens in which peptides are presented on MHC I or MHC II molecules include viral infections, intracellular bacterial infections, protozoan infections, and intracellular fungal infections.

[0556] Examples of viral infections that can be treated using the methods of the invention include HIV, hepatitis (e.g., hepatitis A, B, or C), coronavirus (e.g., SARS-CoV-2), influenza virus, varicella zoster virus, and mumps virus.

[0540]

[0557] Examples of intracellular bacterial infections that can be treated using the methods of the invention include mycobacterial infections (e.g., Mycobacterium tuberculosis), Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Salmonella Typhi, Brucella, Legionella, Nocardia, Neisseria, Rhodococcus, Yersinia, Staphylococcus aureus, Chlamydia, Rickettsia, Coxiella, and Chlamydophila pneumoniae.

[0541]

[0558] Examples of intracellular infections by fungal pathogens: Histoplasma capsulatum, Cryptococcus neoformans, and Pneumocystitis jirovecii.

[0542]

[0559] Examples of obligate intracellular protozoan pathogens include Apicomplexans (Plasmodium spp., Toxoplasma gondii, and Cryptosporidium parvum), and Trypanosomatids (Leishmania spp. and Trypanosoma cruzi).

[0543]

[0560] Immune cells that can be targeted to modulate the immune system in the context of cancer and / or autoimmune disease can be B cells (CD19, CD20, CD22), plasma cells (BCMA, CD38, CD138), T cell subsets (TRBC1 or TRBC2, α4β7 and αEβ7, CD7), macrophages and TAMs (CD163 and CD206). In the context of allogeneic stem cell transplantation, immune-based conditioning can be achieved by targeting (CD34, CD117, CD133, CD33 and CD38), particularly in the case of non-malignant diseases, such as thalassemia major or sickle cell anemia, and / or DNA repair deficiencies such as Fanconi anemia.

[0544]

[0561] Targeting senescent tumor cells through a marker (uPAR) likely promotes faster proliferation of cancer cells at later stages by aiding in the elimination of dormant tumor cells, which expand at later time points, secrete tumor-promoting cytokines, and create a tumor-suppressive environment that protects new cancerous subclones.

[0545]

[0562] CAR T cells are designed to bind other immune cells, such as T REG CAR T cells can be made immunosuppressive or can be constructed to secrete immunosuppressive cytokines (TGF beta, IL10), and chemokines by introducing corresponding inducible expression cassettes [NFAT-dependent cytokine secretion] and signaling into the construct.

[0546]

[0563] The antigen-binding proteins and bridging molecules of the present invention can be formulated for administration to a subject using techniques known to those skilled in the art.The formulation of the bridging molecule can include pharma-ceutically acceptable excipient(s) (carriers or diluents).Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, osmotic agents, swelling agents, and lubricants.

[0547]

[0564] Formulations of antigen binding proteins and bridging molecules may contain one type of antigen binding protein and / or bridging molecule, or more than one type of antigen binding protein and / or bridging molecule (i.e., the bridging molecules may have the same or different targeted and / or dysfunctional P2X7 receptor epitope portions).

[0548]

[0565] The crosslinking molecule can be administered to the subject using modes and techniques known to those skilled in the art.Exemplary modes include, but are not limited to, intravenous, intraperitoneal, and intratumoral injection.Other modes include, but are not limited to, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluid).

[0549]

[0566] A formulation containing the antigen-binding protein(s) or bridging molecule(s) is administered to a subject in an amount that is effective to treat a particular indication or disorder. In general, a formulation containing at least about 0.01 μg / kg to about 100 mg / kg body weight of the antigen-binding protein or bridging molecule can be administered to a subject in need of treatment. In most cases, the dosage can be about 100 μg / kg to about 10 mg / kg body weight of the antigen-binding protein or bridging molecule daily, taking into account the route of administration, symptoms, etc. However, the amount of the antigen-binding protein or bridging molecule in the formulation administered to a subject can vary between wide limits, depending on the location, source, identity, extent and severity of the disorder, the age and condition of the individual being treated, etc. A physician can ultimately determine the appropriate dose to be used. The antigen-binding protein or bridging molecule can be administered as a continuous infusion or a bolus dose.

[0550]

[0567] The timing between administration of the CAR T cells and the antigen-binding protein and / or bridging molecule preparation can vary widely depending on factors including the type of (immune) cell used, the binding specificity of the CAR, the identity of the targeting moiety, and the identity of the target cell, e.g., the cancer cell being treated, the location of the target cell in the subject, the means used to administer the preparation to the subject, and the health, age, and weight of the subject being treated. Indeed, the preparation can be administered before, simultaneously with, or after the engineered (immune) cell preparation.

