Novel cell therapy system

Abstract

The present invention relates to therapeutic agents, compositions, kits and methods of treatment comprising: (a) an immune cell or a precursor thereof expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface, preferably a dysfunctional P2X7 receptor; and (b) (i) a targeting moiety that binds to a cell surface molecule on the target cell; and (ii) a bridging molecule comprising a tumor-specific epitope moiety that is bound by the antigen recognition domain, preferably a dysfunctional P2X7 receptor epitope moiety.

Description

[Technical field]

[0001] The present invention relates to chimeric antigen receptors, effector cells expressing the antigen receptors, and methods of using the antigen receptors for the prevention and / or treatment of various conditions, including cancer. Related Applications

[0002] This application claims priority from Australian Provisional Applications Nos. 2021900708, 2021902565 and 2021902830, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002]

[0003] Cancer immunotherapy is a rapidly growing field. The development of T cells expressing chimeric antigen receptors (CARs) is revolutionizing adoptive cell therapy.

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

[0003]

[0005] Several challenges remain for the clinical application of CAR T cell therapy. These include the difficulty in achieving sufficient therapeutic responses in the context of solid tumors, which may be due to insufficient activation, expansion and persistence of CAR T cells, and / or an immunosuppressive tumor microenvironment. Furthermore, the long-term clinical application of CAR T therapy may be negatively affected by the selection pressure of monospecific CAR T cells, antigen-negative escape variants, and antigen depletion. Furthermore, low expression levels of target antigens in healthy tissues may result in severe "on-target, off-tumor" toxicity. Finally, cytokine release syndrome (CRS) and CAR T cell-associated encephalopathy syndrome (CRES) are frequently observed side effects of CAR T cell therapy. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0006] As a result, new and improved approaches to CAR therapy, preferably CAR-T therapy, are needed. [Means for solving the problem]

[0005]

[0007] 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.

[0006]

[0008] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (a) an immune cell or a precursor thereof that expresses a receptor comprising an antigen recognition domain and a signal transduction domain, the antigen recognition domain binding to a tumor-specific antigen expressed on the cell surface; (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 antigen recognition domain A bridging molecule comprising The present invention provides a two-component therapeutic agent comprising:

[0007]

[0009] In any aspect, the tumor-specific antigen is an antigen expressed on a solid tumor or a liquid tumor. In one embodiment, the tumor-specific antigen is any one of nfP2X7, EGFRvIII, or CLDN6.

[0008]

[0010] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (a) an immune cell or a precursor thereof, expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a dysfunctional P2X7 receptor expressed on the cell surface; (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) the epitope portion of the dysfunctional P2X7 receptor that is bound by the antigen recognition domain A bridging molecule comprising The present invention provides a two-component therapeutic agent comprising:

[0009]

[0011] In another aspect, the present invention provides a method for producing a composition comprising: (a) an immune cell or a precursor thereof, expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a dysfunctional P2X7 receptor expressed on the cell surface; (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) the epitope portion of the dysfunctional P2X7 receptor that is bound by the antigen recognition domain A bridging molecule comprising A composition comprising:

[0010]

[0012] In another aspect, the present invention provides a method for producing a composition comprising: (a) an immune cell or a precursor thereof, expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a dysfunctional P2X7 receptor expressed on the cell surface; (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) the epitope portion of the dysfunctional P2X7 receptor that is bound by the antigen recognition domain A bridging molecule comprising A kit comprising:

[0011]

[0013] 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 that codes for the polypeptide.

[0012]

[0014] In a further aspect, the present invention provides (i) a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a first tumor antigen, and the first tumor antigen is a tumor-specific antigen (preferably a dysfunctional P2X7 receptor); and (ii) an inducible expression construct encoding a bridging molecule in the form of a fusion protein comprising (a) an antibody or antigen-binding fragment thereof that binds to a second tumor antigen, and (b) a peptide or fragment of a tumor-specific antigen, preferably a dysfunctional P2X7 receptor. The present invention provides an immune cell or a precursor thereof comprising:

[0013]

[0015] In another aspect, the present invention provides a method for producing a composition comprising: (i) a targeting moiety that binds to a cell surface molecule on a target cell; (ii) a tumor-specific antigen epitope portion (preferably a dysfunctional P2X7 receptor epitope portion) capable of being recognized or bound by an antigen recognition domain of a receptor, the receptor being optionally a chimeric antigen receptor; and The present invention provides a cross-linking molecule comprising:

[0014]

[0016] 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.

[0015]

[0017] In another aspect, the present invention provides nucleic acid comprising the nucleotide sequence encoding the bridging molecule described herein.Preferably, the nucleic acid comprises a first nucleotide sequence encoding targeting moiety and a second nucleotide sequence encoding tumor specific antigen epitope moiety.Preferably, the tumor specific antigen epitope moiety is dysfunctional P2X7 receptor epitope moiety.

[0016]

[0018] In another aspect, the present invention provides the vector or expression construct comprising the nucleic acid of the present invention.In one aspect, the vector or expression construct further comprises the nucleotide sequence encoding the receptor comprising antigen recognition domain and signal transduction domain, and the antigen recognition domain recognizes the dysfunctional P2X7 receptor expressed on cell surface as described herein.

[0017]

[0019] In another aspect, the invention provides a method of treating a disorder in a subject, the method comprising administering to the subject: (a) a cell expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface (preferably, the tumor-specific antigen is a dysfunctional P2X7 receptor); (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific antigen epitope portion, preferably a dysfunctional P2X7 receptor epitope portion, that is bound by the antigen recognition domain A bridging molecule comprising thereby treating the disorder in the subject.

[0018]

[0020] In another aspect, the invention provides a method of treating cancer in a subject, the method comprising: (a) an immune cell expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface, preferably a dysfunctional P2X7 receptor; (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific epitope portion, preferably a dysfunctional P2X7 receptor epitope portion, that is bound by the antigen recognition domain A bridging molecule comprising The method includes administering

[0019]

[0021] In another aspect, the invention provides a method of killing a target cell, the method comprising: (a) an immune cell expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface, preferably a dysfunctional P2X7 receptor; (b)(i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumor-specific antigen epitope portion, preferably a dysfunctional P2X7 receptor epitope portion, that is bound by the antigen recognition domain A cross-linking molecule comprising The method includes exposing the target cells to a

[0020]

[0022] 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.

[0021]

[0023] In any embodiment, the target cell can be any cell that expresses a dysfunctional P2X7 receptor, for example a cancer cell.

[0024] 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.

[0022]

[0025] 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.

[0023]

[0026] 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.

[0024]

[0027] Furthermore, the present invention provides a method in which bridging molecules for redirecting CAR T cells to different cancer antigens can be synchronously administered to a subject in need thereof, allowing for fine-tuning of the therapeutic approach, such that CAR T cells can be directed to bind to cancer cells via different antigens at different times during the course of a patient's treatment regimen.

[0025]

[0028] In a further embodiment, 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.

[0026]

[0029] 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.

[0027]

[0030] In any of the embodiments of the aspects directed to methods of treatment herein, the bridging molecule may be delivered via injection into the subject or may be expressed by immune cells expressing the chimeric antigen receptor. The bridging molecule may be a polypeptide and is encoded in an inducible or constitutive expression construct contained in the immune cell.

[0028]

[0031] In any embodiment, antigen recognition domain binds to the epitope 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.

[0029]

[0032] 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 proline at amino acid position 210 of dysfunctional P2X7 receptor.

[0030]

[0033] In any embodiment, the antigen recognition domain binds to an epitope that includes a proline at amino acid position 210 of a dysfunctional P2X7 receptor. In some embodiments, the antigen recognition domain binds to an epitope that includes one or more amino acid residues spanning from a glycine at amino acid position 200 to a cysteine ​​at amino acid position 216 (inclusive) of a dysfunctional P2X7 receptor.

[0031]

[0034] The antigen recognition domain of receptor can be any suitable molecule that can interact with and specifically bind to dysfunctional P2X7 receptor.However, in some embodiments, antigen recognition domain comprises amino acid sequence homology with the amino acid sequence of antibody or its fragment that binds to dysfunctional P2X7 receptor.In some embodiments, antigen recognition domain comprises amino acid sequence homology with the amino acid sequence of the fragment-antigen binding (Fab) part of antibody that binds to dysfunctional P2X7 receptor.In some embodiments, antibody is a humanized antibody.

[0032]

[0035] In any embodiment, the antigen recognition 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.

[0033]

[0036] In any embodiment, the antigen recognition domain comprises amino acid sequence homology to a single antibody domain (sdAb) that binds to a dysfunctional P2X7 receptor.

[0037] In any embodiment, 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 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.

[0034]

[0038] In some embodiments, the signaling domain comprises a portion derived from an activating receptor. In some embodiments, the activating receptor is a member of the CD3 coreceptor complex or is an Fc receptor. In some embodiments, the portion derived from the CD3 coreceptor complex is CD3-zeta. In some embodiments, the portion derived from an Fc receptor is FcεRI or FcγRI.

[0035]

[0039] In some embodiments, the signaling domain comprises a portion derived from a costimulatory receptor. In some embodiments, the signaling domain comprises a portion derived from an activating receptor and a portion derived from a costimulatory receptor. In some embodiments, the costimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137) and ICOS.

[0036]

[0040] In any embodiment, the cell expressing the antigen receptor can be an immune cell, such as an immune cell described herein, or a cell that can be differentiated into an immune cell (e.g., a precursor of 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.

[0037]

[0041] In any embodiment of the foregoing aspects, the receptor comprising an antigen recognition domain and a signaling domain is a chimeric receptor antigen (CAR).

[0042] 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.

[0038]

[0043] 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.

[0039]

[0044] In any embodiment, the cell surface molecule may comprise an antigen, preferably an antigen described herein.

[0045] 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.

[0040]

[0046] 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.

[0041]

[0047] 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.

[0042]

[0048] 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.

[0043]

[0049] 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 modifications thereof. The immunoglobulin can be IgG. The IgG can be IgG1. The IgG can be any IgG subclass.

[0044]

[0050] 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.

[0045]

[0051] The dysfunctional P2X7 receptor epitope portion can be provided in the form of P2X7 receptor or a fragment of P2X7 receptor that 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.

[0046]

[0052] In any embodiment, the dysfunctional P2X7 receptor epitope portion comprises or consists of a fragment of the dysfunctional P2X7 receptor. Exemplary fragments include GHNYTTRNILPGLNITC (SEQ ID NO: 2; also referred to herein as the "E200 epitope") and variants thereof (exemplary variants are provided in SEQ ID NOs: 3-10 and 15-30 and 168); KYYKENNVEKRTLIKVF (SEQ ID NOs: 12 and 13; also referred to herein as the "E300" epitope); or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14; also referred to herein as the "E200 / E300" or "composite" epitope). Further examples are provided in Table 1 herein.

[0047]

[0053] 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.

[0048]

[0054] 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.

[0049]

[0055] 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.

[0050]

[0056] 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]

[0051] [Figure 1]

[0057] 1 shows a schematic diagram of an exemplary cross-linking molecule. [Diagram 2]

[0058] FIG. 1 shows a schematic diagram of an exemplary embodiment of the invention utilizing nfP2X7-CAR and Fab-based E200 bridging molecules targeting cancer, infection and immune regulation related target antigens. [Diagram 3]

[0059] FIG. 1 shows a schematic diagram of an exemplary embodiment of the invention utilizing nfP2X7-CAR and single-chain TCR-based E200 bridging molecule targeting cancer- and infection-associated peptides. [Figure 4]

[0060] 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, making the E200 epitope 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 the dysfunctional P2X7 receptor epitope. [Diagram 5]

[0061] Bridging molecules in scFv format with a single E200 epitope linked either 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 6]

[0062] Bridging molecules in Fab format with a single E200 epitope linked either 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 7]

[0063] Bridging molecules in scFv format with a single E200 epitope linked either 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 8-1]

[0064] The cross-linking molecules bind 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 epitopes. [Figure 8-2]The cross-linking molecules bind 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, (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitopes. [Figure 8-3] The cross-linking molecules bind 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, (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitopes. [Figure 8-4] The cross-linking molecules bind 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, (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitopes. [Figure 8-5]The cross-linking molecules bind 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, (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitopes. [Figure 8-6] The cross-linking molecules bind 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, (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitopes. [Figure 8-7] The cross-linking molecules bind 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, (b), (d), (f), (h), (j), (l), (n), (p), (r), (t), (v), (x) and (z) show antibody binding to dysfunctional P2X7 receptor epitopes. [Figure 9]

[0065] Figure 1 shows "painting" of JeKo-1 cells with CD19-targeting 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-targeting Fab cross-linking molecules served as negative controls at 10 ng / mL and 1000 ng / mL of JeKo-1. CD19-targeting Fab cross-linking molecules were used at 1 ng / mL, 10 ng / mL, 100 ng / mL and 1000 ng / mL. [Figure 10]

[0066] Figure 1 shows "painting" of MOLM-13 cells with CD33-targeting 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-targeting Fab cross-linking molecules served as negative controls at 10 ng / mL and 1000 ng / mL of JeKo-1. CD33-targeting Fab cross-linking molecules were used at 1 ng / mL, 10 ng / mL, 100 ng / mL and 1000 ng / mL. [Figure 11-1]

[0067] Figure 1 shows "painting" of MOLM-13 (AML) cells via CD33-targeting Fab bridge molecule. Flow data shows isotype control in black, staining with BIL03 2-2-1 sd-mAb only at 1ug / mL in blue, and increased staining with the combination of CD33-targeting Fab bridge molecule and BIL03 2-2-1 sd-mAb in green. Increasing the number of target molecules that can be recognized by effector cells expressing nfP2X7 BRIDGE CAR leads to CAR-mediated effector function. The use of bridge molecules increases CAR function by increasing the targeting epitopes on cancer cells. [Figure 11-2]

[0067] Figure 1 shows "painting" of MOLM-13 (AML) cells via CD33-targeting Fab bridge molecules. Flow data shows isotype control in black, staining with BIL03 2-2-1 sd-mAb only at 1ug / mL in blue, and increased staining with the combination of CD33-targeting Fab bridge molecules and BIL03 2-2-1 sd-mAb in green. Increasing the number of target molecules that can be recognized by effector cells expressing nfP2X7 BRIDGE CAR leads to CAR-mediated effector function. The use of bridge molecules increases CAR function by increasing the targeting epitopes on cancer cells. [Figure 12]

[0068] Representative flow cytometry plots with direct comparison of non-transduced T cells (left panel), CAR0007_hPGK (middle panel) and CAR0007_EF1a (right panel) expressing T cells. Both CAR T cell containing conditions showed a significant increase in expression of activation markers CD25 and CD69 when incubated with MOLM-13 at an ET ratio of 20:1 and after 48 hours with CD33-targeted Fab cross-linking molecule at 1000 ng / mL. [Figure 13]

[0069] Flow cytometry plots of T cells and MOLM-13 at an ET ratio of 20:1, corresponding to FIG. 12, and CD33-targeted Fab cross-linking molecule at 1000 ng / mL after 48 hours showed complete clearance of leukemic cells under conditions containing CAR0007_hPGK and CAR0007_EF1a, but no effect on leukemic cell numbers under non-transduced T cell conditions. [Figure 14]

[0070] Figure 13 shows flow cytometry plots showing dose-dependent clearance of leukemia cells at the indicated CD33-targeted Fab cross-linking molecule concentrations of CAR0007_EF1a containing T cells and MOLM-13 at an ET ratio of 20:1 after 48 hours. Values ​​as low as 40 ng / mL showed near complete elimination of leukemia cells and at 200 and 1000 ng / mL complete elimination of leukemia cells. [Figure 15-1]

