Molecules and methods for activating car t cells

Abstract

The present invention relates to molecules comprising a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, and wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor. Methods for activating immune cells expressing chimeric antigen receptors and for the prevention and / or treatment of various conditions, including cancer, are also contemplated.

Description

Molecules and methods for activating CAR T cellsField of the invention

[0001] The present invention relates to molecules and methods for activating immune cells expressing an exogenous cell surface receptor comprising an intracellular signalling domain (eg chimeric antigen receptors), for the prevention and / or treatment of various conditions, including cancer.Related application

[0002] This application claims priority from Australian provisional application AU 2023902090, the entire contents of which are hereby incorporated by reference.Background of the invention

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

[0004] The potential of this approach has been demonstrated in clinical trials, wherein CAR T cells were infused into adult and paediatric patients with B-cell malignancies, neuroblastoma, and sarcoma. To date, over 500 clinical trials have emerged worldwide, designed at testing the efficacy of CAR T cells targeted to bind 64 different tumour associated antigens. Among these, three CD19-specific CAR T cell products have been approved for the treatment of acute lymphoblastic leukaemia (ALL), large B cell lymphoma and mantle cell lymphoma. To date, most of the success with CAR T therapies has been observed in the context of so-called “liquid” tumours, or where the CARs are directed to CD19, CD22 or the B cell maturation antigen (BCMA).

[0005] Several challenges remain in the clinical application of CAR T cell therapies. These include difficulties with achieving sufficient therapeutic responses in the context of solid tumours, which may be due to insufficient activation, expansion and persistence of CAR T cells and / or the immunosuppressive tumour microenvironment. In addition, the long-term clinical application of CAR T therapies can be negatively impacted by the selective pressure of mono-specific CAR T cells, antigen-negative escape variants, and antigen depletion. Moreover, low levels of target antigen expression in healthy tissues can result in severe “on-target, off-tumour” toxicities. Finally, cytokine release syndrome(CRS) and CAR T cell-related encephalopathy syndrome (CRES) are frequently observed side effects of CAR T cell therapy.

[0006] There is consequently a need for new and improved approaches to CAR, preferably CAR-T, therapies.

[0007] Reference to any prior art in the specification 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 could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0008] The present invention is based on the surprising finding by the inventors that molecules having a particular architecture are especially useful for activating immune cells expressing an exogenous cell surface receptor comprising an intracellular signalling domain (eg chimeric antigen receptors, including expressed on T cells). These molecules may be referring to herein as “bridging molecules” (or may also be referred to as adapter molecules).

[0009] Accordingly, in a first aspect, the invention provides a molecule capable of simultaneously binding to a target cell (such as a tumour cell) and to an immune cell expressing an exogenous cell surface receptor comprising an intracellular signalling domain, the molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor andwherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0010] In a further aspect, the invention provides a molecule capable of simultaneously binding to a target cell (such as a tumour cell) and to an immune cell expressing an exogenous cell surface receptor comprising an intracellular signalling domain, the molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least two moieties capable of being bound by the exogenous immune cell receptor, and wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor. Preferably, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0011] In a third aspect there is provided a two-component therapeutic comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor,wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0012] In a fourth aspect there is provided a composition comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0013] The invention also provides a molecule capable of simultaneously binding to a target cell (such as a tumour cell) and to an immune cell expressing an exogenous cell surface receptor comprising an intracellular signalling domain, the molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise means for binding to a cell surface molecule on a target cell,wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0014] In a further aspect, the invention provides a molecule capable of simultaneously binding to a target cell (such as a tumour cell) and to an immune cell expressing an exogenous cell surface receptor comprising an intracellular signalling domain, the molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise means for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least two moieties capable of being bound by the exogenous immune cell receptor, and wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor. Preferably, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0015] Further, there is provided a two-component therapeutic comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody,wherein the first and second Fab arms each comprise means for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0016] In a further aspect there is provided a composition comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise means for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0017] In any embodiment, a means for binding to a cell surface molecule on a target cell comprises or consists of an antigen binding domain for binding to a cell surface molecule on a target cell.

[0018] Further still there is provided a kit comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

[0019] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a heterodimer (bridging molecule) as described herein. Preferably, the nucleic acid comprises a first nucleotide sequence encoding the first portion of the molecule and a second nucleotide sequence encoding the second portion of the molecule.

[0020] In another aspect, the present invention provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject:(a) an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm,wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, thereby treating the disease or disorder in the subject.

[0021] In another aspect, the present invention provides a method of treating a cancer in a subject, the method comprising administering to the subject:(a) an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a tumour cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, thereby treating the cancer in the subject.

[0022] In another aspect, the present invention provides a method of killing a target cell, the method comprising exposing or contacting the target cell with:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a tumour cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second the Fab arm portion does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, thereby killing the target cell.

[0023] Further still there is provided use of a heterodimer as described herein, in the manufacture of a medicament for treating a disorder, wherein optionally the disorder is cancer or a disease or condition requiring depletion of T cells or B cells (such as an autoimmune disease or condition or a transplant related condition such as graft versus host disease).

[0024] Preferably, the use comprises the administration of the medicament comprising the heterodimer and the administration of an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain, wherein the binding domain is capable of binding to the heterodimer.

[0025] In yet a further aspect there is provided a pharmaceutical composition comprising a heterodimer as described herein, optionally comprising a pharmaceutically acceptable excipient. The pharmaceutical composition may optionally further comprise an immunecell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain.

[0026] In a further aspect there is provided a heterodimer as described herein, optionally in combination with an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain; or a pharmaceutical composition as described herein, for use in the treatment of a disease or disorder in a subject. Preferably the disease is cancer. Alternatively, the disease or condition may be one requiring depletion of T cells or B cells (such as an autoimmune disease or condition or a transplant related condition such as graft versus host disease).

[0027] Further still, the invention provides for methods wherein the bridging molecules described herein can be administered synchronously to a subject in need thereof. This allows for fine-tuning of the therapeutic approach, such that a therapeutic immune cell may be directed to binding target cells (such as cancer cells) via different antigens (including cancer-associated antigens), at different times during the course of the patient’s therapeutic regimen. In other words, the molecules can be administered sequentially or at the same time (simultaneously) or separately to the administration of the therapeutic immune cells.

[0028] In any embodiment of aspects directed to methods of treatment herein, the bridging molecule may be delivered via infusion to the subject or may be expressed by the immune cell expressing the receptor or an immune cell designed for the expression of the molecule with or without an inducible expression system. The bridging molecule may be a polypeptide, which is encoded in an inducible or a constitutive expression construct contained in the immune cell.

[0029] As used herein, the term “Fab arm” (or “Fab region”) will be understood to refer to a “fragment antigen-binding region” or Fab region of an antibody. The skilled person will be familiar with the use of this term to describe a region of an antibody that comprises an antigen binding domain and is composed of one constant and one variable domain of each of the heavy and light chains of an antibody.

[0030] In preferred embodiments of any aspect herein, the first and second portions comprise an Fc region of an antibody, such as a CH2, and / or a CH3 region of an antibody, wherein the Fc region is linked to the Fab arms of the molecule. Accordingly, in preferredembodiments of any aspect, the molecule is in the form of heterodimeric Fab-Fc fusion protein (such as a conventional antibody structure) comprising the at least one moiety capable of being bound by an exogenous immune cell receptor (such as a chimeric antigen receptor), linked to the first Fab region / arm of the antibody.

[0031] It will be appreciated that the “first” Fab region / arm of the heterodimeric molecule of the invention can be oriented to the “left” or “right” side of the molecule, as depicted in Figure 2B herein.