[0551]

[0568] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features which are mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative embodiments of the invention.

[0552]

[0569] It is understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features which are mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative embodiments of the invention. EXAMPLES

[0553] Example 1 - Materials and methods including production of antigen binding proteins (OR molecules) and bridging molecules

[0570] Cultivation, transfection and protein production were performed according to the ExpiCHO Expression System User Guide (Thermo-ExpiCHO™ Expression System USER GUIDE. For transfection of ExpiCHO-S™ cells in defined serum-free medium, Catalog No. A29133, Publication No. MAN0014337). In summary, ExpiCHO are routinely passaged in ExpiCHO medium and cultured at 4–6 × 10 6 The cell count was maintained at less than 5–7 × 10 cells / mL. 6 Cells in mid-log phase growth were transfected when the DNA concentration was in the range of 1000xg / mL. For transfection, liposome complexes were prepared with 1 μg of DNA for each 1 mL of culture. For co-transfection of vectors encoding heavy and light chains separately (either for OR or bridging molecule production), the vector ratio was set at 1:1 unless otherwise stated. The "high titer" or "maximum titer" expression protocol was followed after transfection and cultures were harvested when cell viability dropped below 70%. Harvesting was performed by centrifugation at 300 x g for 5 min at 20°C. Cells were discarded and the supernatant was centrifuged again at 4000 x g for 30 min at 4°C. The harvested supernatant was clarified by 0.2 μm filtration using a PES membrane and then frozen for storage.

[0554]

[0571] The collected samples can be concentrated and buffer exchanged by spin columns or TFF cassettes with nominal molecular size cutoffs of 5, 10 or 30 kDa.

[0572] For HIS-tagged column purification, the collected supernatant was dialyzed through SnakeSkin dialysis tubing with a nominal molecular size cutoff of 5, 10 or 30 kDa, depending on the protein of interest. For large-scale production, the supernatant was washed through a TFF cassette with a certain molecular size cutoff membrane. Buffer exchange to the desired column loading buffer was also achieved through the procedure described above to prepare the sample for His-tagged column purification. Purification was performed on either a HisTrap Excel column (Cytiva) or a PureCube 100 compact cartridge Ni-INDOGO affinity column (Cat#75302, Cube Biotech) or other equivalent columns. Purification was performed on an AKTA pure system (Cytiva) equipped with a UV detector at 280 nm wavelength, a conductivity detector and a pH probe. Loading and washing buffers consisted of 50 mM sodium phosphate monobasic and 0.3 M sodium chloride, pH 8.0. Elution buffer contained 500 mM imidazole. The eluted proteins were buffer exchanged into PBS using a Vivaspin (Sartorius) and stored at 4°C.

[0555]

[0573] Protein was quantified by Nanodrop at 280 nm wavelength and standard bicinchoninic acid (BCA) protein assay. Protein purity was confirmed by SDS PAGE gel electrophoresis.

[0556]

[0574] The detailed experimental data generated by the inventor(s) and described herein includes the generation of a wide range of antigen binding proteins or OR molecules, including: 1. Antigen binding proteins produced in multiple antibody formats, e.g., Fab, scFv, IgG; 2. The antigen-binding domain of an antigen-binding protein that binds to a wide range of cell surface antigens present on immune cells; 3. The antigen-binding domain of an antigen-binding protein that binds to a tumor-specific antigen.

[0557]

[0575] The detailed experimental data generated by the inventor(s) and described herein includes the generation of a wide range of cross-linking molecules including: 1. Targeting moieties produced in multiple antibody formats, e.g., Fab and scFv; 2. For example, V L and V H Various positioning of dysfunctional P2X7 receptor epitope moieties on the targeting moiety, including: 3. Inclusio...

Claims

A pharmaceutical composition for use in a method for treating cancer in a subject, comprising: (i) a first antigen-binding domain that binds to a dysfunctional P2X7 receptor; and (ii) a second antigen-binding domain comprising a variable region of an antibody that binds to a cell surface molecule on an immune cell wherein the pharmaceutical composition comprises an antigen-binding protein. The pharmaceutical composition according to claim 1, wherein the dysfunctional P2X7 receptor is expressed on a solid tumor.

3. The first antigen-binding site binds to an epitope comprising one or more amino acid residues from glycine at amino acid position 200 to cysteine at amino acid position 216 (both ends included) of the dysfunctional P2X ７ receptor, the pharmaceutical composition according to claim 1 or 2.

4. The pharmaceutical composition according to claim 1 or 2, wherein the cell surface molecule on the immune cell is present on the surface of a lymphoid cell (such as a natural killer cell, or a lymphocyte such as a cytotoxic T cell, γδ T cell, NKT cell or B lymphocyte) or a myeloid cell (such as a monocyte).