[0071] (a) Specific lysis of MOLM-13 leukemia cells by CAR0007_hPGK T cells at an ET ratio of 20:1 after 48 hours of incubation in the presence and absence of EGFR- and CD33-targeted bridging molecules at the indicated concentrations. Significant lysis was found in a dose-dependent manner for increasing concentrations of CD33-targeted Fab bridging molecules (40, 200 and 1000 ng / mL). (b) Specific lysis of MOLM-13 leukemia cells by CAR0007_hEF1a T cells at an ET ratio of 20:1 after 48 hours of incubation in the presence and absence of EGFR- and CD33-targeted bridging molecules at the indicated concentrations. Significant lysis was found in a dose-dependent manner for increasing concentrations of CD33-targeted Fab bridging molecules (40, 200 and 1000 ng / mL). [Figure 15-2] (a) Specific lysis of MOLM-13 leukemia cells by CAR0007_hPGK T cells at an ET ratio of 20:1 after 48 hours of incubation in the presence and absence of EGFR and CD33-targeting bridging molecules at the indicated concentrations. Significant lysis was found in a dose-dependent manner for increasing concentrations of CD33-targeting Fab bridging molecules (40, 200 and 1000 ng / mL). (b) Specific lysis of MOLM-13 leukemia cells by CAR0007_hEF1a T cells at an ET ratio of 20:1 after 48 hours of incubation in the presence and absence of EGFR and CD33-targeting bridging molecules at the indicated concentrations. Significant lysis was found in a dose-dependent manner for increasing concentrations of CD33-targeting Fab bridging molecules (40, 200 and 1000 ng / mL). [Figure 16]

[0072] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_hPGK. There was a significant effect on MOLM-13 viability after 24 hours of incubation at an ET ratio of 10:1 between conditions using EGFR targeted bridge molecules and CD33 targeted bridge molecules at 1000ng / mL. Statistical analysis was performed by t-test. [Figure 17]

[0073] Figure 17 shows an alternative representation of the data from Figure 16. There were no significant differences in the titration of EGFR cross-linking molecules (data not shown). There were significant differences in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test. [Figure 18]

[0074] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_hPGK. There was a significant effect on MOLM-13 viability after 24 hours of incubation at an ET ratio of 20:1 between EGFR targeted bridge molecule conditions and CD33 targeted bridge molecule at 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Figure 19]

[0075] Figure 19 shows an alternative representation of the data from Figure 18. There were no significant differences in the titration of EGFR cross-linking molecules (data not shown). There were significant differences in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test. [Figure 20]

[0076] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_hPGK. There was a significant effect on MOLM-13 viability after 48 hours of incubation at a 10:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Figure 21]

[0077] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_hPGK. There was a significant effect on MOLM-13 viability after 48 hours of incubation at an ET ratio of 20:1 between EGFR targeted bridge molecule conditions and CD33 targeted bridge molecules at 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Figure 22]

[0078] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_EF1a. There was a significant effect on MOLM-13 viability after 24 hours of incubation at a 10:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Figure 23]

[0079] Figure 23 shows an alternative representation of the data from Figure 22. There were no significant differences in the titration of EGFR cross-linking molecules (data not shown). There were significant differences in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test. [Figure 24]

[0080] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_EF1a. There was a significant effect on MOLM-13 viability after 24 hours of incubation at a 20:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 40ng / mL, 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Diagram 25]

[0081] Figure 25 shows an alternative representation of the data from Figure 24. There were no significant differences in the titration of EGFR cross-linking molecules (data not shown). There were significant differences in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test. [Figure 26]

[0082] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_EF1a. There was a significant effect on MOLM-13 viability after 48 hours of incubation at a 10:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 40ng / mL, 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Figure 27]

[0083] Titration experiments of EGFR and CD33 targeted Fab bridge molecules were used to test the effect on MOLM-13 killing by CAR0007_EF1a. There was a significant effect on MOLM-13 viability after 48 hours of incubation at an ET ratio of 20:1 between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 40ng / mL, 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. [Figure 28]

[0084] (a) In killing assays (cytotoxicity measured by quantification of residual leukemic cells compared to control), elimination of leukemic cells at EGFR bridge molecule concentrations of 40, 200 and 1000 ng / mL showed no significant difference between non-transduced T cells compared to CAR T cells. (b) Elimination of leukemic cells at CD33 bridge molecule concentrations of 40, 200 and 1000 ng / mL showed significant difference between non-transduced T cells compared to CAR0007-transduced T cells (hPGK and EF1a). [Figure 29]

[0085] A titration experiment of CD33-targeted Fab cross-linking molecules was used to test their efficacy in killing MOLM-13 by non-transduced T cells, CAR0007_hPGK and CAR0007_EF1a effector cells at an ET ratio of 10:1 after 48 hours of incubation. [Figure 30-1]

[0086] Analysis of the results in Figure 29. There were no consistent significant differences between the three effector cell populations at 40ng / mL, 200ng / mL and 1000ng / mL titration of CD33 cross-linking molecule. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test comparing the three conditions indicated per concentration of EGFR cross-linking molecule. [Figure 30-2]Analysis of the results in Figure 29. There were no consistent significant differences between the three effector cell populations at 40ng / mL, 200ng / mL, and 1000ng / mL titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test comparing the three conditions indicated per concentration of EGFR cross-linking molecules. [Diagram 31]

[0087] A titration experiment of CD33-targeted Fab cross-linking molecules was used to test their efficacy in killing MOLM-13 by non-transduced T cells, CAR0007_hPGK and CAR0007_EF1a effector cells at an ET ratio of 20:1 after 48 hours of incubation. [Figure 32-1]

[0088] Analysis of the results in Figure 31. There were consistent significant differences in titration of CD33 cross-linking molecules between the three effector cell populations at 8 ng / mL, 40 ng / mL, 200 ng / mL and 1000 ng / mL. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test comparing the three conditions indicated per concentration of EGFR cross-linking molecules. [Figure 32-2] Analysis of the results in Figure 31. There were consistent significant differences in the titration of CD33 cross-linking molecules between the three effector cell populations at 8 ng / mL, 40 ng / mL, 200 ng / mL and 1000 ng / mL. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test comparing the three conditions indicated per concentration of EGFR cross-linking molecules. [Diagram 33]

[0089] Figure 1 shows cell killing by nfP2X7 CAR-T cells in the presence of CD19-targeted bridging molecules with different dysfunctional P2X7 receptor epitope moieties in Fab and IgG1 formats. CAR10 vs. JeKo-1 effector cell / target cell (ET) ratio 10:1. CAR10 vs. target 2.9:1. N=1 healthy donor. 6 replicates. CAR10 expression (d12)=28.6%. Significance is shown for groups that are all significantly different from control vs. control (****). [Diagram 34]

[0090] Figure 1 shows cell killing by nfP2X7CAR-T cells in the presence of CD19-targeted bridge molecules with different dysfunctional P2X7 receptor epitope portions. CAR10 vs. JeKo-1 effector cell / target cell (ET) ratio of 20:1. N=1 healthy donor. 100ng / mL Fab CD19 bridge molecule. 6 replicates. In all CAR conditions, cells expressing CAR were normalized to an ET ratio of 1.48:1. Significance is shown for groups that are all significantly different from control vs. control (****). [Diagram 35]

[0091] Figure 1 shows cell killing by three different classes of nfP2X7 CAR-T cells (i.e., with different CAR structures) in the presence of CD19-targeting bridge molecules in Fab and IgG1 format. CAR7, CAR10 and CAR16 vs. JeKo-1 effector cell / target cell (ET) ratio 10:1. CAR7 vs. target 3.3:1; CAR10 vs. target 2.8:1. CAR16 vs. target 2.2:1. N=1 healthy donor. 6 replicates. CAR7 expression (d12) 33.3%; CAR10 expression (d12) = 28.6%; CAR16 expression (d12) = 22.6%. Significance is shown for groups vs. control (****) all significantly different from control. [Diagram 36]

[0092] Figure 1 shows cell killing by nfP2X7 CAR-T cells in the presence of (A) CD19-targeted bridging molecule in Fab format (killing of JeKo-1 cells), or (b) CD33-targeted bridging molecule in Fab format (killing of MOLM-13 cells). [Figure 37]

[0093] FIG. 1 shows a schematic diagram of the in vivo evaluation of cross-linking molecules. [Figure 38]

[0094] Figure 1 shows bioluminescence (total flux, p / s) as an indicator of tumor burden in mice after inoculation with Jeko-1_LUC_eGFP cells. Mice were treated with: Group 1 = no treatment; Group 2 = activated non-transduced T cells; Group 3 = bridging molecule (E200 epitope + anti-CD19 Fab); Group 4 = activated non-transduced T cells + bridging molecule; Group 5 = 3rd generation anti-CD19 CAR T cells; Group 6 = T cells expressing anti-nfP2X7 receptor CAR1; Group 7 = CAR1-T cells + bridging molecule; Group 8 = T cells expressing nfP2X7 receptor CAR2; Group 9 = CAR2-T cells + bridging molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] [Table 1-1]

[0053] [Table 1-2]

[0054] [Table 1-3]

[0055] [Table 1-4]

[0056] [Table 1-5]

[0057] [Table 1-6]

[0058] [Table 1-7]

[0059]

Table 1-8

[0060]

Table 1-9

[0061]

Table 1-10

[0062]

Table 1-11

[0063]

Table 1-12

[0064]

Table 1-13

[0065]

Table 1-14

[0066]

Table 1-15

[0067]

Table 1-16

[0068]

Table 1-17

[0069]

Table 1-18

[0070]

Table 1-19

[0071]

Table 1-20

[0072]

Table 1-21

[0073]

Table 1-22

[0074]

Table 1-23

[0075]

Table 1-24

[0076]

Table 1-25

[0077]

Table 1-26

[0078]

Table 1-27

[0079]

Table 1-28

[0080]

Table 1-29

[0081]

Table 1-30

[0082]

Table 1-31

[0083]

Table 1-32

[0084]

Table 1-33

[0085]

Table 1-34

[0086]

Table 1-35

[0087]

Table 1-36

[0088]

Table 1-37

[0089]

Table 1-38

[0090]

Table 1-39

[0091]

Table 1-40

[0092]

Table 1-41

[0093]

Table 1-42

[0094]

Table 1-43

[0095]

Table 1-44

[0096]

Table 1-45

[0097]

Table 1-46

[0098]

Table 1-47

[0099]

Table 1-48

[0100]

Table 1-49

[0101]

Table 1-50

[0102]

Table 1-51

[0103]

Table 1-52

[0104]

Table 1-53

[0105]

Table 1-54

[0106]

Table 1-55

[0107]

Table 1-56

[0108]

Table 1-57

[0109]

Table 1-58

[0110]

Table 1-59

[0111]

Table 1-60

[0112]

Table 1-61

[0113] [Table 1-62]

[0114] [Table 1-63]

[0115] [Table 1-64]

[0116] [Table 1-65]

[0117] [Table 1-66]

[0118] Detailed Description of the Preferred Embodiments

[0095] 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.

[0119]

[0096] 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.

[0120]

[0097] 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.

[0121]

[0098] All patents and publications referred to herein are incorporated by reference in their entirety.

[0099] The present invention aims to address 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) expanding the targeting of immune cells expressing chimeric antigen receptors (CARs) that bind to tumor-specific antigens, such as dysfunctional P2X7 receptors; and / or b) provide a tunable, "switchable" approach to targeted cell killing in a variety of settings that minimizes on-target and off-tumor effects; and c) Minimizing aberrant immune responses against adaptor molecules.

[0122] The present invention provides a novel treatment modality that includes a first component and a second component. The first component is the administration of immune cells that include a CAR (also referred to herein as "nfP2X7-directed CAR") that binds to dysfunctional P2X7 receptor expressed on the cell surface. The second component is the administration (or expression) of a bridge molecule that can redirect tumor-specific CAR (e.g., nfP2X7-directed CAR) to other cell surface molecules such as cancer-associated antigens, e.g., CD19, CD20, CD33, GD2, intracellularly processed proteins that are presented as peptides of various lengths via MHC I and II or any other mechanism of accessible surface antigen exposure.

[0123] The targeted cell surface molecule may be associated with cancer (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 targeting peptides for cancer-specific proteins (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).

[0124]

[0102] Furthermore, the present invention offers several advantages over existing "adapter CAR" approaches. More specifically, adapter CAR T cell technology is based on an approach that uncouples the tumor targeting and signaling parts of conventional CARs, resulting in a bifurcated system consisting of an adapter CAR and a soluble tumor-specific adapter molecule. The basic structure of the adapter CAR corresponds to the conventional CAR design, but the extracellular domain does not interact with tumor-associated antigens, but instead with a binding partner incorporated in the adapter molecule. The bifunctional adapter molecule, in turn, provides target cell (tumor cell) specificity and acts as a linker at the interface between the tumor and the adapter CAR T cell. This complex can then mediate an anti-tumor response, similar to conventional CAR T cells.

[0125]

[0103] Although the dual principle of the adaptor CAR system provides an important molecular safety switch for precisely controlling adaptor CAR T cell activity, several limitations have been identified in the systems developed to date.

[0126] For example, one system relies on scFv-based α-FITC CARs that target the synthetic dye FITC coupled to various tumor-specific adaptor molecules. Despite the full humanization of the α-FITC CAR T cell product, the immunogenicity of FITC is a concern for clinical translation, as highlighted by the emergence of anti-α-FITC antibodies in therapeutic mouse models.

[0127] In another example, the adapter molecule contains a small peptide tag to redirect standard scFv-based adapter CAR. However, concerns have been raised again regarding the immunogenicity of small peptide tags contained in the adapter molecule, especially when the tag contains a non-human sequence or a sequence derived from a human nuclear protein.

[0128]

[0106] In contrast to prior art approaches, the present invention exploits both the specificity of the CAR binding to dysfunctional P2X7 and the unique properties of the epitope derived from the dysfunctional P2X7 receptor.

[0129]

[0107] The present invention provides advantages over existing mono-antigen directed CAR T therapies in that: Utilizing CAR T cells that are functional (i.e., there is no need for the use of a switch molecule to activate the CAR T cell); · Utilizing CAR T cells that do not bind to healthy cells because dysfunctional P2X7 receptors are only exposed on the surface of cancer cells; This means that there is no need for the administration of immunosuppressants; This also means that side effects are significantly reduced compared to other approaches, including the use of CARs that bind to antigens that are also found on non-cancerous cells; Contains non-immunogenic, naturally occurring human epitopes on the bridging molecule in the form of epitopes derived from dysfunctional P2X7 receptors.

[0130]

[0108] Thus, the present invention provides a new concept and approach in the use of adaptor / bridging molecules in conjunction with CAR T therapy.

[0109] More specifically, the specificity of CAR (also referred to herein as nfP2X7CAR) binding to dysfunctional nfP2X7 receptor 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, CAR-mediated recognition can be broadened to include any target antigen of interest through the corresponding bridging molecule. Although nfP2X7CAR only recognizes dysfunctional P2X7 receptor, e.g., E200 (or E300 or E200-300 complex) epitope, the use of the bridging molecule, however, facilitates unlimited targeting by any accessible recognition site expressed on the cell surface. For example, nfP2X7CAR can be further directed (or redirected) to bind to CD19-positive cells through the use of a bridging molecule that includes a targeting moiety for CD19 binding on the cell surface and the E200 epitope from nfP2X7.

[0131] 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.

[0132] The advantage of non-immunogenic recognition sites in the bridge molecules, 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.

[0133]

[0112] 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 CAR. Thus, the present invention exploits the specificity of nfP2X7CAR in two ways: first, it relies on the fact that nfP2X7CAR only targets cells that express nfP2X7 (cancer cells only), and second, it relies on the fact that immune cells expressing nfP2X7CAR can be redirected to other tumor-associated antigens and / or specific target antigens in a switchable and adjustable manner using a bridging molecule engineered to receive nfP2X7 binding.