[0032] The skilled person will be familiar with standard techniques for designing a heterodimeric (asymmetric) molecule as described herein, including the use of recombinant techniques to ensure the proper dimerisation of non-identical Fab and Fab- Fc fusion proteins (ie for forcing a pairing of a light chain polypeptide with its correct heavy chain counterpart). More specifically, the skilled person will appreciate that the correct assembly of the heavy chain / light chain to achieve the asymmetric design of the molecules required the following heavy chain / light chain pairing:• A. heterodimeric-heavy chain pairing techniques and• B. homologic light chain / heavy chain pairing techniques.

[0033] Examples of such techniques include the use of so-called “CrossMab” and “DuetMab” systems, such as described in Mazor et al., (2015) mAbs, 7:377-389 and Klein et al., (2016) mAbs, 8:1010-1020, incorporated herein by reference. These references discuss techniques for enabling the correct association of generic light chains using immunoglobulin domain crossover (CrossMAb technology), which can be combined with approaches enabling correct heavy chain association such as knob-into-holes (KIH) (Ridgway et al., (1996), Protein Eng, 9: 617-621 ) technology or electrostatic steering. WO 2009 / 080251 , WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254 and Schaefer, W. et al, PNAS, 108 (201 1 ) 1 1187-1 191 disclose methods for obtaining bivalent, bispecific IgG antibodies with a domain crossover. WO 2010 / 145792 discloses methods for obtaining tetravalent antigen binding proteins with a domain crossover. The multispecific antibodies with a VH / VL replacement / exchange in one binding site to prevent light chain mispairing (CrossMab VHA L), which are described in WO 2009 / 080252 (see also Schaefer, W. et al, PNAS, 108 (201 1 ) 1 1 : 187-1 191 ), clearly reduce the production of mispaired variants caused by the mismatch of a light chain against a first antigen with thewrong heavy chain against the second antigen (compared to approaches without such domain exchange). The methods disclosed in the aforementioned documents for preventing mispairing of light and heavy chains (and thereby ensuring that the moiety capable of being bound by the exogenous immune cell receptor is on one portion only of the heterodimeric molecules of the invention) are incorporated herein by reference.

[0034] Similarly, the “DuetMab” technology uses knobs-into-holes (KIH) technology for heterodimersiation of two distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulphide bond in one of the CH1 - CL interfaces with an engineered disulphide bond.

[0035] The skilled person will also be familiar with other technologies that can be used to achieve the same outcome. Some examples, include, but are not limited to: A) for heterodimeric heavy chain technology: KiHS-S: Mutations T366S / L368A / Y407V / Y349C KiH + inter-CH3 domain S-S bond (Carter P. Bispecific human IgG by design. J Immunol Methods (2001 ) 248(1 -2):7-15); EW-RVTS-S : Mutations of K360E / K409W / Y349C Q347R / D399V / F405T / S354C EW-RVT + inter-CH3 domain S-S bond (Choi HJ, Seok SH, Kim YJ, Seo MD, Kim YS. Crystal structures of immunoglobulin Fc heterodimers reveal the molecular basis for heterodimer formation. Mol Immunol (2015) 65(2):377-83); B) for homologic light chain / heavy chain pairing: Common light chain (Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, et al. An efficient route to human bispecific IgG. Nat Biotechnol (1998) 16(7):677— 81 ) ; Ortho-Fab IgG technology (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat Biotechnol (2014) 32(2):191 — 8).

[0036] It is well within the purview of the skilled person to be able to determine which of KIH, DuetMab, or CrossMab or other technology to employ in order to generate molecules falling within the scope of the invention. More specifically, the skilled person will recognise that in order to generate a monotagged molecule on the heavy chain of the molecule, a knob-in-hole technology would be sufficient to achieve this molecule since the light chains would be identical and no DuetMab, or CrossMab design would be required. Using DuetMab, or CrossMab alone could also be used to provide such a molecule (such as XmAbl ).

[0037] For the generation of monotagged antibodies or dual-tagged or triple-tagged, or quadrouple-tagged, or quinttrouple-tagged etc. molecules that have at least one tag on the light chain (N-TERMINAL, between VL and CL, or C-TERMINAL) the DuetMab, or CrossMab are required to be used with a knob-in-hole (KIH) technology. Otherwise, the molecule cannot be generated intentionally and will be subject to a stochastic distribution of protein expression and assembly. The combination of KIH and DuetMab, or CrossMab reliably facilitate generation of molecules that show the wanted properties of having the tag only on one side of the molecule.

[0038] In any aspect, the antigen binding domains of the first and second Fab arms of the heterodimeric molecule may be identical in amino acid sequence. Alternatively, the antigen binding domains may be non-identical but may bind to the same epitope of the antigen, or may bind to different epitopes of the same antigen. In further embodiments, the antigen binding domains may be non-identical and may bind to distinct (different) antigens.

[0039] As will be understood herein, the first and second Fab arms are preferably for binding to an antigen which is an antigen of a target cell. In other words, the antigen binding domains of the Fabs are capable of specifically binding to an antigen of a target cell.

[0040] In certain embodiments, the target cell is a cancer cell, or a cell capable of presenting a peptide from an infectious agent on an MHC class receptor.

[0041] In especially preferred embodiments, the target cell is a cancer cell, such that the first and second Fab arms therefore comprise antigen binding domains for binding to antigens on the surface of cancer cells. Such antigens are further described herein.

[0042] The heterodimeric molecule comprises at least one moiety capable of being bound by an exogenous immune cell receptor (such as a chimeric antigen receptor or universal CAR system receptor). The use of appropriate heavy and / or light chain pairing approaches such as discussed above, enables the design of bridging molecules which comprise a single moiety (tag) in a heterodimeric molecule. The skilled person will appreciate that without using this approach, manufacture of a dimeric molecule would result in the generation of homodimeric species, resulting in molecules which comprise an epitope moiety on each arm of the Fab. Thus, using appropriate heavy and / or lightchain pairing (such as using KIH in combination with Cross-Mab and DuetMab), ensures that only one arm of the Fab in the dimeric molecule comprises the one or more moieties capable of being bound by the exogenous immune cell receptor.

[0043] Further still, the location of the one or more moieties on a Fab arm of the heterodimeric molecule, ensures the binding to a single CAR molecule on an immune cell only and thereby reduces likelihood of transactivation of different CAR receptors on individual CAR T cells.

[0044] In further embodiments, the heterodimeric molecule comprises 2, 3, or 4 or more moieties capable of being bound by an exogenous immune cell receptor (such as a chimeric antigen receptor or universal CAR system receptor) wherein the moieties are present on one Fab arm, but not on the other Fab arm. Preferably, the heterodimeric molecule comprises no more than 5, no more than 3, or no more than 2 moieties capable of being bound by an exogenous immune cell receptor as described herein, wherein the moieties are present on one Fab arm of the heterodimeric molecule, but not on the other Fab arm.

[0045] In any aspect, the heterodimeric molecule may comprise 3 or more moieties capable of being bound by the exogenous immune cell receptor.

[0046] In any aspect, the heterodimeric molecule may comprise 4 or more moieties capable of being bound by the exogenous immune cell receptor.