5. The pharmaceutical composition according to claim 1 or 2, wherein the cell surface molecule on the immune cell is present only on the immune cell and not on the non-immune cell.

6. The pharmaceutical composition according to claim 1 or 2, wherein the second antigen-binding domain binds to a T cell receptor or a molecule associated with the T cell receptor.

7. The method further comprises administering a bridging molecule comprising: (i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) an epitope of a dysfunctional P2X7 receptor that receives binding of the first antigen-binding domain of the antigen-binding protein wherein the pharmaceutical composition is according to claim 1 or 2.

8. The pharmaceutical composition according to claim 7, wherein the bridging molecule is a polypeptide.

9. The pharmaceutical composition according to claim 7, wherein the bridging molecule is a polypeptide in the form of a fusion protein or a chimeric protein.

10. The pharmaceutical composition according to claim 7, wherein the targeting moiety of the bridging molecule comprises an antibody or an antibody fragment. The pharmaceutical composition according to claim 7, wherein the targeting moiety binds to an antigen on a cancer cell. **Claim 12**: The pharmaceutical composition according to claim 11, wherein the targeting moiety binds to a tumor-associated antigen selected from the group consisting of CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (L1 CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, CD22, CD37, CD38, CD79B, CD276, uPAR, GD2, IL13Ra, PSMA, PSCA, EpCAM, ROR1, ROR2, CD117, CD70, CD30, Her2, Her3, BCMA, PDL1, MET-R, PDGFR alpha, GPC3, SLAMF7, GPNMB, VEGFR2, a4β7, aEβ7, CSPG4, CD80, CCR4, ENOX-2, MSLN, EphA2, IGF1R, FAP, AXL, Her4, Claudin 18.2, o-acetylated GD-2, GD3, CD147, CD163, podoplanin, WT1, GPC2, NY-ESO-1, FGFR4, EphB4, STEAP-1, STEAP-2, IL1RAP, MAGE-A1, TRBC1, TRBC2, CD105, CD138, CEACAM5, IL11Ra, nectin-4, and EGFR. **Claim 13** Dysfunctional P2X ７ The epitope portion of the receptor is a fragment of the dysfunctional P2X ７ receptor, preferably GHNYTTRNILPGLNITC (SEQ ID NO: 2), or a variant thereof (e.g., any one defined in SEQ ID NOs: 3-10 and 15-30, 168, 361-396, 437, 438), KYYKENNVECKRTLIKVF (SEQ ID NOs: 12 and 13), or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14), the pharmaceutical composition according to claim 7, comprising or consisting of. **Claim 14**: The pharmaceutical composition according to claim 1 or 2, wherein the method further comprises administering an immune cell or a precursor thereof that expresses a receptor comprising an antigen recognition domain and a signaling domain, for example, the immune cell is a T cell that expresses a chimeric antigen receptor (CAR), i.e., a CAR-T cell. **Claim 15** (i) a first antigen-binding domain that binds to a dysfunctional P2X7 receptor; (ii) a second antigen-binding domain comprising a variable region of an antigen-binding domain of an antibody that binds to a cell surface molecule on an immune cell, and an antigen-binding protein comprising the same.

16. Dysfunctional P2X ７ The antigen-binding protein according to claim 15, wherein the receptor has a conformational change that renders the receptor dysfunctional.

17. The first antigen-binding site binds to an epitope comprising one or more amino acid residues from glycine at amino acid position 200 to cysteine at amino acid position 216 (both ends included) of the dysfunctional P2X ７ receptor, the antigen-binding protein according to claim 15 or 16.

18. The antigen-binding protein according to claim 15 or 16, wherein the second antigen-binding domain binds to a cell surface antigen present on a T lymphocyte (e.g., a cytotoxic T cell, a γδ T cell, or an NKT cell), a B lymphocyte, or a natural killer cell.

19. The antigen-binding protein according to claim 15 or 16, wherein the second antigen-binding domain binds to a T cell receptor or a molecule associated with the T cell receptor.

20. The antigen-binding protein according to claim 15 or 16, wherein the second antigen-binding domain binds to CD3.

21. The antigen-binding protein according to claim 15 or 16, wherein the second binding domain is a polypeptide comprising an Fc region of an antibody or an Fc receptor-binding domain.

22. The antigen-binding protein according to claim 15 or 16, wherein the first and / or second antigen-binding domain is an immunoglobulin, or comprises an immunoglobulin domain, or the first and / or second antigen-binding domain comprises an antigen-binding fragment of an antibody.