[0134]

[0113] Thus, the use of the bridging molecules of the present invention allows the redirection of immune cells expressing nfP2X7CAR for targeting of any surface-expressed target antigen. A particular advantage of this approach is that targeting is limited to the period during which the bridge molecule therapeutic agent in vivo persists in circulation. This means that any toxicity resulting from "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 nfP2X7CAR cells can only target nfP2X7 in a tumor-specific manner again. 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 implement 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 CAR T cells can be temporarily directed to cancer cells via antigens other than nfP2X7. Furthermore, the length of time that CAR T cells are redirected to other cancer antigens can be regulated by the time that the bridge molecule is administered to the patient in need.

[0135]

[0115] 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 CAR molecule (i.e., for binding to dysfunctional P2X7 receptors present on cancer cells) to target multiple antigens present on cancer cells. More specifically, using a bridging molecule that includes a targeting moiety for CD19 binding, CAR-T cells can be targeted to cancer cells at both dysfunctional P2X7 receptors and CD19. This maximizes the chances that cancer cells will be killed because multiple sites are targeted. 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 for the use of a variety of different bridging molecules, each of which includes an epitope for receiving the binding of a single type of CAR T cell, but includes different targeting moieties, such that CAR T cells can be directed to bind to cancer cells via multiple cancer antigens. In this way, cancer cells can be targeted and bound by CAR T cells at multiple sites, increasing the efficacy of cell killing.

[0136] 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 that arise 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 that have been 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.

[0137] 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 CAR T cells that bind to dysfunctional P2X7 receptors can be simultaneously administered a bridging molecule that facilitates redirecting CAR T cells to cells that present peptides derived from infectious agents on their cell surface MHC molecules. In other words, the present invention provides a platform for the simultaneous or sequential treatment of cancer and infectious diseases.

[0138]

[0118] 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.

[0139]

[0119] When using nfP2X7R CAR without the presence of a bridging molecule, effector cells expressing nfP2X7CAR show cancer-specific targeting (Scenario I, see Figure 11).

[0140] To broaden the applicability to cancers that are functionally negative for nfP2X7 (very low or negative for nfP2X7), effector cells expressing nfP2X7CAR 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 bridging molecules means that, without limitation, any surface-expressed target antigen or presented antigen can engage effector cells expressing nfP2X7CAR in the same manner of action, in the context of MHC peptide presentation (class I and II) via TcR-like mAbs or ligands (Scenario II, see Figure 11).

[0141] In most cases, the dual function of effector cells expressing nfP2X7CAR is utilized (Scenario III, see Figure 11). This is a combination of scenarios I and II, meaning that effector cells expressing nfP2X7CAR directly engage with cancer cells via nfP2X7 expressed on cancer cells, and are further recruited to cancer cells via bridging molecules targeting cancer-associated antigens, such as CD33, or cancer-specific antigens via TcR-like mAbs.

[0142] definition

[0122] 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.

[0143]

[0123] 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.

[0124] "Purinergic receptor" generally refers to a receptor that uses purine (eg, ATP) as a ligand.

[0144]

[0125] "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.

[0145] SEQ ID NO: 1 MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVISSVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRLCPEYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVYEGNQKTCEVSAWCPIEAVEEAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIFRETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNFRYAKYYKENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLIDTYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRDSPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQYSGFKSPY

[0127] Insofar as P2X7 receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X7 receptor" encompasses naturally occurring variants of P2X7 receptor, for example, P2X7 monomer isoforms (e.g., forms consisting of extracellular domain sequence or truncated forms thereof), naturally occurring variant forms (e.g., alternative splice forms) and naturally occurring allelic variants, including splice variants, allelic variants, SNPs, and naturally occurring truncated or secreted forms of monomers forming P2X7 receptor. In certain embodiments of the present invention, the native sequence P2X7 monomer polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in SEQ ID NO:1. In certain embodiments, P2X7 receptor can be modified amino acid sequence, for example, various amino acids in the sequence shown in SEQ ID NO:1 are replaced, deleted, or residues are inserted.

[0146]

[0128] "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, which 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.

[0147]

[0129] "Dysfunctional P2X7 receptor" generally refers to a form of P2X7 receptor that has a conformation different from functional P2X7, whereby the receptor cannot form an apoptotic pore, but can still operate as a nonselective 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 cannot bind ATP at one, more specifically two, of the ATP binding sites on the trimer, and as a result, cannot extend the opening of the channel. In such a situation, the receptor cannot form a pore, which limits the extent to which calcium ions can enter the cytoplasm. Dysfunctional P2X7 receptors are expressed in a wide range of epithelial and hematopoietic cancers. As used herein, the term "dysfunctional P2X7 receptor" can be used interchangeably with the terms "non-functional P2X7 receptor" or "nfP2X7 receptor."

[0148]

[0130] 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.

[0149]

[0131] "E200 epitope" generally refers to an epitope having the sequence GHNYTTNILPGLNITC, and variants thereof (eg, SEQ ID NOs: 2-11, 15-30, and 168).

[0150]

[0132] "E300 epitope" generally refers to the epitope having the sequence KYYKENNVEKRTLIK, and variants thereof (SEQ ID NOs: 12 and 13).

[0133] "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).

[0151]

[0134] 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.

[0152]

[0135] 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 disulfide bridges.

[0153]

[0136] 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).

[0154]

[0137] 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.

[0155]

[0138] 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.

[0156]

[0139] 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.

[0157] "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).

[0158]

[0141] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0142] An "antigen-binding site" generally refers to a molecule that includes at least the hypervariable and framework regions necessary to confer antigen-binding function to a V domain. The antigen-binding site may be in the form of an antibody or antibody fragment (e.g., mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD-Fv, Fv, F(ab')2 or scFv) in the manner described herein.

[0159] 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.

[0160]

[0144] "Whole antibody fragments containing the variable domain" includes SD-mAbs, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0161]

[0145] 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.

[0162] "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.

[0163]

[0147] 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.

[0148] "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.

[0164] 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.

[0165]

[0150] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and refers to V chains 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.

[0166]

[0151] 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.

[0167]

[0152] "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 that are spaced apart such that inter-chain, but not intra-chain, 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.

[0168] 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.

[0169] 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.

[0170]

[0155] "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 modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled.

[0171] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, 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.

[0172]

[0157] "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.

[0173] 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.

[0174]

[0159] 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.

[0175]

[0160] 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.

[0176]

[0161] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0162] "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.

[0177]

[0163] 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, a scFv or multimer thereof, a 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.

[0178]

[0164] "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.

[0179]

[0165] As used herein, the term "target cell" refers to the cell that expresses dysfunctional P2X7 receptor (e.g., nfP2X7 receptor) or cell surface molecule that the targeting portion of the bridging molecule binds to. Target cell can be cancer cell or any other disease cell.

[0180]

[0166] 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.

[0181] 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.

[0182]

[0168] 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.

[0183]

[0169] The term "autologous" as used herein refers to any material from the same subject into which it is subsequently reintroduced.

[0170] 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.

[0184]

[0171] 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.

[0185]

[0173] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. These terms mean to contain and / or express a foreign gene or nucleic acid sequence that in turn modifies the genotype or phenotype of the cell or its 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).

[0186]

[0174] The term "immune cell" or "immune effector cell" refers to a cell that is part of the immune system and performs 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.

[0187]

[0175] 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.

[0188]

[0176] 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. 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.

[0189] 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.

[0190]

[0179] 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 comprise 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.

[0191] 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 (or E300 or E200-300 complex) 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 NYVVTDH at amino acid sequence 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.

[0192] 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 LIt contains amino acid sequence homology with the CDR1, 2 and 3 domains of the chain.

[0193]

[0182] 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 described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or corresponding U.S. Pat. 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, 8,102,432, 8,112,432, 8,122,432, 8,136,432, 8,140,432, 8,152,432, 8,162,432, 8,172,432, 8,182,432, 8,192,432, 8,202,432, 8,232,432, 8,242,432, 8,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 8,252,432, 8,326,415, 7,888,473, 7,531,171, 8,080,635, 8,399,617, 8,709,425, 8,136,432, 8,122,432, 8,242,432, 8,136,432, 8,142,432, 8,242,432, 8,1 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 9,944,701). No. 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Patents No. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Patents No. 8,597,643, 9,328,155 or 10,238,716), PCT / AU2010 / 001070 (or any one of the corresponding 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 (or any one of the corresponding WO 2011 / 075789 or U.S. Pat. No. 8,835,609). H and / or V LPreferably, the binding polypeptide comprises the amino acid sequence of the CDR of the V chain of antibody 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 V chain 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. H and / or V L Alternatively, the binding polypeptide of the CAR may comprise the amino acid sequence of the CDRs of antibodies sdAb2-2-3, 2-472-2, or 2-2-12, which are described in WO 2017 / 041143 (also published as U.S. Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Application No. 17 / 057,060), which are incorporated herein by reference.

[0194] CAR binding polypeptides can 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, 9,621, 9,731, 9,821, 9,971, 9,991, 10,102, 10,104, 10,106, 10,108, 10,112, 10,116, 10,122, 10,130, 10,140, ​​10,152, 10,162, 10,170, 10,182, 10,190, 10,192, 10,194, 10,196, 10,198, 10,199, 10,183, 10,19 ... Nos. 63,584, ... No. 10,597,451), PCT / AU2008 / 001365 (or any one of the corresponding U.S. Patents No. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Patents No. 8,597,643, 9,328,155 or 10,238,716), PCT / AU 2010 / 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 binding polypeptide comprises the amino acid sequence of antibody 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 V 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 No. 06080101. H and / or V L Alternatively, the binding polypeptide of the CAR comprises the amino acid sequence of the V chain of antibodies sdAb2-2-3, 2-472-2, or 2-2-12, described in WO 2017 / 041143 (also published as U.S. Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Application No. 17 / 057,060), which are incorporated herein by reference. H and / or V L The amino acid sequence of the chain may be included.

[0195] 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, and 9,663,584), the entire contents of which are incorporated herein by reference. 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 10,5 Nos. 8,293,491 or 8,658,385), PCT / AU2009 / 000869 (or any one of the corresponding U.S. Patents. 8,597,643, 9,328,155 or 10,238,716), PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patents. 8,293,491, 8,658,385, and 10,238,716), PCT / AU2010 / 001070 (or any one of the corresponding U.S. Patents. 8,597,643, 9,328,155, and 10,238,716), PCT / AU2012 / 001070 (or any one of the corresponding U.S. Patents. 8,293,491, 8,658,385, and 10,238,716), PCT / AU2014 / 001070 (or any one of the corresponding U.S. Patents. 8,293,491, 8,658,385, and 10,238,716), PCT / AU2016 / 001070 (or any one of the corresponding U.S. Patents. 8,293,491, 8,658,385, and 10,238,716), PCT / AU2018 ... may comprise the amino acid sequence of an antibody or fragment thereof described in any one of corresponding 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 (or any one of corresponding WO 2011 / 075789 or U.S. Pat. No. 8,835,609).Preferably, the binding polypeptide comprises the amino acid sequence of sdAb2-2-1, 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 antibody BPM09, as described in WO2013185010A1 or WO2019056023, as described in PCT / AU2007 / 001541 (or the corresponding U.S. Patent Application Publication No. 2010-0036101) and produced by hybridoma AB253 deposited at the European Collection of Cultures (ECACC) under Accession No. 06080101. Alternatively, the binding polypeptide may comprise the amino acid sequence of sdAb2-2-3, 2-472-2, or 2-2-12, as described in WO 2017 / 041143 (also published as U.S. Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Application No. 17 / 057,060), which are incorporated by reference herein.

[0196]

[0186] "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.

[0197]

[0187] 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.

[0198] 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.

[0199]

[0189] 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.

[0200] 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).

[0201] 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, for example, 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 that is sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0202]

[0192] The function of the intracellular domain may be pro-inflammatory and / or immunomodulatory, or a combination of such.

[0193] 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.

[0203]

[0194] 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.

[0204]

[0203] Primary cytoplasmic signaling sequences that act in a stimulatory manner are likely to contain ITAM (immunoreceptor tyrosine-based activation motif) signaling motifs.

[0196] 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 notable is the sequence derived from CD3 zeta.

[0205] 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. 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.

[0206]

[0198] 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.

[0207]

[0199] As one example, the cytoplasmic domain can include the signaling domain of CD3-zeta and the signaling domain of CD28. In another example, the cytoplasmic domain can include the signaling domain of CD3-zeta and the signaling domain of CD27. In a further example, the cytoplasmic domain can include the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD27.

[0208]

[0200] As mentioned above, the extracellular portion or transmembrane or cytoplasmic domain of the CAR may also contain a heterodimerization domain for the purpose of dividing the key signaling and antigen recognition modules of the CAR.

[0209]

[0201] Non-limiting examples of CARs that can be used in accordance with the present invention are set forth in SEQ ID NOs: 165-167, 266 or 272. Examples of structures of various CAR molecules are also provided herein in Figure 35. Further examples of CARs that can be used in accordance with the present invention are provided in WO 2017 / 041143 (also published as U.S. Patent Application Publication No. 2019 / 0365805), and WO 2019 / 222796 (corresponding to U.S. Patent Application Publication No. 17 / 057,060), which are incorporated herein by reference.

[0210]

[0202] CARs for use in accordance with the present invention, i.e., CARs comprising an nfP2X7E200 binding domain, can be designed to comprise any portion or parts of the above domains described herein in any order and / or combination, resulting in a functional CAR.

[0211]

[0203] 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 are normally associated 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.

[0212]

[0204] 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.

[0213] EGFRvIII-targeted CAR T cells 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 is also expressed 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.

[0214] EGFRvIII-targeted CAR T cells can be used to treat glioblastoma in a conventional manner by targeting EGFRvIII on cancer cells, but can also be redirected to other cancer-associated target antigens via the bridging molecule described herein, when the EGFRvIII epitope portion is incorporated into the sequence of the bridging molecule.The EGFRvIII CAR T cells can then be used in the same manner 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.

[0215]

[0207] 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.

[0216]

[0208] 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.

[0217] crosslinking molecule It is understood that the bridging molecule can be in any form, provided that it comprises a) a targeting portion for binding to target cells, and b) a tumor-specific antigen epitope portion.Preferably, the tumor-specific antigen epitope portion is or comprises a dysfunctional P2X7 receptor epitope portion, an EGFRvIII epitope portion or a CLDN6 epitope portion, and preferably, according to the method of the present invention, the epitope portion is recognized by the tumor-specific CAR that is redirected.

[0218]

[0210] 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.

[0219] It is understood that the bridging molecule can have any of a number of different architectures. For example, and as further described herein, the targeting moiety for binding to the target cell can be in the form of any suitable binding domain, such as from an antibody, or a fragment thereof. The targeting moiety can be linked to the tumor-specific antigen epitope moiety in any configuration. For example, the tumor-specific antigen epitope moiety can be linked to the N- or C-terminus of the targeting moiety. Examples of suitable architectures are provided in the figures herein. In a particularly preferred embodiment, the tumor-specific antigen epitope moiety is linked to the targeting moiety via its C-terminal region, such that the N-terminal region of the tumor-specific antigen epitope moiety is freely accessible for binding by the tumor-specific CAR.

[0220]

[0212] Any suitable linker can be used to link the targeting moiety to the tumor-specific antigen epitope moiety. The linker can include a polypeptide, peptide, or a chemical group.

[0221] The linker may be a peptide having a length of up to 20 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 may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. For example, the bridging molecule provided herein may 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 may increase the likelihood that different polypeptides of the fusion protein will fold independently and behave predictably.