[0047] The at least 2 moieties (or 3 or 4 or more moieties) may comprise or consist of identical sequences, or of non-identical sequences. Non-identical sequences may comprise overlapping identical amino acid sequence. Non-identical sequences may comprise amino acid sequences of a single epitope capable of being bound by an immune cell receptor. Alternatively, non-identical sequences may comprise amino acid sequences of more than one epitope capable of being bound by an immune cell receptor. For example, in any aspect, a bridging molecule may comprise a moiety comprising an amino acid sequence from a dysfunctional P2X? receptor epitope in the form of the E200 epitope and a further moiety comprising an amino acid sequence from a dysfunctional P2X? receptor epitope in the form of the E300 epitope. Alternatively, in any aspect, a bridging molecule may comprise a dysfunctional P2X? receptor epitope moiety in the form of the E200 epitope and a further dysfunctional P2X? receptor epitope moiety in the form of thecomposite epitope. Still further, in any aspect, a bridging molecule may comprise a first dysfunctional P2X? receptor epitope moiety in the form of the E200 epitope and a further dysfunctional P2X? receptor epitope moiety in the form of the E200 epitope.

[0048] It will be appreciated by the skilled person that the need to ensure proper pairing of heavy and light chains of the bridging molecule will be dictated to a certain extent by the location of the epitope moiety on the Fab and the number of moieties. For example, where the first and second Fab arms comprise identical antigen binding domain sequences (eg in the scenario where the bridging molecule is bivalent and monospecific) and the first Fab arm comprises a single moiety (or indeed additional moieties) capable of being bound by the exogenous immune cell receptor, it is necessary to ensure proper pairing of the heavy chains only, but not of the light chains, if the moiety is linked to the heavy chain of the first Fab.

[0049] On the other hand, if the first and second Fab arms comprise non-identical antigen binding sequences (eg in the scenario where the bridging molecule is bispecific) then it is necessary to ensure proper pairing of distinct heavy chains and correct pairing of cognate light chains (ie, pairing of the cognate light chains to the respective heavy chain pair).

[0050] Similarly, if the first and second Fab arms comprise identical antigen binding domain sequences (eg the molecule is bivalent and monospecific), and the first Fab arm comprises a single moiety (or indeed additional moieties) capable of being bound by the exogenous immune cell receptor, it is necessary to ensure proper pairing of distinct heavy chains and proper pairing of cognated light chains, if the moiety is linked to the light chain of the Fab.

[0051] In any aspect, the moiety may comprise or consist of a peptide or 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.

[0052] In any aspect, the moiety capable of being bound by the exogenous immune cell receptor may be any peptide, polysaccharide or lipid or other moiety capable of being bound by a receptor that is useful in a “universal CAR” or “switchable CAR” system or similar. Such systems are known to the skilled person and are described for example in Wermke et al., (2021 ), Blood, 137: 3145-3148; Lin et al., (2021 ) Frontiers in Immunology,12: https: / / doi.Org / 10.3389 / fimmu.2021 .744823 and Liu et al., (2019) J. Hematol. & Oncol. 12:69; and WO 2019 / 238722, all incorporated herein by reference. In certain embodiments, the receptor is capable of binding to a neo-epitope or exogenous epitope or peptide “tag”, such as a dye (eg fluroscein isothiocyanate, FITC), biotin moiety, “AZip” tag (or Leucine zipper moiety), “Latch with Blm peptide”, “SpyTag”, the 5B9 tag (ie comprising 10 amino acids derived from the human nuclear autoantigen La / SS-B) the PNE tag (ie a 14 amino acid peptide neo-epitope derived from a yeast transcription factor), or any peptide or tag capable of being used and bound by an extracellular receptor for use in a UniCAR, OmniCar (SpyCatcher), D-Domain (ARC-SparX) based CAR T cells or other similar universal CAR system. In the art, such neo-epitopes or tags may also be referred to as “switch elements” in the context of so-called “Universal CAR” systems. The skilled person will appreciate that such neo-epitopes are not typically epitopes associated with cancer antigens.

[0053] In any aspect, in scenarios where the exogenous immune cell receptor is a chimeric antigen receptor, the moiety capable of being bound by the exogenous immune cell receptor is a moiety capable of being bound by the binding domain or other extracellular domain of chimeric antigen receptor (CAR). Typically, CARs are designed for binding to tumour antigens, and accordingly, the moiety capable of being bound by the CAR is preferably a tumour antigen or a sequence derived from a tumour antigen. The tumour antigen may be expressed on a solid tumour or liquid tumour. Accordingly, in such embodiments, the moieties on the heterodimeric molecule comprise an amino acid sequence of an epitope of a solid or liquid tumour antigen.

[0054] In any aspect, the CAR recognises an epitope of a tumour-specific or tumour- associated antigen and the moiety capable of being bound by the exogenous immune cell is the epitope of the tumour-specific or tumour-associated antigen. Accordingly, in such embodiments, the moieties on the heterodimeric molecule comprise an amino acid sequence of an epitope of a tumour-specific antigen or tumour-associated antigen, or a neo-epitope or other peptide sequence (such as described herein in the context of universal CAR systems).

[0055] In one embodiment, the tumour-associated or tumour-specific antigen is an epitope derived from the antigens P2X?, EGFRvlll or CLDN6. Accordingly, in such embodiments, the moieties on the heterodimeric molecule comprise an amino acidsequence of an epitope derived from the antigens P2X?, EGFRvlll or CLDN6. Examples of such amino acid sequences are provided in the Tables herein.

[0056] In one example, the CAR recognises an epitope of the P2X? receptor and the moieties present on the heterodimeric bridging molecule of the invention therefore comprise at least the amino acid sequence as set forth in SEQ ID NO: 162. In any aspect, the P2X? receptor epitope moiety comprises or consists of a fragment of a dysfunctional P2X? receptor. Exemplary fragments include GHNYTTRNILPGLNITC (SEQ ID NO: 153; also referred to herein as the “E200 epitope”) and variants thereof (exemplary variants are provided in SEQ ID NOs: 154 to 162 and 166 to 218; KYYKENNVEKRTLIK and variants thereof (SEQ ID NO: 163 and 164; also referred to herein as the “E300” epitope); or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 165; also referred to herein as the “E200 / E300” or “composite” epitope). Further examples are provided in Table 3 herein.

[0057] In further preferred embodiments, the CAR recognises an epitope of the EGFRvlll receptor and the moieties present on the heterodimeric bridging molecule of the invention therefore comprise at least the amino acid sequence as set forth in SEQ ID NO: 134.

[0058] In further preferred embodiments, the CAR recognises an epitope of CLDN6 and the moieties present on the heterodimeric bridging molecule of the invention therefore comprise at least the amino acid sequence as set forth in SEQ ID NO: 143.

[0059] In certain embodiments, the heterodimeric bridging molecule of the invention is bivalent and bispecific. In such embodiments, the epitope moieties located on the molecule are preferably located on a Fab portion which is capable of binding to the target cell.

[0060] In particularly preferred embodiments, the heterodimeric bridging molecule of the invention is bivalent and monospecific. In other words, the molecule preferably comprises two binding sites, each for binding to the same target antigen.

[0061] Exemplary sequences of certain heterodimeric bridging molecules of the invention are bivalent and monospecific and are provided in Tables 1 and 2. Table 1 provides examples of sequences of molecules comprising moieties capable of being bound by an anti-E200 CAR (ie comprise peptide moiety tags derived from the E200 epitope of the P2X7 receptor, for being bound by a CAR capable of binding to the E200epitope). The molecules defined in Table 1 include various molecules capable of binding to a wide range of different cancer antigens including CD19, CD33, and others.

[0062] Table 2 provides sequences of alternative tags and alternative CAR / bridging molecule systems, such as bridging molecules capable of binding to CD19 or CD33 and comprising: epitope moieties comprising the amino acid sequence of PNE (for use in a PNE-type universal CAR system), epitope moieties comprising the amino acid sequence of a fragment of EGFRvlll or epitope moieties comprising the amino acid sequence of a fragment of CLDN6.