[0222]

[0214] Those skilled in the art are familiar with the design and use of various peptide linkers, including various amino acids and lengths, that are suitable for use as linkers according to the present invention. The linker may include various combinations of repeated amino acid sequences. The linker may be a flexible linker (e.g., one that includes repeats of glycine and serine residues), a rigid linker (e.g., one that includes glutamic acid and lysine residues, adjacent alanine repeats), and / or a cleavable linker (e.g., a sequence that is more susceptible to protease cleavage).

[0223]

[0215] The peptide linker can be any one or more repeats of Gly-Ser (GS), Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS), or variations thereof. In any embodiment, the linker can comprise or consist of the sequence GGGGSGGGSGGGGGS, i.e., (G4S)3.

[0224] In any 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.

[0225]

[0217] In further embodiments, the linker may include the inclusion of amino acids that provide rigidity, such as lysine. For example, in certain embodiments, the linker region may also include the sequence GSGK.

[0226]

[0218] 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).

[0227] In a further embodiment, alternatively or in addition to the above glycine-serine based linker region, the targeting moiety can be in the form of a fusion protein, in which the targeting moiety is linked to the tumor-specific antigen epitope moiety, preferably the dysfunctional P2X7 receptor epitope moiety, via a hinge region. The link between the targeting moiety and the tumor-specific antigen epitope moiety can include a combination of a hinge region and a linker region.

[0228]

[0220] Examples of suitable hinge regions include those derived from immunoglobulins. The hinge region may be derived from IgG1, IgG2, IgG3 or IgG4 and may contain one or more amino acid substitutions (e.g., to prevent or reduce the possibility of disulfide bridge formation). Alternative hinge sequences may be derived from alternative immunoglobulin domains, CD8A, CD8B, CD4 or CD28, TRAC, TRBC, TRGC, TRDC.

[0229]

[0221] Table 2 below provides non-limiting examples of suitable hinge regions for use in joining targeting moieties and tumor-specific antigen epitope moieties in the bridge molecules of the invention. It is understood that the targeting moiety may be connected to the tumor-specific antigen epitope moiety by more than one linker and / or more than one hinge region. For example, the fusion protein may contain a tumor-specific antigen epitope moiety directly conjugated (N-to-C-terminus) to the targeting moiety. Alternatively, the fusion protein may contain a tumor-specific antigen epitope moiety, followed by a linker region, then a targeting moiety. Still further, the fusion protein may contain a tumor-specific antigen epitope moiety, followed by a linker region, then a hinge region, then a targeting moiety. In still further embodiments, the fusion protein may contain a tumor-specific antigen epitope moiety, followed by a linker region, then a hinge region, further linker region, then a targeting moiety. Of course, those skilled in the art will understand that alternative structures are possible (i.e., the tumor-specific antigen epitope moiety is connected to the C-terminus of the targeting moiety via one or more linkers and / or hinge regions).

[0230]

[0223]

[0231] [Table 2-1]

[0232] [Table 2-2]

[0233] Targeting moiety of the bridging molecule The targeting portion of the bridging 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.

[0234]

[0225] The bridging 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 invading organism (e.g., yeast, nematode, bacteria, fungi). Further disclosed herein are bridging molecules that interact with molecules on non-cellular targets. The non-cellular target may be a virus or 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.

[0235]

[0226] The target cells can be derived from a tissue. The tissue 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.

[0236]

[0227] 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.

[0228] The target cell may be a cancer stem cell. The target cell may be a cancer cell. The cancer cell may be derived from a tissue. The tissue may 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 cell may be derived from bone. The cancer cell may be derived from blood. The cancer cell may be derived from B cells, T cells, monocytes, platelets, leukocytes, neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, or endothelial cell precursor cells. The cancer cell may be derived from CD19 positive B lymphocytes. The cancer cell may be derived from a stem cell. The cancer cell may be derived from a pluripotent cell. The cancer cell may be derived from one or more endocrine glands. The endocrine glands may be lymphatic, pituitary, thyroid, parathyroid, pancreas, gonads, or pineal gland.

[0237] 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 non-limiting examples, 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 non-limiting examples, 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.

[0238] 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.

[0239]

[0231] The cell surface molecule of the target cell may be a peptide-free 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.

[0240]

[0232] The cell surface molecule of the target cell can be an antigen. The antigen can be at least a part of a surface antigen or a cell surface marker on the cell. The antigen can be a receptor or a co-receptor on the cell. The antigen can refer to a molecule or a molecular fragment that can be bound by the major histocompatibility complex (MHC) and presented to a T cell receptor. The term "antigen" can also refer to an immunogen. An immunogen can provoke an adaptive immune response when injected into a subject alone. An immunogen can induce an immune response by itself. The 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.

[0241] The present invention may also find application for treating specific B or T cell lineage-related autoimmune diseases, for example, 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, acute, chronic or mixed rejection of solid organ transplants, rejection of bone marrow or stem cell transplants, graft-versus-host disease.

[0242] The cell surface molecule of the target cell may be an antigen designated as a tumor antigen. The tumor antigen or neoantigen may be an antigen presented by MHC I or MHC II molecules on the surface of the tumor cell. 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). TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors may occur under conditions that allow the immune system to respond to the antigen. TAA may be an antigen expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells. Cytotoxic T lymphocytes that recognize these antigens can destroy tumor cells before they can proliferate or metastasize. Tumor antigens may also be on the surface of the tumor, for example in the form of mutated receptors, in which case they can be recognized by B cells.

[0243]

[0235] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, 20 These include CD68\Pl, C0-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-C0-1, RCASl, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0244] 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 ... CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD184 (CXCR4), CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD44V6, CD47, CD 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. Other target antigens include TCR, sugars, lipids, carbohydrates, or any other molecule expressed on the surface of a target cell. The antigen can be any antigen mentioned in Table 1 in the context of a bridging molecule or binding construct.

[0245] In a preferred embodiment, the target cell is a cancer cell and the cell surface molecule of the cancer cell is an antigen associated with the cancer. The antigen may be a tumor-specific antigen or a tumor-associated antigen. The antigen may be associated with a particular type of cancer. For example, overexpression of an antigen may be associated with a particular cancer or a particular class of cancer. For example, if the cancer is breast cancer, the antigen may be associated with breast cancer but not with another type of cancer. Alternatively, the antigen may be associated with a class of cancer, such as solid tumors, but not with hematological (i.e., "liquid") tumors, or vice versa. The antigen may be associated with a particular lineage of cancer but not with other cancers. For example, the antigen may be associated with sarcomas but not with lymphomas or carcinomas. As used herein, the term "associated with" in reference to cancer is understood to mean that expression of the antigen (whether increased or decreased) is considered to be a marker of the cancer. It is understood that there may be a low level of expression of an antigen, but this does not equate to an antigen being "associated" with a given cancer.

[0246] 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 endothelial cell line (EGFR-specific endothelial cell line). 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), HER3, 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 (M uc-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 is understood that these or other cancer antigens can be utilized for targeting by the bridging molecules in the present invention.

[0247]

[0239] The targeting portion 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.

[0248] 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 modifications 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.

[0249]

[0241] The targeting antibody or antibody fragment may be an Fc null immunoglobulin or a fragment thereof.

[0242] As used herein, the term "antibody fragment" refers to an antibody in any form 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."

[0250] 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.

[0251]

[0244] The targeting antibody or antibody fragment may be based on or derived from an antibody or antibody fragment from a mammal, bird, fish, amphibian or reptile. Mammals include, but are not limited to, carnivores, rodents, elephants, marsupials, rabbits, bats, primates, seals, anteaters, whales, dung beetles, and ungulates. Mammals may be humans, non-human primates, mice, sheep, cats, dogs, cows, horses, goats, or pigs.

[0252]

[0245] 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.

[0253]

[0246] 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. The targeting polypeptide may comprise an anti-CD30 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD79B antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD37 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD38 antibody or fragment thereof. The targeting polypeptide may comprise an anti-CD70 antibody or fragment thereof. The targeting polypeptide may comprise an anti-CD276 antibody or fragment thereof. The targeting polypeptide may comprise an anti-GD2 antibody or fragment thereof. The targeting polypeptide may comprise an anti-CD371 antibody or fragment thereof. The targeting polypeptide may comprise an anti-CD135 antibody or fragment thereof. The targeting polypeptide may comprise an anti-CD105 antibody or fragment thereof. The targeting polypeptide may comprise an anti-CD123 antibody or fragment thereof. The targeting polypeptide may comprise an anti-ROR-1 antibody or fragment thereof. The targeting polypeptide may comprise an anti-PD-L1 antibody or fragment thereof. The targeting polypeptide may comprise an anti-MET-R antibody or fragment thereof. The targeting polypeptide may comprise an anti-PDGFRalpha antibody or fragment thereof. The targeting polypeptide may comprise an anti-Her3 antibody or fragment thereof. The targeting polypeptide may comprise an anti-FRalpha antibody or fragment thereof. The targeting polypeptide may comprise an anti-GPC3 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-SLAMf7 antibody or a fragment thereof.The targeting polypeptide may comprise an anti-TNFRSF10B antibody or a fragment thereof. The targeting polypeptide may comprise an anti-GPNMB antibody or a fragment thereof. The targeting polypeptide may comprise an anti-VEGFR2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-alpha4beta7 / alphaEbeta7 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CSPG4 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD80 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CCR4 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD115 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-ENOX-2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD56 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-huVH1-69 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD117 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD133 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MUC1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MSLN antibody or a fragment thereof. The targeting polypeptide may comprise an anti-ROR-2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-IL13Ra2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-EPHA2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-EGFRvIII antibody or a fragment thereof. The targeting polypeptide may comprise an anti-PSMA antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CEA antibody or a fragment thereof. The targeting polypeptide may comprise an anti-Lewis Y antibody or a fragment thereof. The targeting polypeptide may comprise an anti-PSCA antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MUC1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD181L1CAM antibody or a fragment thereof. The targeting polypeptide may comprise an anti-EpCAM antibody or a fragment thereof. The targeting polypeptide may comprise an anti-ALK antibody or a fragment thereof. The targeting polypeptide may comprise an anti-IGF-1R CD221 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-Nectin 4 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-FAP antibody or a fragment thereof.The targeting polypeptide may comprise an anti-AXL antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD138 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CLDN6 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-Her4 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-claudin 18.2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-O-acetylated GD2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-GD3 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-GM2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-TM4SF1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD147 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CEACAM5 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-VEGFR-1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-PDPN antibody or a fragment thereof. The targeting polypeptide may comprise an anti-WT1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-GPC2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-FGFR4 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-EphB4 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-STEAP-1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-STEAP-2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MUC1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-chlorotoxin antibody or a fragment thereof. The targeting polypeptide may comprise an anti-206 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-ILRAP1 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MICA antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MAGE-A1 scTcR antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MAGE-A1 sTCR antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MICA antibody or a fragment thereof. The targeting polypeptide may comprise an anti-TRBC1 / 2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-B7-H7 antibody or a fragment thereof.The targeting polypeptide may comprise an anti-CD34 antibody or a fragment thereof.The targeting polypeptide may comprise an anti-CD7 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-TIM3 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD191 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD66b antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD11b antibody or a fragment thereof. The targeting polypeptide may comprise an anti-EMR2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-MUC16 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-NYESO-1 HLA-A2 antibody or a fragment thereof, or a soluble TcR or a variant thereof. The targeting polypeptide may comprise an anti-survivin HLA-A2 antibody or a fragment thereof, or a soluble TcR or a variant thereof. The targeting polypeptide may comprise an anti-CD200 antibody or a fragment thereof.

[0254]

[0247] Targeting antibody or antibody fragment can be selected from any commercially available antibody. Targeting antibody or antibody fragment can be selected from trastuzumab (for Her2 binding), alemtuzumab (for CD52 binding), bevacizumab (for VEGF-A binding), brentuximab (for CD30 binding), gemtuzumab (for CD33 binding), ipilimumab (for VTLA-4 binding), ibritumomab (for CD20 binding), panitumumab (for EGFR binding), cetuximab (for EGFR binding), rituximab (for CD20 binding), and fragments thereof.

[0255]

[0248] The targeting antibody or antibody fragment can be any of those referred to in Table 1, or an antigen-binding fragment thereof 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.

[0256]

[0249] 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.

[0257]

[0250] Non-limiting examples of sequences of various targeting antibodies, or antigen-binding fragments thereof, are provided herein in Table 1.

[0251] It is well within the skill of one in the art to be able to determine the appropriate design of the cross-linking molecules described herein, including the selection of the targeting portion of the cross-linking molecule.

[0258]

[0252] More specifically, by having knowledge of the CAR that requires "redirection" as described herein, one of skill in the art can select an alternative targeting moiety to which the CAR should be directed, facilitating treatment of the cancer or disease in need of treatment.

[0259]

[0253] As described herein, the targeting moiety can be for binding to any cell surface molecule on the target cell, such as a tumor-associated antigen expressed on a cancer cell. A person skilled in the art can select an appropriate cell surface molecule depending on the disease (e.g., cancer) that requires treatment. For example, if the disease is a Her2+ cancer, a person skilled in the art can select a targeting moiety that can specifically bind to Her2 on the cancer cell. Similarly, if the disease is a CD19+ cancer cell, a person skilled in the art can select a targeting moiety that can specifically bind to CD19 on the cancer. A person skilled in the art is familiar with how to determine the antigen expression profile of the cancer or condition to be treated in order to identify the appropriate surface molecule expressed on the target cell.

[0260]

[0254] Having identified a suitable cell surface molecule (target antigen) on a target cell (e.g., CD19, Her2, CLL-1, CD33, EGFRvIII, CD20, CD22, BCMA, or any other tumor-associated antigen described herein or that can be selected for targeting), one skilled in the art can easily determine the sequence of a binding domain capable of binding to said target antigen, including by reference to commercially available antibodies or published literature that describes the antigen-binding domain of known antibodies that bind to the antigen. One skilled in the art can then formulate a structure of a bridging molecule (fusion protein) as described herein that includes a targeting portion that binds to the cell surface molecule on the target cell. The composition of the remaining components of the bridging molecule (e.g., tumor-specific antigen epitope portion) is further described herein.

[0261] Finally, when a person skilled in the art identifies a suitable cell surface molecule (target antigen) and tumor-specific antigen epitope portion on a target cell to arrive at a bridging molecule of the present invention, he or she can easily determine using routine techniques whether the bridging molecule a) binds to the target cell, b) binds to the CAR T cell, and c) successfully redirects the CAR T cell to the target cell to induce cell killing. Methods for determining binding to a target antigen and cytotoxicity are well known in the art. Non-limiting methods and various experimental protocols for determining binding to a target cell, CAR T cell, and induction of cell killing are described in detail in the examples herein.

[0262] Dysfunctional P2X7 receptor epitope region

[0256] The present invention contemplates the use of a tumor-specific antigen epitope portion in the form of an epitope from a dysfunctional P2X7 receptor (ie, the tumor-specific antigen is a dysfunctional P2X7 receptor).

[0263]

[0257] The dysfunctional P2X7 receptor epitope portion can be provided in the form of a dysfunctional P2X7 receptor or a fragment of a dysfunctional P2X7 receptor, which has at least one of the three ATP binding sites formed at the interface between adjacent correctly packed monomers that cannot bind ATP. Such receptors cannot extend the opening of non-selective calcium channels to the apoptotic pore.

[0264] A range of peptide fragments of dysfunctional P2X7 receptors are known and are described 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 U.S. Pat. No. 1,621,616, the entire contents of which are incorporated herein by reference in their entirety. No. 0,450,380), 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) In any embodiment, the amino acid sequence of the dysfunctional P2X7 receptor epitope portion of any of the bridging molecules described herein comprises or consists of a sequence shown in any of SEQ ID NOs: 2-30, 168, and 365-400, 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.

[0265]

[0260] 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.