[0063] It will be understood that in accordance with the molecules and methods of the invention, the exogenous immune cell receptor is a cell surface receptor comprising an extracellular domain capable of binding to our being bound by a bridging molecule as described herein, and wherein the receptor further comprises an intracellular signalling domain. Typically, wherein the extracellular domain is an antigen binding domain, cell surface receptors may be referred to a chimeric antigen receptors (or CARs). In the scenario where the extracellular domain is one which comprises a peptide or other moiety capable of being bound (eg a so-called neo-epitope), the receptor may instead be referred to as a “universal CAR” (meaning that the CAR is not one which is limited to recognising and binding to a specific antigen via an antigen binding domain).

[0064] In certain embodiments, where the immune cell receptor is a CAR, the binding domain may be capable of binding to a tumour-specific or tumour-associated antigen

[0065] The immune cell comprising the exogenous immune cell surface receptor (such as a CAR or universal CAR), may be any immune cell capable of eliciting a cell-killing response, such as a T cell, NK cell, NKT cell, B cell, cytokine induced killer (CIK) cell, lymphokine activated killer (LAK) cell or gamma-delta T cell. Other examples of immune cells are further described herein. Preferably the immune cell is a CAR T cell or a universal CAR T cell.

[0066] In any aspect, the immune cell or a cell that is capable of differentiating into an immune cell (e.g., a progenitor of an immune cell) is a cell that is capable of differentiating into an immune cell (e.g. T cell that will express the receptor) and may be a stem cell, multi-lineage progenitor cell or induced pluripotent stem cell.

[0067] In any embodiment, the exogenous immune cell receptor may be a CAR comprising an antigen recognition domain for binding to a tumour-associated or tumourspecific antigen. Examples of such tumour-associated and tumour-specific antigens are well known in the art, as are antigen recognition domains for binding to the same in the context of a CAR. Some examples of tumour-associated and tumour-specific antigens are further described herein, although the skilled person will appreciate that the present invention finds application for improving the utility and reducing unwanted toxicities for any CAR construct.

[0068] An antigen recognition domain of a CAR typically comprises an antigen binding domain or fragment derived from, or that is an antibody.

[0069] In non-limiting examples which exemplify the utility of the invention, the exogenous immune cell receptor may be a CAR comprising an antigen recognition domain for binding to an epitope of the P2X? receptor, (such as dysfunctional or nonfunctional forms of the receptor as further defined herein). In any embodiment, the binding domain (antigen recognition domain) of the exogenous receptor binds to an epitope associated with an adenosine triphosphate (ATP)-binding site of the P2X? receptor. In especially preferred embodiments, the epitope of P2X? comprises or is a derivative of the E200 epitope as herein defined, including in Table 3. In particularly preferred embodiments, the epitope of the P2X? receptor comprises at least the amino acid sequence as set forth in SEQ ID NO: 162.

[0070] In any embodiment, the antigen-recognition domain binds to an epitope that includes the proline at amino acid position 210 of the P2X? receptor. In some embodiments, the antigen-recognition domain binds to an epitope that includes one or more amino acid residues spanning from glycine at amino acid position 200 to cysteine at amino acid position 216, inclusive, of the P2X? receptor, or a variant thereof. .

[0071] The antigen-recognition domain of the receptor can be any suitable molecule that can interact with and specifically binds to a P2X? receptor. However, in some embodiments, the antigen-recognition domain includes amino acid sequence homology to the amino acid sequence of an antibody, or a fragment thereof, which binds to the dysfunctional P2X? receptor. In some embodiments, the antigen-recognition domain includes amino acid sequence homology to the amino acid sequence of a fragment-antigen binding (Fab) portion of an antibody that binds to a dysfunctional P2X? receptor. In some embodiments, the antibody is a humanised antibody.

[0072] In any embodiment, the antigen-recognition domain includes amino acid sequence homology to the amino acid sequence of a VHH (ie a heavy chain antibody or nanobody), a d-domain based recognition protein, a single-chain variable fragment (scFv) or a multivalent scFv that binds to a the E200 (or variant thereof) or other epitope of the (dysfunctional) P2X? receptor. In some embodiments, the multivalent scFv is a divalent or trivalent scFv.

[0073] In any embodiment, the antigen-recognition domain includes amino acid sequence homology to a single-antibody domain (sdAb) that binds to a dysfunctional P2X? receptor.

[0074] In any embodiment, the antigen-recognition domain includes a binding polypeptide that includes amino acid sequence homology to one or more complementarity determining regions (CDRs) of an antibody that binds to the E200 epitope (or variant thereof) or other epitope of a (dysfunctional) P2X? receptor. In any embodiment, the antigen-recognition domain includes amino acid sequence homology to the CDR1 , 2 and 3 domains of the VH and / or VL chain of an antibody that binds to a dysfunctional P2X? receptor. In preferred embodiments, the antigen-recognition domain comprises the amino acid sequence of the CDRs of the VH and / or VL chain of an antibody, or the amino acid sequence of the VH and / or VL chains of an antibody, or the amino acid sequence of an antibody or fragment thereof, wherein the antibody or fragment thereof comprises the amino acid sequences of any antibody described in PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531 ,171 , US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2007 / 001540 (or in corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in corresponding US publication US 2010- 0036101 ), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181 ,320, US 9,944,701 or US 10,597,451 ), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 , or US 10,053,508), andPCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609) the entire contents of which are hereby incorporated by reference.

[0075] Preferably the antigen recognition domain comprises the CDR amino acid sequences of the 2-2-1 antigen binding domain described in PCT / AU2010 / 001070 (or in any one of the corresponding US patents US 9,127,059, US 9,688,771 , or US 10,053,508) or BPM09 described in PCT / AU2007 / 001541 (or in corresponding US publication US 2010-0036101 ) and produced by the hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under Accession no. 06080101 .

[0076] In further preferred embodiments, the antigen-recognition domain is a binding domain as described in WO 2023 / 028653.

[0077] In some embodiments, the signalling domain of the immune cell receptor (such as a CAR) includes a portion derived from an activation receptor. In some embodiments, the activation receptor is a member of the CD3 co-receptor complex or is an Fc receptor. In some embodiments, the portion derived from the CD3 co-receptor complex is CD3- . In some embodiments, the portion derived from the Fc receptor is FcsRI or FcyRI.

[0078] In some embodiments, the signalling domain includes a portion derived from a co-stimulatory receptor. In some embodiments, the signalling domain includes a portion derived from an activation receptor and a portion derived from a co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, 0X40, 4-1 BB (CD137) and ICOS.

[0079] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0080] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0081] Figure 1 : Schematics showing examples of target antigen independent toxicity arising from suboptimal bridging molecule format (ie comprising epitope tags capable of being bound by the CAR T cells on both arms of the bridging molecule). A: chronicactivation, exhaustion of T cells and induction of apoptosis upon binding of a sub-optimal bridging molecule to a CAR-T cell; B: fratricide arising from toxic events between two CAR expressing T cells.

[0082] Figure 2: Proposed solution to problem: design of mono-tagged and dual-tagged heterodimeric antibody molecule where epitope (tag) bound by CAR is present only on one arm of the molecule.

[0083] Figure 3: Exemplary formats of heterodimeric molecules of the invention showing location of epitope (tag) bound by CAR on antibody molecules. Dark green and light green colouring denotes different antibody chains. The figure indicates possible combinations of tags and tag locations, however does not show all possible combinations. The main sites for peptide tag positioning are indicated in the figure and are: N-terminal light chain, N-terminal heavy chain, C-terminal light chain, C-terminal heavy chain, between VL and CL, between VH and CH1. All tag locations and all combinations can be utilised and optimised in an target antigen individualised manner.