[0266]

[0261] Various examples of dysfunctional P2X7 receptor epitope portions are described herein, in particular in Table 1. As is evident from Table 1, the minimal E200 sequence comprises at least the sequence of SEQ ID NO: 11. In a preferred embodiment, the minimal E200 sequence comprises at least the sequence of SEQ ID NO: 2 or a variant thereof, such as the sequence of SEQ ID NO: 4.

[0267] In certain instances, the E200 epitope for inclusion in the bridging molecule of the present invention may include an extension in the N-terminal and / or C-terminal region. The extension may be derived from the naturally occurring nfP2X7 receptor sequence and may include an extension of at least one amino acid residue, at least two amino acid residues, at least three, at least four, at least five or more amino acid residues. For example, the E200 epitope defined in SEQ ID NO:4 may include an N-terminal extension of the sequence DFP, which corresponds to the three amino acid residues immediately N-terminal to the E200 sequence of SEQ ID NO:1. In addition, the E200 epitope defined in SEQ ID NO:4 may include a C-terminal extension, for example, as shown in SEQ ID NOs:6 and 7. It is understood that such N-terminal and C-terminal extensions may help improve the effectiveness of CAR-T cell binding to the epitope on the bridging molecule.

[0268] In any embodiment, dysfunctional P2X7 receptor epitope part is conjugated to targeting part so as to allow sufficient accessibility for binding by nfP2X7 receptor CAR.Thus, the minimum dysfunctional P2X7 receptor epitope part sequence comprises the minimum E200 sequence at least the sequence of SEQ ID NO:11, but in a preferred embodiment, the minimum E200 sequence comprises at least the sequence of SEQ ID NO:2 or its variant, such as the sequence of SEQ ID NO:4, in addition to one or more linkers or hinge regions to allow binding by nfP2X7 receptor CAR. In certain embodiments, the total length of the dysfunctional P2X7 receptor epitope portion (including the linker and hinge regions) is at least 17 amino acids in length, or at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Preferably, the dysfunctional P2X7 receptor epitope portion is about 100 amino acids or less in length, more preferably about 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or 50 amino acids or less in length.

[0269]

[0264] In the context of a bridging molecule for targeting CD19, preferably the dysfunctional P2X7 receptor epitope portion is no more than about 35 amino acids in length, at least 20 amino acids in length, preferably at least 21, 22, 23, 24, 25 or 26 amino acids in length.

[0270]

[0265] In the context of a bridging molecule for targeting CD33, preferably the dysfunctional P2X7 receptor epitope portion is no more than about 45 amino acids in length, at least 20 amino acids in length, preferably at least 21, 22, 23, 24, 25 or 26 amino acids in length.

[0271] EGFRvIII epitope portion In any embodiment, the amino acid sequence of the EGFRvIII epitope portion of any of the bridging molecules described herein comprises or consists of any of the sequences set forth in SEQ ID NO: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.

[0272] CLDN6 epitope portion In any embodiment, the amino acid sequence of the CLDN6 epitope portion of any of the bridge molecules described herein comprises or consists of the sequence shown 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 NOs: 273, 274 or 275.

[0273] Exemplary Cross-Linking Molecules

[0268] The present specification provides various non-limiting examples of tumor-specific antigen epitope moiety (eg, dysfunctional P2X7 receptor epitope moiety) / targeting moiety pairs.

[0274]

[0269] Exemplary cross-linking molecules of the invention are set forth in Table 1. For those cross-linking molecules set forth in Table 1 that include an nfP2X7 epitope portion, the present specification includes those cross-links but with the nfP2X7 epitope portion replaced with an EGFRvIII or CLDN6 epitope portion.

[0275]

[0270] In examples in which the bridging molecule comprises a targeting moiety for binding to CD19, the targeting moiety can comprise or consist of the heavy chain and paired light chain variable chain combination 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.

[0276] In the above embodiment, the bridging molecule may comprise a tumor-specific antigen epitope portion (e.g., dysfunctional P2X7 receptor epitope portion) conjugated to the heavy chain, or a tumor-specific antigen epitope portion (e.g., dysfunctional P2X7 receptor epitope portion) conjugated to the light chain.Preferably, the tumor-specific antigen epitope portion (e.g., dysfunctional P2X7 receptor epitope portion) is conjugated to the light chain of the target binding portion.

[0277] In any embodiment where the bridging molecule comprises a CD19-binding heavy / light chain pair, where the heavy chain comprises or consists of 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.

[0278] In any embodiment in which the bridging molecule comprises a CD19-binding heavy / light chain pair, where the light chain comprises or consists of a dysfunctional P2X7 receptor epitope portion, the sequences of the variable sequences of the heavy and light chain pair are preferably selected from the group consisting of SEQ ID NOs: 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 156; 143 and 157; 143 and 158; 143 and 159; or 143 and 164 (heavy and light chain sequences as recited, 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.

[0279]

[0274] The targeting moiety can be in the form of an scFv comprising a heavy and a light chain.

[0275] 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, 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.

[0280]

[0276] In a preferred embodiment, the bridging molecule for binding to CD19 comprises a targeting moiety comprising an antigen-binding domain for specifically binding to CD19, preferably as described herein (more preferably comprising the amino acid sequence set forth in SEQ ID NO: 52 / 143 or 143 / 144 (heavy and light chains, 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), and comprises a tumor-specific antigen epitope portion comprising or consisting of the amino acid sequence set forth in any of SEQ ID NOs: 365, 371, 377, 383, 389, and 395.

[0281] 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 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.

[0282]

[0278] 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.

[0283] In any embodiment, a bridging molecule for binding to CD79B can 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 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]

[0280] 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 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]

[0281] 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 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]

[0282] 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 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]

[0283] 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 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]

[0284] 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 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]

[0285] In a preferred embodiment, a bridging molecule for binding to CD33 comprises a targeting moiety comprising an antigen binding domain for specifically binding to CD33, preferably as described herein (more preferably comprising an amino acid sequence set forth in SEQ ID NOs: 73 and 72 or 74 (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), and comprises a tumor-specific antigen epitope portion comprising or consisting of an amino acid sequence set forth in any of SEQ ID NOs: 365, 373, 377, 388, 390, and 395.

[0290]

[0286] 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 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, a bridging molecule for binding to EGFR can comprise or consist of a sequence 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 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]

[0288] 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 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]

[0289] 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.

[0294] 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.

[0295]

[0291] 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 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]

[0292] 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 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]

[0293] 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 chain 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.

[0298]

[0294] 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 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]

[0295] 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.

[0300] 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 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.

[0301]

[0303] 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.

[0302]

[0313] In any embodiment, a bridging molecule for binding to PDGFR alpha 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.

[0303]

[0301] 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 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.

[0304]

[0300] In any embodiment, a bridging molecule for binding to FR alpha can comprise or consist of a sequence set forth in SEQ ID NOs: 111 and 112 (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.

[0305]

[0301] 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.

[0306]

[0302] 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.

[0307]

[0303] 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.

[0308]

[0304] 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.

[0309]

[0305] 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.

[0310]

[0306] 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.

[0311]

[0307] 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.

[0312]

[0308] 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 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.

[0313]

[0309] 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 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.

[0314]

[0310] 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 chain 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.

[0315]

[0311] 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 (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.

[0316] 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 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.

[0317]

[0313] 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 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.

[0318]

[0314] 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 chain 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.

[0319]

[0315] 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.

[0320] In any embodiment, a bridging 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.

[0321] 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.

[0322]

[0318] 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.

[0323]

[0319] In any embodiment, a bridging molecule for binding to IL13Ra2 can comprise or consist of a sequence set forth in SEQ ID NOs: 179 and 180 (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.

[0324]

[0320] In any embodiment, a bridging molecule for binding to IL13Ra2 can comprise or consist of a sequence 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.

[0325]

[0321] 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 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.

[0326] 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.

[0327]

[0323] 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 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.

[0328]

[0324] 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 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.

[0329]

[0325] 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 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.

[0330]

[0326] 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 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.

[0331]

[0327] In any embodiment, a bridging molecule for binding to CD171 LICAM can comprise or consist of a sequence set forth in SEQ ID NOs: 194 and 195 (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.

[0332]

[0328] 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 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.

[0333]

[0329] In any embodiment, a bridging molecule for binding to ALK can comprise or consist of a sequence set forth in SEQ ID NOs:198 and 199 (the light chain 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.

[0334]

[0330] 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.

[0335]

[0331] 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.

[0336]

[0332] 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.

[0337]

[0333] 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.

[0338]

[0334] 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 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.

[0339]

[0335] 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.

[0340]

[0336] 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 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.

[0341]

[0337] 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 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.

[0342]

[0338] 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.

[0343]

[0339] 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.

[0344]

[0340] 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 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.

[0345]

[0341] 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.

[0346]

[0342] 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 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.

[0347]

[0343] In any embodiment, a bridging molecule for binding to CEACAM5 can comprise or consist of a sequence set forth in SEQ ID NOs:226 and 227 (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.

[0348]

[0344] 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.

[0349]

[0345] 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.

[0350]

[0346] 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 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.

[0351] 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 recited 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.

[0352]

[0348] 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.

[0353]

[0349] 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.

[0354]

[0350] 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.

[0355]

[0351] 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.

[0356]

[0352] In any embodiment, a bridging molecule for binding to IL11Ra can comprise or consist of a sequence 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.

[0357]

[0353] 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 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.

[0358]

[0354] 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.

[0359]

[0355] 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 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.

[0360]

[0356] 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.

[0361]

[0357] 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.

[0362]

[0358] In any embodiment, a bridging molecule for binding to MAGE-A1 may comprise or consist of a sequence as 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.

[0363]

[0359] 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.

[0364]

[0360] 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.

[0365]

[0361] 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.

[0366]

[0362] 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 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.

[0367]

[0363] 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 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.

[0368]

[0364] 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.

[0369]

[0365] In any embodiment, a bridging 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 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.

[0370]

[0366] 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.

[0371]

[0367] In any embodiment, a bridging 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 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.

[0372]

[0368] 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.

[0373]

[0369] 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 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.

[0374]

[0370] 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 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.

[0375]

[0371] 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 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.

[0376]

[0372] 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 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.

[0377]

[0373] 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.

[0378]

[0374] 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 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.

[0379]

[0375] 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.

[0380]

[0376] 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 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.

[0381]

[0377] 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.

[0382] In any embodiment, a bridging molecule for binding to MUC16 can comprise or consist of a sequence set forth in SEQ ID NOs:299 and 300 (the recited 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.

[0383]

[0379] 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 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]

[0380] In any embodiment, a bridging molecule for binding survivin to HLA-A2 can comprise or consist of a sequence set forth in SEQ ID NOs: 303 and 304, 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.

[0385] 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.

[0386] 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.

[0387]

[0383] In any embodiment, a bridging molecule for binding to C200 can comprise or consist of a sequence set forth in SEQ ID NOs: 308 and 307 (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] In any aspect, the bridge molecules described herein do not have a HIS tag. Also contemplated are bridge molecules that include an amino acid sequence specified in the sequence information table above, but without the HIS tag specified 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 bridge molecules may include a tag other than a HIS tag, or may include an amino acid sequence specified in the sequence information table above, but with a different tag at the position of the HIS tag specified in the sequence.

[0389] nucleic acid In a second aspect, the present invention provides a nucleic acid molecule encoding a bridge molecule of the present invention, or a portion thereof.

[0390]

[0386] A nucleic acid molecule may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified or modified, RNA or DNA. For example, a nucleic acid molecule may comprise 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 containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, a nucleic acid molecule may comprise a triple-stranded region that contains RNA or DNA, or both RNA and DNA. A nucleic acid molecule may also contain one or more modified bases for stability, or a DNA or RNA backbone modified for other reasons. A variety of modifications can be made to DNA and RNA, and thus the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms.

[0391] In some embodiments of the second aspect of the present invention, the nucleic acid molecule comprises a nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NOs: 2 to 308. Preferably, the nucleic acid comprises a nucleotide sequence encoding the heavy and light chain pair described above.

[0392]

[0388] The present invention further provides a nucleic acid construct comprising a nucleic acid molecule encoding a bridge 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.

[0393]

[0389] 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.

[0394]

[0390] 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 those 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).

[0395]

[0391] 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).

[0396] 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 activating 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.

[0397] 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 purposes herein, a transcription control sequence is considered to be "operably connected" to a given nucleic acid molecule if the transcription control sequence is capable of promoting, inhibiting, or otherwise regulating 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.

[0398] 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.

[0399] 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 particular environmental or physiological condition. The present invention contemplates the use of any promoter that is active in the cells of interest. Thus, a wide range of promoters can be easily ascertained by one of ordinary skill in the art.

[0400]

[0396] 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 β-actin (CAGG).

[0401] Inducible promoters may 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).

[0402]

[0398] In the context of the present invention, it will be appreciated that 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.

[0403] In certain embodiments, and in the case of inducible expression constructs, immune cells expressing CAR can be genetically modified with a) a nucleic acid encoding an antigen-binding receptor, and b) an inducible expression construct encoding a bridging molecule.When dysfunctional P2X7 receptor binds, immune cells induce the expression of a gene encoding a bridging molecule.In certain embodiments, the expression of such a gene promotes and / or improves the treatment of cancer.

[0404]

[0400] As mentioned above, the control sequence may also include a terminator. The term "terminator" refers to a DNA sequence at the end of a transcription unit that signals the termination 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 the 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.

[0405]

[0401] 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).

[0406]

[0402] 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 parts thereof into the genome of a eukaryotic cell.

[0407] Methods for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the third aspect of the invention, into eukaryotic cells are known in the art. As will be appreciated, the most suitable method for introducing the nucleic acid construct into the desired host cell will depend 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 viability 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.

[0408] 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 for transduction of 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. Modified cells As described herein, the present invention includes the use of cells expressing chimeric antigen receptors that include an antigen recognition domain, which recognizes a tumor-specific antigen (such as a dysfunctional P2X7 receptor) expressed on the cell surface. The cells can be "engineered cells," "genetically modified cells," "immune cells," or "immune effector cells," as described herein. Furthermore, the cells can be differentiated into immune cells. The cells that can be differentiated into immune cells (e.g., T cells expressing dysfunctional P2X7CAR) can be stem cells, multilineage progenitor cells, or induced pluripotent stem cells.

[0409]

[0406] In any embodiment, the 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).

[0410]

[0407] 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).

[0411]

[0408] In some embodiments, the genetically modified cells contain two or more different CARs. 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.

[0412]

[0410] As referred to herein, a "genetically modified cell" includes any cell that contains a non-natural 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.

[0413]

[0411] 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. A "genomically integrated form" of a construct can be all or a portion of the construct. However, in some embodiments, a genomically integrated form of a construct includes at least a nucleic acid molecule of the second aspect of the invention.

[0414]

[0412] As used herein, the term "different CARs" or "different chimeric antigen receptors" refers to any two or more CARs that have either non-identical antigen recognition domains and / or non-identical signaling domains. In one example, "different CARs" includes two CARs that have the same antigen recognition domain (e.g., both CARs can recognize a 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 a dysfunctional P2X7 receptor, and the other CAR(s) can take any suitable form and be directed against any suitable antigen.

[0415]

[0413] Thus, in some embodiments of the invention, two or more different CARs have different signaling domains and may have the same or different antigen recognition domains. Specifically, the genetically modified cells of the invention may 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.

[0416]

[0414] 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.

[0415] 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.

[0417]

[0416] 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.

[0417] In some embodiments, the genetically modified cells are further modified to constitutively express a costimulatory receptor.

[0418] As mentioned above, a cellular immune response is typically induced only when an activating signal (typically in response to an antigen) and a costimulatory signal are experienced simultaneously. Thus, having genetically modified cells according to some of the above embodiments, including two or more CARs that provide both an intracellular activating signal and an intracellular costimulatory signal in combination, ensures that a sufficient immune response can be induced in response to recognition by the CAR(s) of their cognate antigen. Alternatively, the genetically modified cells 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 likelihood that costimulation is provided at the same time that the CAR is activated. Alternatively, the genetically modified cells can be further modified to constitutively express both the costimulatory receptor(s) and / or their ligand(s). In this way, the cells experience continuous costimulation and only require activation of the CAR, along with a signaling domain that includes a portion from the activating receptor, for immune activation of the cell.

[0419]

[0419] Thus, in some embodiments, the genetically modified cells expressing the CAR are further modified to constitutively express a costimulatory receptor. In further embodiments, the genetically modified cells are further modified to express a ligand of the costimulatory receptor, thereby promoting self-stimulation of the 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, including, inter alia, those disclosed in Stephen MT. et al. Nat Med, 2007; 13: 1440-9.

[0420]

[0420] The capacity of the genetically modified cells containing a CAR can be enhanced by further modifying the cells to secrete a cytokine, preferably a proinflammatory or growth-promoting cytokine. This cytokine secretion provides autocrine support for the cells expressing the CAR as well as alters the local environment surrounding the cells expressing the 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 invention, the genetically modified cells are further modified to secrete a cytokine. This secretion can be constitutive or inducible by the CAR's recognition of its cognate antigen of the ligand.

[0421]

[0421] 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.

[0422] The immune cells of the present invention can be any suitable immune cell, or precursor thereof, or can 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.

[0423]

[0423] 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).

[0424]

[0424] 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).

[0425] Methods of Treatment and Administration

[0425] 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.

[0426]

[0426] The present invention also contemplates various scenarios for the use of the two components of the therapeutic agents described herein.

[0427] In one scenario, an individual in need of treatment is administered a single composition containing both the CAR T cells and the bridging molecule.

[0427]

[0428] In a further scenario, an individual in need of treatment is administered a population of CAR T cells that includes an expression vector encoding a bridging molecule. The expression vector can facilitate constitutive or inducible expression of the nucleic acid sequence encoding the bridging molecule.

[0428]

[0429] Furthermore, an individual in need of treatment can be administered CAR T cells and later administered a composition containing a bridging molecule (e.g., via infusion), or a nucleic acid sequence encoding 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 a cancer that is positive for a dysfunctional P2X7 receptor, and then administration of a bridging molecule is intended to redirect the CAR to another cancer antigen, or a peptide derived from an infectious agent and presented on the MHC I or II molecules of the cell.

[0429]

[0430] Thus, the bridging molecule can be administered before, simultaneously with, or after the subject undergoes treatment with the CAR T cells.

[0431] When the bridging molecule and the CAR T cell are administered to the 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 cell can be administered by injection into the subject's bloodstream, while the bridging molecule can be administered via another route of administration, such as intramuscular, intradermal, subcutaneous or intraperitoneal.

[0430]

[0432] The cross-linking molecules can be produced or expressed in vivo by genetically engineered cells that spontaneously secrete the cross-linking molecules or upon stimulation via a stimulant, e.g., a small molecule. Alternatively, the cells can continuously secrete the cross-linking molecules and cease their secretion upon application of a stimulant, e.g., a small molecule.

[0431]

[0433] 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.

[0432]

[0434] 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.

[0433]

[0435] 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.

[0434]

[0436] 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.

[0435]

[0437] 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.

[0438] The method is particularly useful for extending the time to disease progression.

[0436]

[0439] The methods are particularly useful for extending human survival, including overall survival and progression-free survival.

[0440] 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.

[0437]

[0441] 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.

[0438]

[0442] 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.

[0439]

[0443] In one embodiment, the subject in need of treatment includes a subject with a benign, pre-cancerous, non-metastatic tumor.

[0444] In one embodiment, the cancer is precancerous or preneoplastic.

[0440]

[0445] 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.

[0441]

[0446] In one embodiment, the cancer may be substantially undetectable.

[0447] "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.

[0442]

[0448] 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).

[0443]

[0449] 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).

[0444]

[0450] 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.

[0445]

[0451] 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.

[0446]

[0452] 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.

[0447]

[0453] 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.

[0454] 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.

[0448]

[0455] 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.

[0449]

[0456] Examples of intracellular infections by fungal pathogens: Histoplasma capsulatum, Cryptococcus neoformans, and Pneumocystitis jirovecii.

[0450]

[0457] Examples of obligate intracellular protozoan pathogens include Apicomplexans (Plasmodium spp., Toxoplasma gondii, and Cryptosporidium parvum), and Trypanosomatids (Leishmania spp. and Trypanosoma cruzi).

[0451]

[0458] 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.

[0452]

[0459] 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.

[0453]

[0460] 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.

[0454]

[0461] The crosslinking molecule of the present invention can be formulated for administration to a subject using techniques known to those skilled in the art.The crosslinking molecule formulation can include pharma-ceutically acceptable excipient(s) (carrier or diluent).The commonly used excipient examples 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.

[0455]

[0462] A formulation of bridging molecules may contain one type of bridging molecule, or more than one type of bridging molecule (i.e., the bridging molecules may have the same or different targeted and / or dysfunctional P2X7 receptor epitope portions).

[0456]

[0463] 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).

[0457]

[0464] The formulation containing the bridging molecule is administered to the 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 bridging molecule can be administered to the subject in need of treatment. In most cases, taking into account the route of administration, symptoms, etc., the dosage can be about 100 μg / kg to about 10 mg / kg body weight / day of the bridging molecule. However, the amount of the bridging molecule in the formulation administered to the 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. The physician can ultimately determine the appropriate dosage to be used. The bridging molecule can be administered as a continuous infusion or a bolus dose.

[0458]

[0465] The timing between administration of the CAR T cells and the bridge 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 TCBM preparation can be administered before, simultaneously with, or after the genetically engineered (immune) cell preparation.

[0459]

[0466] 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 evident from the text or drawings, all of these different combinations constituting various alternative embodiments of the invention. EXAMPLES

[0460] Example 1 - Materials and methods including generation of cross-linking molecules

[0467] 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 growth phase were transfected when the DNA concentration was in the range of 1000×g / 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, 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×g for 5 min at 20°C. Cells were discarded and the supernatant was centrifuged again at 4000×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.

[0461]

[0468] 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.

[0469] 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.

[0462]

[0470] 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.

[0463]

[0471] The detailed experimental data generated by the inventors and described herein includes the generation of a wide range of cross-linking molecules including: 1. Targeting moieties generated 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 targeting moieties, including: 3. Inclusion of a linker between the targeting moiety and the dysfunctional P2X7 receptor epitope moiety; 4. Targeting moieties that bind to a broad range of cell surface antigens present on tumor cells of different tissue origins.

[0464] Antibody / Fab conjugation

[0472] Conjugation of BIL03s 2-2-1-Fc (anti-nfP2X7 receptor antibody) with the fluorescent dye Alexa Fluor® 647 (AF647) was performed according to the manufacturer's instructions (Cat# A20186, ThermoFisher). AF647-labeled BIL03s 2-2-1-Fc antibody was reconstituted in PBS pH 7.2 with 2 mM sodium azide.

[0465] Flow cytometry to test binding of bridging molecules to cells

[0473] reagent Antibodies and Fabs: ·BIL03 pure antibody: pre-dilute to 100ug / mL with PBS · BIL03-AF647 antibody: Pre-dilute to 100ug / mL with PBS ·Anti-His antibody-FITC (1mg / mL) (Abcam Cat# ab1206, Cat# GR3361939-1) Rabbit IgG-FITC isotype control (Abcam Cat# ab3706, Cat# GR3356160-1) For cross-linking molecules produced in-house and recovered from the supernatant, see below.

[0466]

[0474] Cell lines used for binding assays: JeKo-1 (CD19, CD20, CD79B, CD37, CD22, ROR1, Her2), MOLM-13 (CD33, CD38, CD37, CD135, CD123), PC3 (Her2), MDA-MB-231 (EGFR, PD-L1), Raji (CD22, CD70, CD79B), Karpas299 (CD30), U937 (CD105), HL60, RPMI8226 (BCMA, CD38, CD33).

[0467]

[0475] 5 x 10 cells 6The cells were resuspended at a density of cells / ml and 100 μL aliquots were used per well for staining (0.5 × 10 6 / sample).

[0468] [Table 3]

[0469] procedure

[0476] 1. Staining conditions list: Staining of bridging molecules bound to BIL03s antibody and detection of His-tag antibody

[0477] Control condition: Staining of control bridging molecules that bind to BIL03s antibodies and His-tag antibody detection Staining of bridging molecules that do not bind to BIL03s antibodies and detection of His-tag antibodies ·Unstained cells

[0478] 2. Staining Procedure · Block cells with human FcR blocker (20% v / v, Miltenyi) for 10 min on ice and wash off unbound FcR blocker.

[0470] · Incubate cells in 50 μL of crude prep supernatant and stain for 15 min on ice. Wash the cell suspension with FACS buffer x 2 Stain cells with 1 μg / mL BIL03s-AF647 and 1 μL His Tag antibody per 100 μL of cell suspension.

[0471] Wash cells with FACS buffer x2. Cells are prepared for analysis in MacsQuant16. Production of lentiviral vectors using adherent lenti-X HEK293T (Takara) cells and PEI Lenti-X293T medium

[0479] For cell culture prior to transduction:

[0480] 90% Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5 g / L), 4 mM L-glutamine, and sodium bicarbonate (Sigma-Aldrich, D5796); 10% fetal bovine serum (FBS); 1 mM sodium pyruvate (Sigma-Aldrich, S8636).

[0472]

[0481] For cell culture after transduction:

[0482] 90% Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5 g / L), 4 mM L-glutamine, and sodium bicarbonate (Sigma-Aldrich, D5796); 10% fetal bovine serum (FBS); 1 mM sodium pyruvate (Sigma-Aldrich, S8636); and 10 mM sodium butyrate.

[0473] [Table 4]

[0474]

[0483] Protocol, Part I Lenti-X 293T Cell Lentiviral Transfection

[0484] Day 0: 1.7 x 10 per 1.15 cm dish 6 Seed the cells so that they are approximately 80% confluent (approximately 16 × 10 6 cell).

[0475]

[0485] Day 1: 1. Check the cells under a microscope. The cells should be about 75-90% confluent.

[0476] 2. Gently aspirate the medium and add fresh 20 mL of DMEM supplemented with 10% FCS to each 15 cm dish and incubate for at least 2 h prior to transfection.

[0477] 3. Perform the transfection procedure in the afternoon (approximately 2.30-4.30 pm). Warm an aliquot of serum-free DMEM to 37 °C. 4. Prepare mixture A (plasmid DNA solution) and mixture B (PEIpro solution).

[0478] 5. Determine the required volumes of DMEM and plasmid DNA in Mixture A according to the table below.

[0479] [Table 5]

[0480] 6. Determine the volume needed for each plasmid DNA component for the number of plates needed. The area of ​​a 15 cm dish is approximately 175 cm. 2 It is.

[0481] [Table 6]

[0482] 7. Determine the volume needed for each component of Mixture B (PEIpro solution). For each 15cm dish,

[0483] [Table 7]

[0484] 8. Vortex the PEIpro for 5 seconds and, if necessary, spin down to collect the liquid at the bottom of the tube. 9. Prepare mixture B (PEIpro in medium) in a 15 mL tube by adding PEIpro to DMEM high glucose without additives. Add PEI to DMEM, invert several times, and spin down quickly.

[0485] 10. Prepare Mixture A (plasmid DNA dilution in medium) in a 50 mL conical tube by adding DNA to medium. Mix gently by inversion and centrifuge quickly.

[0486] 11. Mix and prepare the transfection mixture (PEIpro / DNA solution) by adding Mixture B (PEIpro solution) to Mixture A (plasmid DNA dilution). Using a p1000 micropipette, add the PEIpro solution dropwise to the DNA solution and immediately mix by inverting 3-4 times. Do not vortex.

[0487] 12. Incubate at room temperature for 15 minutes, but not longer than 30 minutes. Do not agitate the tube during this time. 13. After the 15 minute incubation, add the transfection mix to the flask / dish containing the cells and fresh medium (drop-wise if possible). Gently shake the flask / dish in a back-and-forth motion.

[0488] 14. Incubate the flask / dish overnight at 37°C and 5% CO2.

[0486] Day 2: 15. 16-18 h after transfection, change the medium. Working with two plates at a time, aspirate the old medium. Using a 25 mL pipette, carefully add 15 mL of fresh DMEM supplemented with 10% FCS and 10 mM sodium butyrate.

[0489]

[0487] Day 3, first harvest 24 hours after medium change: 16. Collect the supernatant from the culture flasks / dishes into 50 mL falcon tubes. Carefully replace each plate with 15 mL of fresh DMEM supplemented with 10% FCS and 10 mM sodium butyrate and return the plates to the incubator.

[0490] 17. Centrifuge the supernatant at 500 x g for 10 minutes at 4°C (for concentration with Lentic-X concentrators). For ultracentrifugation, centrifuge at 3800 rpm for 30 minutes at room temperature. 18. Aspirate the virus-containing supernatant with a 20 mL syringe and filter through a 0.45 μm PES filter (Millipore) into a new 50 mL Falcon tube. This can be (a) used as a crude preparation for transduction or (b) proceeded to concentration by ultracentrifugation or (c) concentration in a Lenti-X concentrator. Alternatively, store crude virus 4°C overnight and pool at 48-h harvests. Store concentrated virus in aliquots at -80°C for long term.

[0491] 19. Dispose of plates, tubes and filters in a biohazard bag in the tissue culture hood. Seal the biohazard bag before removing it from the hood and discarding it.

[0492]

[0488] On day 4, harvest for the second time 48 hours after medium change: 20. Collect the supernatant from the culture flask / dish into a 50 mL Falcon tube. 21. Centrifuge the supernatant at 2000 x g for 30 minutes at room temperature.

[0493] 22. Aspirate the virus-containing supernatant into a 20 mL syringe and filter through a 0.45 μm filter (Millipore) into a new 50 mL Falcon tube. This can be pooled with the 24 hour harvest or processed separately and proceeded to (b) concentration by ultracentrifugation, or (c) concentration by Lenti-X concentrator.

[0494] 23. Dispose of plates, tubes and filters in a biohazard bag in the tissue culture hood. Seal the biohazard bag before removing it from the hood and discarding it.

[0495]

[0489] Part II Concentration of lentivirus using the Lenti-X concentrator 24. Harvest the supernatant containing the lentivirus. Note: The supernatant contains live lentivirus. Pool similar stocks together if desired. Centrifuge briefly at 500 x g for 10 minutes or filter through a 0.45 μm filter.

[0496] 25. Transfer the clear supernatant to a sterile container and combine 1 volume of Lenti-X concentrator with 3 volumes of clear supernatant. Mix by gently inverting. Larger volumes can be accommodated by using larger (i.e., 250 mL or 500 mL) centrifuge tubes.

[0497] 26. Perform one incubation with Lenti-X concentrators. Either at harvest (after 1 day) or after 2 days (pool harvest). Incubate for at least 30 minutes or overnight, then centrifuge the pre-incubated fluid.

[0498] 27. Centrifuge the samples at 1,500 x g for 45 minutes at 4° C. After centrifugation, an off-white pellet is visible. 28. Carefully remove the supernatant, taking care not to disturb the pellet. Remaining supernatant can be removed either with a pipette tip or by brief centrifugation at 1,500 x g.

[0499] 29. Gently resuspend the pellet in 1 / 10-1 / 100 of the original volume with complete DMEM, PBS, or TNE. The pellet will quickly become suspended, even if it is somewhat sticky at first.