[0084] Figure 4: Exemplary data showing cell killing of U937 cells (expressing CD33) by anti-E200 P2X?-CAR T cells and a bridging molecule comprising Fabs for binding to CD33 and E200 peptide tag. Mono-tagged (ie comprising single copy of E200 epitope) and dual-tagged (comprising two copies of E200 epitope) bridging molecules were tested. Dual-tagged bridging molecules provide superior cell killing in combination with anti-E200 P2X7-CAR T cells compare to bridging molecules having only one tag for binding by the CAR T cells. Concentration of bridging molecules used (y axis) Concentration of bridging molecules used (y axis) 0, 6.4, 15, 40, 100 ng / ml or 1 pg / ml.

[0085] Figure 5: Exemplary data showing that Jeko-1 cells are not killed by anti-E200 P2X7-CAR T cells when combined with a bridging molecule comprising Fabs for binding to CD33 and E200 peptide tag. These data indicate there is no non-specific activation of the CAR T cells by the bridging molecule. Concentration of bridging molecules used (y axis) 0, 6.4, 15, 40, or 100 ng / ml.

[0086] Figure 6: Further exemplary data showing cell killing of U937 cells (expressing CD33) by a second anti-E200 P2X7-CAR T cells and a bridging molecule comprising Fabs for binding to CD33 and E200 peptide tag. Mono-tagged (ie comprising single copy of E200 epitope) and dual-tagged (comprising two copies of E200 epitope) bridgingmolecules were tested. Dual-tagged bridging molecules provide superior cell killing in combination with anti-E200 P2X?-CAR T cells compared to bridging molecules having only one tag for binding by the CAR T cells. Concentration of bridging molecules used (y axis) 100 ng / ml or 1 ng / ml.

[0087] Figure 7: Schematic showing A) optimum tag presentation (orthogonal presentation of tag) on bridging molecules for enabling engagement of CAR T cells and APCs; B) sub-optimum tag presentation (non-orthogonal) bridging molecules.

[0088] Figure 8: Cell killing of U937 cells (expressing CD33) or Jeko-1 cells (expressing CD19) by anti-E200 P2X?-CAR T cells and various bridging molecules at 24 hours and 48 hours. Bridging molecules are bivalent and may be mono- or bi-specific for binding to CD33 (green) and / or CD19 (red) and one or more E200 peptide tags. Concentration of bridging molecules used (y axis) 0, 1 , 10, 100 ng / ml or 1 pg / ml.

[0089] Figure 9: Unwanted activation induced by the presence of a dual-tagged bridging molecule (where the tags are present on each of the Fab arms of the antibody). The different antibodies used in this assay comprised symmetric antibodies with 2 epitope moieties (peptide tags) on the light chains (mAb2) or heavy chains (mAb2) as well as a symmetric antibody with 4 tags (mAb4) [two on the light chain and 2 on the heavy chain]. Further, the assay comprised Cross-MAB antibody variants with a single tag on the light chain (XmAbl ) of one Fab arm of the antibody or on the heavy chain (XmAbl ) of one Fab arm of the antibody as well as with two tags on the light chain and the heavy chain of one Fab arm of the antibody (XmAb2). All tags tested were positioned at the N-terminal end of the molecules. Concentration of bridging molecules used (y axis) 10, 40, 100 or 400 ng / ml.

[0090] Figure 10: A. Cytolysis of AML cell line U937 at 24 hours, B - at 48 hours and C - at 72 hours. Ai, Bi and Ci: cytolysis of the AML cell line U937 by an anti-E200 CAR was measured using a luciferase-based kill assay at an ET ratio of 0.5:1 (CAR T to target ratio) in conjunction with and without the depicted bridging molecule variants (comprising anti- CD33 binding domains and E200 tags at indicated concentrations. The second condition (Aii, Bii and Cii) makes use of a CAR T product that is unable to bind to the E200 peptide (via a mutated antigen binding domain in the CAR) and serves as a negative control. Concentration of bridging molecules used (y axis) 0, 1 , 2.6, 6.4, 16, 40 or 100 ng / ml.

[0091] Figure 11 : Comparison of cytolysis of anti-E200 CAR versus the AML cell line U937 at 1 ng / mL of the corresponding adapter molecule formats at 24 hours, 48 hours, and 72 hours. Statistical analysis of the various antibodies demonstrated a superior function of the symmetric antibodies mAb2, mAb4, and XmAb2compared to XmAb1and Fab1.

[0092] Figure 12: Kaplan-Meier plot showing overall survival of mice treated with anti- E200 CAR T cells + dual-tagged bridging molecule (XmAb2) comprising anti-CD33 binding domains (based on lintuzumab) + one E200 peptide at the N-terminal light chain and one E200 peptide at the N-terminal heavy chain of one Fab arm of the antibody (the other Fab arm having no E200 peptide tags), compared to untreated mice, or mice treated with untransduced activated and expanded T cells or mice receiving treatment with anti- CD33 CAR T cells (based on lintuzumab).

[0093] Figure 13: Cell killing of U937 cells by a PNE-directed universal CAR, using bridging molecules (0 ng / ml or 100 ng / ml) comprising a PNE peptide and antigen binding domains based on lintuzumab. Similarly to the results shown using E200-based CARs and bridging molecules comprising E200 peptides, the PNE-based bridging molecules and CAR system are useful for killing target cells.

[0094] Figure 14: Bioluminescence in animals of MOLM-13 cancer cells in a xenograft model of AML; animals treated with CAR-T cells. 1 x106effective anti-E200 P2X?-CAR T cells or 25x106untransduced T cells were administered 3 days after 1 x106of MOLM-13 cells. CD33-targeted XmAbl , XmAb2, mAb2 No 1 , or mAb2 No 2 were injected at 25 pg / mouse twice per week (50 pg / mouse per week) intraperitoneally in the E200-targeted CAR T cell group. Bioluminescence imaging was performed twice per week over the course of treatment. (A) Individual animal bioluminescence readings during the treatment course of anti-E200 P2X?-CAR only group, CAR T with CD33-targeted XmAbl , XmAb2 , or mAb2 No 1 . (B) Mean animal bioluminescence readings of each five treated mice group during the treatment course. (C) The peripheral blood cell subpopulation of mice bearing MOLM-13 xenograft (and treated as described in Figure 14) was analyzed by flow cytometry when the animal reached the experiment end-point and was euthanized. The blood cell subpopulations were: mouse blood cells (mouse CD45+), MOLM-13 cancer cells (GFP+), injected CAR T cells (mouse CD45-human CD3 + CAR+), and CAR negative human T cells (mouse CD45- human CD3+ CAR-). The percentage of each population to total blood cells was plotted. Animals treated with the asymmetrical XMab2bridging molecule had better CAR T cell persistence compared to mice treated with the symmetrical mAb2 No 1 molecule.

[0095] Figure 15: Median fluorescence intensity ratio (MFIR) as measured by flow cytometry, following incubation of wild-type and CD33-knockout U937 cells.

[0096] Figure 16: Percentage cell viability of U937 cells following co-incubation with BIL- 03 CAR T cells and various bridging molecules. The results show comparable efficacy of a CLL1 xCD33 bispecific Crossmab to 1 :1 mixed CD33 and CLL1 monospecific Crossmabs on U937 WT cells, which express CD33 and CLL1 . • = CD33 monospecific Crossmab with E200 tag on single Fab arm. ■ = CLL1 monospecific Crossmab with E200 tag on single Fab arm.A= CLL1 x CD33 bispecific Crossmab with E200 tag on anti- CLL1 Fab arm. ▼ = CD33 and CLL1 monospecific Crossmab mixed at 1 :1 ratio.