[0500] 30. Titrate samples immediately or store in single-use aliquots at -80°C. CAR T Cell Generation Protocol

[0490] nfP2PX7BRIDGE CAR T cells were generated by lentiviral transduction (1:1 ratio) of CD4 / CD8 positively selected T cells via magnetic activated cell sorting (MACS) stimulated with TransAct (all according to manufacturer's instructions) cultured in TecsMACS medium supplemented with IL7 / IL15 (both at 10 ng / mL). The donor source was buffy coat.

[0501] CAR T cells were treated in exactly the same way, but were lentivirally transduced to express the nfP2X7BRIDGE CAR. Activated non-transduced T cells (aUT) do not express any receptors that can engage with the EGFR or CD33 bridge molecule.

[0502]

[0492] Hypothesis:

[0493] nfP2X7BRIDGE CAR T cells have superior effector function than aUT because they are directly redirected to cancer cells via nfP2X7 recognition on the cell surface of MOLM-13 leukemia cells.

[0503]

[0494] nfP2X7BRIDGE CAR T cells have superior effector function than aUT because they are indirectly redirected to cancer cells via nfP2X7E200-derived epitopes on CD33 Fab cross-linking molecules on the surface of MOLM-13 leukemia cells.

[0504] Preparation of reagents and equipment CAR T medium: TexMACS with human IL-7 and IL-15. IL-7 stock concentration was 100ug / mL, each vial was 55μL. IL-15 stock concentration was 50μg / mL, each vial was 55μL.

[0505] To prepare TexMACS with a final concentration of 10 ng / mL IL-7, 5 ng / mL IL-15 and 3% FBS, add 50 ul of IL-7, 50 ul of IL-15 stock and 15 mL of FBS to each bottle (500 mL) of TexMACS medium. Mark the medium bottles with the date the cytokines were added.

[0506]

[0497] Preparation of freezing medium on the day of harvest: 10% DMSO, 90% FBS. Note: Add the following reagents in the order DMSO to FBS to a 50 mL falcon tube. Part I: T cell activation and T cell transduction

[0498] Day 1: T cell activation 1. Isolate CD4+ and CD8+ CAR T cells from whole blood or buffy coat. See protocols for PBMC isolation and CD4 and CD8 cell isolation.

[0507] 2. Wash the CD4+ and CD8+ cells twice with pre-warmed TexMACS medium (without cytokine supplements) by filling the Falcon tube to its maximum volume and centrifuging at 300 x g for 5 minutes. Aspirate the supernatant completely.

[0508] 3. Resuspend CD4+ and CD8+ cells in pre-warmed TexMACS medium supplemented with 10 ng / mL IL-7, 5 ng / mL IL-15 and 3% FBS to a final concentration of 10^6 cells / mL.

[0509] 4. CD4+ and CD8+ cells are plated at a 1:1 ratio by adding 0.5ml of CD4+ cells and 0.5ml of CD8+ cells to each well in a 24-well plate.

[0510] 5. Add 10 μL of T cell TransAct to a final dilution of 1:100 in cell culture and resuspend carefully. 6. Incubate at 37°C with 5% CO2 for approximately 36 hours before transduction.

[0511]

[0499] Day 3: T cell transduction 7. If possible, use fresh viral vectors for transduction. If not, thaw frozen viral vectors slowly on ice.

[0512] 8. Slowly remove 800 μL of media from the side of the well with a P1000 pipette, being careful not to disrupt the underlying cell layer. 9. Drop 150 µL (viral vector for 1 plate) or 300 µL (viral vector for 2 plates) onto the T cells. Top up the wells to 600 µL with fresh supplemented TexMACS medium and add polybrene to each well at a final concentration of 4 µg / mL. From this point onwards, the T cells should only be placed in the incubator for lentiviral action.

[0513] 10. After transduction, appropriate clean-up procedures should be performed. Decontaminate the biosafety cabinet and aspirator lines by cleaning surfaces and running the lines with 2% Virkon solution in 70% ethanol. Dispose of contaminated waste such as tips and serological pipettes in a biohazard bag inside the biosafety cabinet and seal the bag before taking the waste outside for disposal.

[0514]

[0500] Day 4 and after - T cell maintenance 11. Transduced T cells were maintained in TexMACS medium containing IL-7, IL-15 and 3% FBS.

[0515] 12. Monitor T cell proliferation in 24-well plates and transfer cells to T75 flasks 2 days after transduction. 13. Monitor lactate levels in the medium daily using a CCS analyzer. If lactate levels exceed 10 mmol / L, replenish with fresh medium; ideally, lactate levels should be kept below 4 mmol / L.

[0516]

[0501] Day 9 - Day 5 after T cell transduction - Expression analysis

[0502] Transduced T cells are counted using a flow cytometer on day 5. Samples are taken for flow cytometry analysis to determine expression efficiency based on standard flow cytometry protocols.

[0517] Generation of luciferase-expressing cancer cell lines

[0503] Firefly luciferase lentiviral transfer plasmid used to generate viral vectors: pRRLsin18.cPPT.EF1a_firefly_luciferase_T2A_EGFP.WPRE pRRLsin18.cPPT.hPGK_firefly_luciferase_T2A_EGFP.WPRE 1. Plate cells at a density of 300,000 cells / well in 24-well plates in a total volume of 850 μL.

[0518] 2. Generate viral vectors containing the firefly luciferase gene using a four-plasmid transfection protocol as described in the lentivirus generation section. If possible, use fresh concentrated viral vectors for transduction. Otherwise, thaw frozen viral vectors slowly on ice.

[0519] 3. Drop 150 μL (one plate's worth of viral vector) onto the cells. From this point onwards the cells should only be in the incubator for the lentivirus to act. 4. Incubate cells at 37°C with 5% CO2.

[0520] 5. After transduction, appropriate clean-up procedures should be performed. Decontaminate the biosafety cabinet and aspirator lines by cleaning surfaces and running the lines with 2% Virkon solution in 70% ethanol. Dispose of contaminated waste such as tips and serological pipettes in a biohazard bag inside the biosafety cabinet and seal the bag before taking the waste outside for disposal.

[0521] 6. Two days after transduction, transfer the cells into a T25 flask. 7. Five days after transduction, cells are counted and samples are taken for expression analysis by flow cytometry.

[0522] 8. Seven days after transduction, the transduced cells are bulk sorted based on eGFP expression using a live cell sorter. 9. After expansion, the bulk selected cells are further selected for single cell clones, which are propagated, expanded and frozen to produce stocks.

[0523] Functional assays Target cells constitutively expressing firefly luciferase and eGFP (selected and grown as single cell clones defined as high-performing cell lines) were used to measure viability via bioluminescence and / or fluorescence in functional assays. The amount of luminescence correlates with the total number of cells in bioluminescence, and fluorescent target cells identified via flow cytometry correlate with the total number of viable cells.

[0524] Effector cells and target cells were seeded according to the indicated effector to target ratio (ET). The indicated ET ratio, e.g. 10:1, always refers to the total number of T cells and the total number of target cells. Since the CAR-expressing fraction is different from the total number of T cells, the ET ratio referred to the cells expressing CAR is shown separately. Target cells were seeded at 25,000 or 50,000 cells per 96-well plate.

[0525] Luciferase killing assay Effector and target cells were seeded according to the indicated effector / target ratio (ET). BRIDGE molecules were added in the indicated formats (Fab, IgG1) and at the indicated concentrations. D-Luciferin was added and bioluminescence was measured under standard conditions in an incubator at 37°C and 5% CO2 on a SpectraMaxi3 at the indicated time points after incubation began.

[0526]

[0507] Cell viability was calculated according to the bioluminescence activity curves from serial dilutions of cells (100%, 75%, 50%, 25%, 10% and 0% target cells) and expressed as the percentage of viable cells. In general, lysis was calculated by (bioluminescence of test condition - 0% bioluminescence) / (100% bioluminescence - 0% bioluminescence).

[0527] Flow-based killing assay Effector and target cells were seeded according to the indicated effector-to-target ratio (ET) and BRIDGE molecules were added in the indicated format (Fab, IgG1) at the indicated concentrations. Cell numbers were measured at the indicated time points (24 or 48 hours) after starting the incubation under standard conditions in an incubator at 37°C and 5% CO2 on a MACSQuant16 flow cytometer according to standard protocols. Staining of cells included a viability dye to exclude all dead cells from the analysis. T cells were clearly differentiated from eGFP-positive cancer cells via CD3. Furthermore, T cells were characterized after 24 or 48 hours by CD25 and CD69 as indicators of specific T cell activation according to standard protocols. Final data analysis was performed by FlowJo10.

[0528]

[0509] Antibody Cocktail

[0529] [Table 8]

[0530] Flow-based acquisition of cytokine secretion Effector and target cells were seeded according to the indicated effector to target ratio (ET) and BRIDGE molecules were added in the indicated formats (Fab, IgG1) at the indicated concentrations. Supernatants were harvested after 24 or 48 hours and incubation was started at the indicated time points and measured under standard conditions in a 37°C and 5% CO2 incubator on a MACSQuant16 flow cytometer following standard protocols using Miltenyi cytokine beads. Final data analysis was performed by FlowJo10.

[0531] Example 2 - Characterization of various cross-linking molecules The results in Figures 4-7 show that bridging molecules containing a targeting moiety in the form of a Fab or scFv (FMC63 clone; amino acid sequence described herein) capable of binding to CD19 can bind to CD19 on the surface of live cells and present a dysfunctional P2X7 receptor epitope moiety (e.g., E200 moiety) that is therefore accessible by anti-P2X7 receptor antibody (BIL03s 2-2-1-Fc). The location of the dysfunctional P2X7 receptor epitope moiety can vary, the targeting moiety can still bind to its target cell surface antigen, and the dysfunctional P2X7 receptor epitope moiety is still available for binding to the antibody. Note that BIL03s 2-2-1-Fc-AF647 / HIS-FITC did not bind to control bridging molecules that did not contain a dysfunctional P2X7 receptor epitope moiety, and anti-HIS antibody did not bind to control bridging molecules that contained a HIS tag (data not shown).

[0532] V H Top Fab format + / - linker-epitope

[0512] FIG. 4 shows V HBridging molecules in Fab format with a single E200 epitope linked directly or via a linker to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, demonstrating that the E200 epitope is available for antibody binding.

[0533] V H Top scFV format + / - linker-epitope

[0513] FIG. 5 shows V H Bridging molecules in scFv format with a single E200 epitope linked directly or via a linker to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, demonstrating that the E200 epitope is available for binding to antibodies.

[0534] V L Top Fab format + / - linker-epitope

[0514] FIG. 6 shows V L Bridging molecules in Fab format with a single E200 epitope linked directly or via a linker to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, demonstrating that the E200 epitope is available for antibody binding.

[0535] V L Top scF V Format + / - linker-epitope

[0515] FIG. 7 shows V L Bridging molecules in scFv format with a single E200 epitope linked directly or via a linker to CD19 on the JeKo-1 (mantle cell lymphoma) cell line, demonstrating that the E200 epitope is available for binding to antibodies.

[0536] Cross-linking molecules targeting various antigens Figure 8: Binding of bridge 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.

[0537] CD37 targeting moiety derived from otlertuzumab, CD79B targeting moiety derived from polatuzumab, ROR1 targeting moiety derived from ROR1 APC (WO20160344A1_D10v3), CD33 targeting moiety derived from lintuzumab, CD38 targeting moiety derived from daratumumab, CD123 targeting moiety derived from clone 32716, CD135 targeting moiety derived from 4G8, clone CA8 BCMA targeting moiety from J9M0, EGFR targeting moiety from necitumumab or matuzumab, PDL1 targeting moiety from atezolizumab, CD22 targeting moiety from m97I-L7 (or inotuzumab (data not shown)), CD70 targeting moiety from cusatuzumab, CD19 targeting moiety from tafasitamab, CD20 targeting moiety from ofatumumab (or ocrelizumab (data not shown)). Exemplary amino acid sequences of bridging molecules targeting these antigens and derived from the immediately mentioned antibodies are set forth in the Sequence Information Table herein.

[0538]

[0518] Figure 8 shows binding of various cross-linking molecules to JeKo-1 (MCL) wild-type cell line (CD37, CD79B, ROR1), Raji (Burkitt's lymphoma) wild-type cell line (CD22, CD70, CD19, CD20, CD22), MOLM-13 (AML) wild-type cell line (CD33, CD38, CD123 and CD135), RPMI8226 (multiple myeloma) wild-type cell line (CD33, BCMA and CD38), MDA-MB231 (breast cancer) wild-type cell line (EGFR and PDL1) and PC-3 (prostate cancer) wild-type cell line (EGFR).

[0539] Example 3 - Dose-dependent increase in cell binding by CD19-targeted bridging molecules JeKo-1 (MCL) CRL-3006™ wild type cell line purchased from ATCC as part of the NCI60 panel. Cells were cultured according to general recommendations and standards for this particular cell line.

[0540]

[0520] Figure 9 shows in a diagrammatic format the "painting" of JeKo-1 with a CD19-targeting Fab cross-linking molecule. Cells were incubated with Fab cross-linking molecules at the indicated concentrations. CD33-targeting Fab cross-linking molecules served as negative controls in JeKo-1 at 10 ng / mL and 1000 ng / mL. CD19-targeting Fab cross-linking molecules were used at 1 ng / mL, 10 ng / mL, 100 ng / mL and 1000 ng / mL.

[0541] Flow cytometry staining was performed in two steps according to the standard in flow cytometry staining using Fc block reagent (Miltenyi). First, target cells were incubated with Fab bridge molecules at the indicated concentrations for 15 minutes, washed three times, and then secondary antibodies anti-HIS FITC and single domain antibody BIL03 2-2-1 AF647 at saturating concentrations (1ug / mL) were used to indirectly stain target cells via 6xHIS and nfP2X7E200 derived epitopes on the bound Fab bridge molecules. After 15 minutes of incubation, samples were washed and analyzed on a MACSQuant16 (Miltenyi). Flow data was analyzed via FlowJo v10.7 (BD).

[0542] There is no expression of CD33 in JeKo-1 cells. CD19 staining showed increased expression with increasing concentrations of CD19-targeted cross-linking molecules. Example 4 - Dose-dependent increase in cell binding by CD33-targeted bridging molecules

[0524] MOLM-13 (AML) wild type cell line purchased from ATCC as part of the NCI60 panel. Cells were cultured according to general recommendations and standards for this particular cell line.

[0543] FIG. 10 “Painting” of MOLM-13 with CD33-targeting Fab cross-linking molecules in the illustrated format. Cells were incubated with Fab cross-linking molecules at the indicated concentrations. CD19-targeting Fab cross-linking molecules served as negative controls in MOLM-13 at 10 ng / mL and 1000 ng / mL, while CD33-targeting Fab cross-linking molecules were used at 1 ng / mL, 10 ng / mL, 100 ng / mL and 1000 ng / mL.

[0544] Flow cytometry staining was performed in two steps according to the standard in flow cytometry staining using Fc block reagent (Miltenyi). First, target cells were incubated with Fab bridge molecules at the indicated concentrations for 15 minutes, washed three times, and then secondary antibodies anti-HIS FITC and single domain antibody BIL03 2-2-1 AF647 at saturating concentrations (1ug / mL) were used to indirectly stain target cells via 6xHIS and nfP2X7 E200 derived epitopes on the bound Fab bridge molecules. After 15 minutes of incubation, samples were washed and then analyzed on a MACSQuand16 (Miltenyi). Flow data was analyzed via FlowJo v10.7 (BD).

[0545] There is no expression of CD19 in MOLM-13 cells. CD33 staining showed increased expression with increasing concentrations of CD33-targeted cross-linking molecules. Example 5 - Detection of marker genes via nfP2X7E200-derived peptides and bridging molecules and direct CAR detection

[0529] nfP2X7 targeting crosslinked CAR effector cells (such as, but not limited to, T cells or NK cells) specifically recognize cancer cells. The epitope targeted in nfP2X7 arises from the conformational change of P2X7 protein trimer, and is only exposed on cancer cells, not on healthy cells.

[0546]

[0530] The basic principle and engagement of effector cells expressing nfPX7CAR via nfP2X7E200-derived peptide-tagged bridging molecules and different formats of bridging molecules are illustrated and outlined in Figures 1-3.

[0547]

[0531] Thus, when nfP2X7CAR is used in the absence of a bridging molecule, effector cells expressing nfP2X7CAR exhibit cancer-specific targeting. To broaden the applicability to cancers that are functionally negative for nfP2X7 (very low or possibly negative for nfP2X7), effector cells expressing nfP2X7CAR can be redirected to cancer cells via bridging molecules that target cancer-associated antigens depicted in CD33 or cancer-specific antigens via TcR-like mAbs. The specificity of the bridging molecule is unlimited, meaning any surface target antigen or presented antigen expressed in the context of MHC peptide presentation (class I and II) via TcR-like mAbs or ligands, and can engage effector cells expressing nfP2X7-bridged CAR with the same mode of action.

[0548] In most cases, the dual function of effector cells expressing nfP2X7-crosslinked CARs is utilized. This is a combination of scenarios I and II, meaning that effector cells expressing nfP2X7-crosslinked CARs are directly engaged to cancer cells via nfP2X7 expressed on the cancer cells, and are further recruited to the cancer cells via bridging molecules targeting cancer-associated antigens as illustrated for CD33 or cancer-specific antigens via TcR-like mAbs.

[0549] Example 6 - "Painting" the MOLM-13 (AML) wild-type cell line with CD33-targeted Fab cross-linking molecules

[0534] Figure 11 shows the "painting" of MOLM-13 (AML) cells via CD33-targeting Fab cross-linking molecules. Flow data shows isotype control (black), staining with BIL03s 2-2-1-Fc sd-mAb only at 1ug / mL (blue), and increased staining with the combination of CD33-targeting Fab cross-linking molecules and BIL03s 2-2-1-Fc (green). The increase in target molecules that can be recognized by effector cells expressing nfP2X7 cross-linked CAR leads to CAR-mediated effector function. Thus, cross-linking technology enhances CAR function by increasing the targeting epitopes on cancer cells.

[0550] Example 7 - Titration of CD33-targeted cross-linking molecules derived from Lintuzumab MOLM-13 cells, which constitutively express luciferase, were co-incubated with increasing concentrations of CD33-targeted Fab cross-linking molecules at the indicated concentrations. However, Fab cross-linking molecules bound to CD33 on MOLM-13 cells do not show direct toxicity due to complement-dependent cytotoxicity (CDC) or recruitment of effector cells (ADCC). No cells other than MOLM-13 were used in the assay. CD33-targeted Fab cross-linking molecules do not have functional sites for CDC or ADCC like full-size antibodies.

[0551]

[0536] There was no significant effect on viability after 4 hours, and no consistent or dose-dependent toxicity was observed after 24 hours of incubation. Example 8 - Activation of nfP2X7CAR-T cells with CD33-targeted Fab bridging molecules

[0537] Figure 12 shows representative flow cytometry plots with a direct comparison of non-transduced T cells (left panel), CAR0007_hPGK (middle panel; CAR0007 is also referred to herein as CAR7) and CAR0007_EF1a (right panel; CAR0007 is also referred to herein as CAR7) expressing T cells. Both CAR T cells increased expression of activation markers CD25 and CD69 when incubated with MOLM-13 at an ET ratio of 20:1 and produced CD33-targeting Fab cross-linking molecules at 1000ng / mL after 48 hours.

[0552] The CD33 Fab cross-linking molecule used in the experiments of Examples 8 and 9 is derived from lintuzumab, and the EGFR-targeting cross-linking molecule is derived from necitumumab. The dysfunctional P2X7 receptor epitope is a V L was directly linked to

[0553] CAR0007_hPGK CAR has the following domain structure from N-terminus to C-terminus: hPGK-CD8a SP-V H BIL03 2-2-1-CD28-CD28T-CD28-CD137-CD3zeta-T2A-tEGFR.

[0554] CAR0007_EF1a CAR has the following domain structure from N-terminus to C-terminus: EF1a-CD8a SP-V H BIL03 2-2-1-CD28-CD28T-CD28-CD137-CD3zeta-T2A-tEGFR.

[0555] Example 9 - Clearance of leukemia cells by nfP2X7 CAR-T cells in the presence of CD33 bridging molecules

[0541] Flow cytometry plots of T cells and MOLM-13 at a 20:1 ET ratio after 48 hours and CD33-targeted Fab cross-linking molecule at 1000 ng / mL (corresponding to Figure 12) showed complete clearance of leukemic cells in the presence of CAR0007_hPGK and CAR0007_EF1a, while leukemic cell numbers were unaffected in non-transduced T cells (Figure 13).

[0556]

[0542] Figure 14 shows flow cytometry plots showing dose-dependent clearance of leukemia cells at the indicated CD33-targeted Fab cross-linking molecule concentrations of T cells containing CAR0007_EF1a and MOLM-13 at an ET ratio of 20:1 after 48 hours. Concentrations as low as 40 ng / mL showed near complete elimination of leukemia cells and at 200 and 1000 ng / mL complete elimination of leukemia cells.

[0557] Specific lysis of MOLM-13 leukemia cells by CAR0007_hPGK T cells at an ET ratio of 20:1 after 48 h incubation in the presence and absence of EGFR and CD33-targeted bridging molecules at the indicated concentrations is shown in Figure 15(a). Significant lysis was found in a dose-dependent manner for increasing concentrations of CD33-targeted Fab bridging molecules (40, 200 and 1000 ng / mL).

[0558] Specific lysis of MOLM-13 leukemia cells by CAR0007_hEF1a T cells at an ET ratio of 20:1 after 48 hours of incubation in the presence and absence of EGFR and CD33-targeted bridging molecules at the indicated concentrations is shown in Figure 15(b). Significant lysis was found in a dose-dependent manner for increasing concentrations of CD33-targeted Fab bridging molecules (40, 200 and 1000 ng / mL).

[0559] In a titration experiment, EGFR and CD33 targeted Fab cross-linking molecules were used to test the effect of CAR0007_hPGK on killing of MOLM-13 (Figure 16). There was a significant difference in the viability of MOLM-13 after 24 hours of incubation at 1000ng / mL with 10:1 ET ratio between EGFR targeted cross-linking molecules and CD33 targeted cross-linking molecules. Statistical analysis was performed by t-test. An alternative representation of the data from Figure 16 is shown in Figure 17. There was no significant difference in the titration of EGFR cross-linking molecules (data not shown). There was a significant difference in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test.

[0560] In a titration experiment, the effect of CAR0007_hPGK in killing MOLM-13 was tested using EGFR and CD33 targeted Fab cross-linking molecules (Figure 18). There was a significant difference in the viability of MOLM-13 after 24 hours of incubation at 200ng / mL and 1000ng / mL with an ET ratio of 20:1 between EGFR and CD33 targeted cross-linking molecules. Statistical analysis was performed by t-test. An alternative representation of the data from Figure 18 is shown in Figure 19. There was no significant difference in the titration of EGFR cross-linking molecules (data not shown). There was a significant difference in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test.

[0561] In a titration experiment, EGFR and CD33 targeted Fab bridge molecules were used to test the effect of CAR0007_hPGK on killing of MOLM-13 (Figure 20). There was a significant difference in the viability of MOLM-13 after 48 hours of incubation at 10:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test.

[0562] In a titration experiment, the difference in killing of MOLM-13 by CAR0007_hPGK was examined using EGFR and CD33 targeted Fab bridge molecules (Figure 21). There was a significant effect on MOLM-13 viability after 48 hours of incubation at a 20:1 ET ratio between EGFR and CD33 targeted bridge molecules at 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test.

[0563] In a titration experiment, the effect of CAR0007_EF1a in killing MOLM-13 was tested using EGFR and CD33 targeted Fab cross-linking molecules (Figure 22). There was a significant difference in the viability of MOLM-13 at 200ng / mL and 1000ng / mL after 24 hours of incubation at 10:1 ET ratio between EGFR targeted cross-linking molecules and CD33 targeted cross-linking molecules. Statistical analysis was performed by t-test. An alternative representation of the data from Figure 22 is shown in Figure 23. There was no significant difference in the titration of EGFR cross-linking molecules (data not shown). There was a significant difference in the titration of CD33 cross-linking molecules. Statistical analysis was performed by One-Way ANOVA and post-hoc Tukey test.

[0564] In a titration experiment, the effect of CAR0007_EF1a in killing MOLM-13 was tested using EGFR and CD33 targeted Fab bridge molecules (Figure 24). There was a significant difference in the survival rate of MOLM-13 after 24 hours of incubation at 20:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 40ng / mL, 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test. An alternative representation of the data from Figure 24 is shown in Figure 25. There was no significant difference in the titration of EGFR bridge molecule (data not shown). There was a significant difference in the titration of CD33 bridge molecule. Statistical analysis was performed by One-Way ANOVA and post-hoc test Tukey.

[0565] In a titration experiment, the effect of CAR0007_EF1a on killing of MOLM-13 was tested using EGFR and CD33 targeted Fab bridge molecules (Figure 26). There was a significant effect on MOLM-13 viability after 48 hours of incubation at 10:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 40ng / mL, 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test.

[0566] In a titration experiment, the effect of CAR0007_EF1a in killing MOLM-13 was tested using EGFR and CD33 targeted Fab bridge molecules (Figure 27). There was a significant difference in the survival rate of MOLM-13 after 48 hours of incubation at 20:1 ET ratio between EGFR targeted bridge molecule and CD33 targeted bridge molecule at 40ng / mL, 200ng / mL and 1000ng / mL. Statistical analysis was performed by t-test.

[0567] In a titration experiment, we tested the efficacy of non-transduced T cells, CAR0007_hPGK and CAR0007_EF1a effector cells in killing MOLM-13 with EGFR-targeted Fab cross-linking molecules at an ET ratio of 10:1 after 24 hours of incubation. There were no significant differences in the titration of EGFR cross-linking molecules between the three effector cell populations (data not shown).

[0568] Example 10 - Activation of different nfP2X7CAR-T cells with CD33-targeted Fab bridge molecules - titration

[0554] In a flow-based assay, activation was assessed by measuring the upregulation of CD25 and CD69 expression on MOLM-13 induced by non-transduced T cells, and T cells expressing CAR0007_hPGK and CAR0007_EF1a, at an ET ratio of 20:1 after 48 hours of incubation, both in the presence and absence of EGFR- or CD33-targeted Fab cross-linking molecules.

[0569]

[0555] Both CAR0007_hPGK and CAR0007_EF1a expressing T cells showed significantly increased expression of activation markers CD25 and CD69 in the presence of EGFR and CD33 cross-linking molecules compared to non-transduced T cel...

Claims

1. (a) an immune cell or a precursor thereof that expresses a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain binds to a tumor-specific antigen expressed on the cell surface; (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 an antigen recognition domain A bridging molecule comprising A two-component treatment comprising:

2. The therapeutic agent of claim 1 , wherein the tumor-specific antigen is an antigen expressed on a solid tumor.

3. The tumor-specific antigen is nfP2X ７ 2. The therapeutic agent of claim 1, which is any one of EGFRvIII and CLDN6.

4. The tumor-specific antigen epitope portion is a dysfunctional P2X ７ The therapeutic agent of claim 1 which is a receptor epitope moiety.

5. The therapeutic agent of claim 1 , wherein the bridging molecule is a polypeptide or an antibody or antibody fragment.

6. The therapeutic agent of claim 5 , wherein the polypeptide is expressed by an immune cell or a precursor thereof.

7. (i) comprising an antigen recognition domain that binds to a first tumor antigen, preferably the first tumor antigen is a dysfunctional P2X ７ a chimeric antigen receptor, (ii) (a) an antibody or antigen-binding fragment thereof that binds to a second tumor antigen; and (b) a first tumor antigen, preferably dysfunctional P2X. ７ an inducible expression construct encoding a bridging molecule in the form of a fusion protein comprising a peptide or fragment of the receptor; An immune cell or a precursor thereof comprising:

8. (i) a targeting moiety that binds to a cell surface molecule on a target cell; (ii) a tumor-specific antigen epitope portion that is bound by an antigen recognition domain, preferably a dysfunctional P2X ７ Receptor epitope portion A bridging molecule comprising:

9. A nucleic acid comprising a nucleotide sequence encoding the cross-linking molecule of claim 8.

10. A first nucleotide sequence encoding a targeting moiety, and a tumor-specific antigen epitope moiety, preferably a dysfunctional P2X ７ The nucleic acid of claim 9, further comprising a second nucleotide sequence encoding a receptor epitope portion.

11. For treating a disorder in a subject, A cell expressing a receptor comprising an antigen recognition domain and a signal transduction domain, wherein the antigen recognition domain is a tumor-specific antigen expressed on the cell surface, preferably a dysfunctional P2X ７ Cells that bind to the receptor, and (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 an antigen recognition domain, preferably a dysfunctional P2X ７ Receptor epitope portion A bridging molecule comprising:

12. The cells and bridging molecules described in claim 11, wherein the disorder is cancer.

13. To kill target cells, The tumor expresses a receptor comprising an antigen recognition domain and a signal transduction domain, the antigen recognition domain being a tumor-specific antigen expressed on the cell surface, preferably a dysfunctional P2X ７ Binding to receptors, immune cells, and (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 an antigen recognition domain, preferably a dysfunctional P2X ７ Receptor epitope portion A bridging molecule comprising:

14. The immune cell and bridging molecule of claim 13, wherein the target cell is a cancer cell or a cell whose MHC I or II molecule presents a peptide derived from an infectious agent.

15. Target cells have dysfunctional P2X ７ The immune cells and bridging molecules of claim 13, which do not express the receptor.

16. The immune cells and bridging molecules of claim 13, wherein two or more bridging molecules are administered to a subject, each bridging molecule comprising a targeting moiety that binds to a different cell surface molecule on a target cell.

17. The therapeutic agent of claim 1 , wherein the bridging molecule comprises a targeting moiety for more than one class of cell surface molecule on the target cell.

18. The therapeutic agent of claim 1 , wherein the bridging molecule comprises targeting moieties to more than one epitope of the same cell surface molecule on the target cell.

19. The antigen recognition domain of CAR binds to dysfunctional P2X ７ The therapeutic agent of claim 1, which binds to an epitope associated with the adenosine triphosphate (ATP) binding site of a receptor.

20. Dysfunctional P2X ７ Receptor is functional P2X ７ The therapeutic agent of claim 19, which has a reduced ability to bind ATP at the ATP binding site compared to the ATP binding ability of a receptor (e.g., a receptor having a wild-type sequence and the conformation or fold of an ATP-binding receptor).

21. The therapeutic agent of claim 1 , wherein the targeting portion of the bridging molecule comprises a ligand or binding partner of a protein or receptor present on the surface of a target cell.

22. The therapeutic agent described in claim 1, wherein the cell surface molecule is a tumor-associated antigen.

23. Tumor-associated antigens include 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 (LI The therapeutic agent of claim 22, wherein the therapeutic agent is selected from the group consisting of CD19, CD20, GD2, CD276, PSMA, and EGFR.

24. Dysfunctional P2X ７ The receptor epitope portion is a dysfunctional P2X ７ Receptor-binding antibodies bind functional P2X ７ The therapeutic agent of claim 23, which is not bound by an antibody that binds to the receptor.

25. Dysfunctional P2X with two or more cross-linking molecules ７ The therapeutic agent of claim 23 , comprising a receptor epitope portion.

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