[0097] Figure 17: Percentage cell viability of U937 CLL1 KO cells following coincubation with BIL-03 CAR T cells and various bridging molecules. • = CD33 monospecific Crossmab with E200 tag on single Fab arm. ■ = CLL1 monospecific Crossmab with E200 tag on single Fab arm.= CLL1 x CD33 bispecific Crossmab with E200 tag on anti-CLL1 Fab arm. ▼ = CD33 and CLL1 monospecific Crossmab mixed at 1 :1 ratio.

[0098] Figure 18: Percentage cell viability of U937 CD33 KO cells following coincubation with BIL-03 CAR T cells and various bridging molecules. • = CD33 monospecific Crossmab with E200 tag on single Fab arm. ■ = CLL1 monospecific Crossmab with E200 tag on single Fab arm.= CLL1 x CD33 bispecific Crossmab with E200 tag on anti-CLL1 Fab arm. ▼ = CD33 and CLL1 monospecific Crossmab mixed at 1 :1 ratio.

[0099] Figure 19: Percentage cell viability of mixed U937 cells following co-incubation with BIL-03 CAR T cells and various bridging molecules. U937 WT, CD33KO and CLL1 KO were mixed at a ratio of 1 :1 :1 . • = CD33 monospecific Crossmab with E200 tag on single Fab arm. ■ = CLL1 monospecific Crossmab with E200 tag on single Fab arm.A= CLL1 x CD33 bispecific Crossmab with E200 tag on anti-CLL1 Fab arm. ▼ = CD33 and CLL1 monospecific Crossmab mixed at 1 :1 ratio.Sequence information

[0100] Table 1 : Sequence information for exemplary heterodimeric molecules (bridging molecules) of the invention comprising E200-based epitope moieties for use with an anti-E200 CAR system.

[0101] The skilled person will appreciate that the pairing of sequences in the below table is: Left arm sequences = KNOB side sequences are paired with each other (always one light chain (LC) and one heavy chain (HC). Right arm sequences = HOLE side sequences are paired with each other (always one LC and one HC). HC CROSS- MAB sequences are for pairing with LC CROSS-MAB sequences only. HC DUET-MAB sequences are for pairing with LC DUET-MAB sequences only.

[0102] In the following, the superscript number indicates the number of peptide tags on the KNOB side or “left-sided” Fab of the antibody which can theoretically be 1 , 2, 3, 4, 5, 6 or more. The XmAb1, XmAb2, XmAb3, XmAb4, XmAb5, XmAb6, XmAbn or DUETMAB1 , DUETMAB2, DUETMAB3, DUETMAB4, DUETMAB5, DUETMAB6, DUETMABn molecule formats provide XmAb and DUETMAB with similar potency to an symmetric mAb in mediating specific effector functions but having much less unwanted target-independent activation, exhaustion, and fratricide than symmetric mAbs.

[0103] Table 2: Exemplary sequences of epitope moieties and exemplary heterodimeric molecules for use with “universal CAR” or alternative CAR systems

[0104] Table 3: Sequences of P2X7 and epitope moieties thereofTable 4: exemplary sequences of symmetrical molecules (used as controls)Detailed description of the embodiments

[0105] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0106] One skilled in the art will recognise 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.

[0107] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0108] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0109] The present invention is based on the recognition by the inventors that unwanted toxicity may arise using molecules previously described in the art as being useful for bringing together CAR T cells and target cells (such as cancer cells). Such molecules are typically referred to as “bridging molecules” or “adapter molecules” in the art. More specifically, the inventors have recognised that symmetrical bridging molecules having more than one moiety for binding to receptors on immune cells (such as CARs on CAR T cells), can elicit chronic activation, exhaustion, and induction of apoptosis. Without wishing to be bound by theory, the inventors believe this is the result of transactivation of different CAR receptors on individual CAR T cells leading to signalling without a target cell being present and / or the bridging molecules engaging with two CAR T cells simultaneously, and thereby eliciting target independent toxicity and fratricide.

[0110] The inventors have surprisingly found that the optimal architecture for a molecule for engaging both CAR T cells and cancer cells, is one in which the moieties for binding to the CAR T cells are present only on a single “arm” of the bridging molecule (for example as depicted in Figure2B) and preferably, wherein the moiety is linked or joined to the Fab portion of the bridging molecule (as distinct from linking to the constant chain of an antibody). In other words, the preferred architecture of a bridging molecule is one which minimises or prevents the likelihood of the molecule binding to two independent CAR receptors on a single CAR T cell, and / or to two or more individual CAR T cells simultaneously.

[0111] The inventors believe that bridging molecules having this optimal architecture:1 . Show similar potency like a symmetric antibody-based bridging molecules2. Have similar pharmacokinetics to a symmetric mAb, but3. Have much less unwanted target-independent activation, exhaustion and fratricide compared to symmetric mAb-based bridging molecules.

[0112] The findings of the inventors have implications for a broad range of CAR technologies, including in indirect CAR technologies and technologies which “redirect” CAR T cells, as the ongoing toxicity of the adapter molecule using the symmetric mAbs compromises the quality of the CAR T cell product with regard to i) increased unwanted toxicity for the patient, ii) increased toxicity on the CAR T cell product.Definitions

[0113] Unless defined otherwise, 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.

[0114] For purposes of interpreting this specification, the following definitions will generally apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.

[0115] "P2X? receptor" generally refers to a purinergic receptor formed from three protein subunits or monomers, with at least one of the monomers having an amino acid sequence substantially as shown in SEQ ID NO: 152 below:MPACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVIIFSYVCFALVSDKLYQRKEPVIS SVHTKVKGIAEVKEEIVENGVKKLVHSVFDTADYTFPLQGNSFFVMTNFLKTEGQEQRL CPEYPTRRTLCSSDRGCKKGWMDPQSKGIQTGRCVVYEGNQKTCEVSAWCPIEAVE EAPRPALLNSAENFTVLIKNNIDFPGHNYTTRNILPGLNITCTFHKTQNPQCPIFRLGDIF RETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNF RYAKYYKENNVEKRTLIKVFGIRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLID TYSSNCCRSHIYPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESHIRMVNQ QLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRD SPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQ EPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQ YSGFKSPY

[0116] To the extent that P2X? receptor is formed from three monomers, it is a "trimer" or "trimeric". "P2X? receptor" encompasses naturally occurring variants of P2X? receptor,e.g., wherein the P2X? monomers are splice variants, allelic variants, SNPs and isoforms including naturally-occurring truncated or secreted forms of the monomers forming the P2X? receptor (e.g., a form consisting of the extracellular domain sequence or truncated form of it), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants. In certain embodiments of the invention, the native sequence P2X? monomeric polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequence shown in SEQ ID NO: 152. In certain embodiments the P2X? receptor may have an amino acid sequence that is modified, for example various of the amino acids in the sequence shown in SEQ ID NO: 152 may be substituted, deleted, or a residue may be inserted.

[0117] " Functional P2X? receptor" generally refers to a form of the P2X? receptor having three intact binding sites or clefts for binding to ATP. When bound to ATP, the functional receptor forms a non-selective sodium / calcium channel that converts to a porelike structure that enables the ingress of calcium ions and molecules of up to 900 Da into the cytosol, one consequence of which may be induction of programmed cell death. In normal homeostasis, expression of functional P2X? receptors is generally limited to cells that undergo programmed cell death such as thymocytes, dendritic cells, lymphocytes, macrophages and monocytes. There may also be some expression of functional P2X? receptors on erythrocytes and other cell types.

[0118] "Dysfunctional P2X? receptor" generally refers to a form of a P2X? receptor having a conformation, distinct from functional P2X?, whereby the receptor is unable to form an apoptotic pore, but which is still able to operate as a non-selective channel through the maintenance of a single functional ATP binding site located between adjacent monomers. One example arises where one or more of the monomers has a cis isomerisation at Pro210 (according to SEQ ID NO: 152). The isomerisation may arise from any molecular event that leads to misfolding of the monomer, including for example, mutation of monomer primary sequence or abnormal post translational processing. One consequence of the isomerisation is that the receptor is unable to bind to ATP at one, or more particularly two, ATP binding sites on the trimer and as a consequence not be able to extend the opening of the channel. In the circumstances, the receptor cannot form a pore and this limits the extent to which calcium ions may enter the cytosol. Dysfunctional P2X? receptors are expressed on a wide range of epithelial and haematopoietic cancers.As used herein, the term “dysfunctional P2X? receptors” may be used interchangeably with the term “non-functional P2X? receptors” or “nfP2X? receptors”.

[0119] "Cancer associated-P2X? receptors" are generally P2X? receptors that are found on cancer cells (including, pre-neoplastic, neoplastic, malignant, benign or metastatic cells), but not on non-cancer or normal cells.

[0120] "E200 epitope" generally refers to an epitope having the sequence GHNYTTRNILPGLNITC and variants thereof (e.g. SEQ ID NOs: 153 to 162 and 166 to 218).

[0121] "E300 epitope" generally refers to an epitope having the sequence KYYKENNVEKRTLIK and variants thereof (SEQ ID NOs: 163 and 164).

[0122] A "composite epitope" generally refers to an epitope that is formed from the juxtaposition of the E200 and E300 epitopes or parts of these epitopes. An example of a composite epitope comprising E200 and E300 epitopes is GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 165).

[0123] "Antibodies" or "immunoglobulins" or "Igs" are gamma globulin proteins that are found in blood, or other bodily fluids of vertebrates that function in the immune system to bind antigen, hence identifying and / or neutralising foreign objects.

[0124] Antibodies are generally a heterotetrameric glycoprotein 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.

[0125] H and L chains define specific Ig domains. More particularly, each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and £ isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1 ).

[0126] Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designateda, 6, s, Y, and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in % sequence and function, e.g., humans express the following subclasses: lgG1 , lgG2, lgG3, lgG4, IgAI, and lgA2. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0127] The constant domain includes the Fc portion that comprises the carboxyterminal portions of both H chains held together by disulfides. The effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which region is also the part recognised by Fc receptors (FcR) found on certain types of cells.

[0128] The pairing of a VH and VL together forms a "variable region" or "variable domain" including the amino -terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." The V domain contains an "antigen binding site" that affects antigen binding and defines specificity of a particular antibody for its particular antigen. V regions span about 1 10 amino acid residues and consist of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3) that are each generally 9-12 amino acids long. The FRs largely adopt a [3-sheet configuration and the hypervariable regions form loops connecting, and in some cases forming part of, the [3-sheet structure.

[0129] "Hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3).

[0130] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues herein defined.

[0131] An "antigen binding site" generally refers to a molecule that includes at least the hypervariable and framework regions that are required for imparting antigen binding function to a V domain. An antigen binding site may be in the form of an antibody or an antibody fragment, (such as a mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab, Fd, SD- Fv, Fv, F(ab')2 or scFv) in a method described herein.

[0132] An "intact" or "whole" antibody is one that comprises an antigen-binding site as well as a CL 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 variant thereof.

[0133] "Whole antibody fragments including a variable domain" include SD-mAb, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0134] The "Fab fragment" consists of an entire L chain along with the variable region domain of the H chain (VH), 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 antigenbinding site.

[0135] A "Fab' fragment" differs 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.

[0136] A "F(ab')2 fragment" roughly corresponds to two disulphide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.

[0137] An "Fv" is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association.

[0138] 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 analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody.

[0139] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected to form a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker betweenthe VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0140] A "single variable domain" is half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognise and bind antigen, although generally at a lower affinity than the entire binding site.

[0141] "Diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). The small antibody fragments are prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that interchain but not intrachain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.

[0142] Diabodies may be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0143] An "isolated antibody" is one that has been identified and separated and / or recovered from a component of its pre-existing environment. Contaminant components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0144] A "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human. Human antibodies can be produced using various techniques known in the art, including phage - display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

[0145] "Humanised' forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanised 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 capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanised antibody will 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 a human immunoglobulin sequence. The humanised antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0146] "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, the monoclonal antibodies are advantageous in that they may be synthesised uncontaminated by other antibodies. Monoclonal antibodies may be prepared by the hybridoma methodology. The "monoclonal antibodies" may also be isolated from phage antibody libraries using molecular engineering techniques.

[0147] The term "anti-R2X? receptor antibody" or "an antibody that binds to P2X? receptor" refers to an antibody that is capable of binding P2X? receptor with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting P2X? receptor, typically non-functional P2X? receptor or a cancer associated P2X? receptor. Preferably, the extent of binding of a P2X? receptor antibody to an unrelated protein is less than about 10% of the binding of the antibody to P2X? receptor as measured, e.g., by a radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Biacore or Flow Cytometry. In certain embodiments, an antibody that binds to P2X? receptor has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. An anti nfP2X7 receptor antibody is generally one having some or all of these serological characteristics and that binds to dysfunctional P2X7 receptors but not to functional P2X7 receptors.

[0148] An "affinity matured' antibody is one with one or more alterations in one or more hypervariable regions thereof that result in an improvement in the affinity of the antibody for the antigen, compared to a parent antibody that does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art.

[0149] A "blocking" antibody" or an "antagonist" antibody is one that inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0150] An "agonist antibody", as used herein, is an antibody, which mimics at least one of the functional activities of a polypeptide of interest.

[0151] "Binding affinity" generally refers to the strength of the sum total 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 indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0152] As used herein, the term "antigen" is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates, and combinations thereof, for example a glycosylated protein or a glycolipid. The term "antigen" as used herein refers to a molecular entity that may be expressed on a target cell and that can be recognised by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0153] "Epitope" generally refers to that part 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 a primary protein structure. In certain embodiments, the epitope is a "conformational epitope" i.e. one in which the hypervariable loops of the CDRs bind to residues as they are presented in the tertiary or quaternary protein structure.

[0154] The term "target cell" as used herein refers to a cell that expresses a cell surface molecule to which the targeting moiety of the bridging molecule binds. The target cell may be a cancer cell or any other diseased cell.

[0155] The term "disorder" or “condition” means a functional abnormality or disturbance in a subject such as a cancer, an autoimmune disorder, or an infection by virus, bacteria, parasite, or others.

[0156] For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide 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, in a host cell.

[0157] As used herein, the term "subject" refers to a mammal such as mouse, rat, cow, pig, goat, chicken, dog, monkey or human. Preferentially, the subject is a human. The subject may be a subject suffering from a disorder such as cancer (a patient), but the subject also may be a healthy subject.

[0158] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later re-introduced.

[0159] The term "allogeneic" as used herein refers to any material derived from a different subject of the same species as the subject to whom the material is re-introduced.

[0160] The terms "therapeutically effective amount" or "therapeutically effective population" mean an amount of, for example, a cell population that provides a therapeutic benefit in a subject.

[0161] The terms "binds to", “specifically binds to” or "specific for" with respect to a targeting moiety, as used e.g. in the bridging molecule as disclosed herein, or of a CAR referring to an antigen-binding domain that recognises and binds to a specific antigen,does not substantially recognise or bind to other molecules in a sample. An antigenbinding domain or targeting moiety that binds specifically to an antigen from one species also may bind to that antigen from another species. This cross-species reactivity is typical of many antibodies and therefore not contrary to the definition that the antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may bind also 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 not contrary to the definition that the antigen-binding domain is specific.

[0162] The terms "engineered cell" and "genetically modified cell" as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acid sequence that in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to the fact that cells, preferentially immune cells, can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins that are not expressed in these cells in the natural state. For example, immune cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface. For example, the CAR sequences may be delivered into cells using an adenoviral, adeno-associated viral (AAV)-based, retroviral or lentiviral vector 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. The gene delivery may be in the form of mRNA (transient) or DNA (transient or permanent).

[0163] The terms "immune cell" or "immune effector cell" refer to a cell that may be part of the immune system and executes a particular effector function such as alpha-beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes or macrophages or any hematopoietic progenitor cells such as pluripotent stem cells and early progenitor subsets that may mature or differentiate into somatic cells. The cells may be naturally occurring or generated by cytokine exposure, artificial / genetically modified cells (such as iPSCs and other artificial cell types). The immune cell may be an artificial cell subset including induced pluripotent stem cells and cells maturated therefrom. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta Tcells, NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells. "Effector function" means a specialised function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper cell activity including the secretion of cytokines.

[0164] The term "treat" (treatment of) a disorder as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0165] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.Exogenous immune cell receptors (eg: CARs)General

[0166] The present invention finds utility in the context of redirecting immune cells expressing exogenous receptors (such as chimeric antigen receptors, or CARs), towards a target cell. In its simplest form, the invention relates to use of a bridging molecule as described herein for bringing a cytotoxic immune cell (such as a T cell and especially a CAR T cell), into proximity with a target cell (such as a cancer cell). The bridging molecules of the invention may enable the redirection of a CAR T cell to a target cell that comprises a different antigen than is recognised by the CAR, or may increase the binding affinity of the CAR to the target cell.

[0167] The skilled person will appreciate that the exogenous immune cell receptors contemplated for use in accordance with the invention are typically exogenous receptors (ie do not normally occur on the surface of the immune cell or are recombination / heterologous), which typically comprise an antigen binding (or recognition) domain and a signalling domain which is stimulated to activate the cytotoxic activity of the cell upon binding of the antigen binding domain. Further contemplated in accordance with the invention is the use of “switch” or “tag” based so-called universal CAR systems where the CAR does not necessarily comprise an antigen binding domain, but another means for engaging with a target cell, via an intermediate molecule.

[0168] The terms cancer- or tumour-associated or tumour-specific CAR targeting refers to the use of a CAR T cell for binding to a target antigen that is presented on the cell surface of tumour cells, but is not typically found on the surface of a healthy cell. In otherwords, normal cells under normal circumstances may be characterised by low expression or the absence of the target antigen on the extracellular membrane (and therefore the presence of the antigen on the cell surface cannot be detected). In still further examples, such cells may express mRNA encoding the antigen at an intracellular level. As CAR T cells only recognize surface-expressed antigens, the intracellular expression of the targeted proteins will not lead to CAR engagement. Alternatively (and particularly in the case of tumour-associated CAR targeting), the CAR may be for binding to a target antigen that is overexpressed by cancer cells compared to healthy cells.

[0169] One example of a surface-expressed epitope on cancer cells is the P2X? receptor. The targeted epitopes E200 and E300 of the P2X? receptor are not exposed on the form of the receptor found in healthy tissue and thus these epitopes can be regarded as cancer specific. In other words, the E200 and E300 epitopes are only exposed, and available for binding when the P2X? receptor has an altered non-functional conformation, such as occurs in the context of cancer (in which case the receptor may be referred to as nfP2X? receptor). Another example of a cancer-specific targeted epitope may be derived from the splice variant EGFRvlll. Still another example is the antigen CLDN6 which is mostly restricted to embryonic and foetal life and has very limited expression in healthy cells after the early phase in life and may be regarded as highly restricted and relatively overexpressed in cancer. The present invention contemplates the binding to any such tumour-specific or tumour-associated antigen, including P2X?, EGFRvlll and CLDN6 for cancer-specific targeting and engaging CAR T cells via the bridging molecules described herein to cancer-associated antigens.

[0170] The present invention also contemplates the binding of a CAR to a tumour- associated antigen. Tumour associated antigens are known in the art (and are typically associated with increased expression in the context of cancer).

[0171] In general, a CAR may comprise an extracellular domain (extracellular part) comprising the binding domain (also referred to as an antigen binding domain or antigen recognition domain, or tag-binding domain in the context of a universal CAR), a transmembrane domain and an intracellular signaling domain.

[0172] Generally, an "antigen recognition domain" refers to the region of the CAR that specifically binds to an antigen (and thereby is able to target a cell containing the antigen). The CARs of the invention may comprise one or more antigen binding domains.Generally, the targeting regions on the CAR are extracellular. The antigen binding domain may comprise an antibody or an antibody binding fragment thereof. The antigen binding domain may comprise, for example, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the "(GyS s-linker" and variations thereof but the skilled person will appreciate that various linker sequences and formats may be used.

[0173] In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will be used in. For example, when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanised antibody or antigen binding fragment thereof. Human or humanised antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art. The CAR as disclosed herein has an extracellular linker / label epitope binding domain as an antigen binding domain allowing it to bind indirectly via a target cell binding molecule as disclosed herein to an antigen expressed on a target cell.

[0174] The extracellular domain of a CAR may be linked to the transmembrane domain by a linker. The extracellular domain may also comprise a signal peptide. The extracellular part of the CAR for use according to the present invention, preferably comprises a tumour-associated antigen binding domain. For example, the antigen may be any one described herein, including P2X?, EGFRvlll or CLDN6.

[0175] The antigen binding domain may be a P2X? binding domain that recognises the E200 (or E300 or E200-300 composite) epitope as disclosed herein. Specifically, the CAR as disclosed herein has an extracellular nfP2X? E200 (or E300 or E200-300 composite) binding domain as an antigen binding domain. Alternatively, the antigen binding domain may be an EGFRvlll binding domain that recognises an epitope resulting out of the fusion of the amino acid sequence starting at position 25-29 LEEKK (SEQ ID NO: 280), followed by the insertion of G and the subsequent amino acid sequence 298-304 NYVVTDH (SEQ ID NO: 281 ), the total epitope is a 13-mer comprised of the sequence LEEKKGNYVVTDH (SEQ ID NO: 134). Alternatively, the antigen binding domain may be a CLDN6 bindingdomain that recognises an epitope in the second extracellular domain of CLDN6 [UniProtKB: P56747 (CLDN6_HUMAN)] via the amino acid sequence of SEQ ID NO: 143.

[0176] Typically, the antigen-recognition domain includes a binding polypeptide that includes amino acid sequence homology to one or more complementarity determining regions (CDRs) of an antibody that binds to a tumour-specific or tumour-associated antigen (such as a dysfunctional P2X? receptor, EGFRvlll or CLDN6). In any embodiment, the binding polypeptide includes amino acid sequence homology to the CDR1 , 2 and 3 domains of the VH and / or VL chain of an antibody that binds to a tumourspecific or tumour-associated antigen (such as a dysfunctional P2X? receptor, EGFRvlll or CLDN6).

[0177] In particularly preferred embodiments of the invention, the antigen-recognition domain of the CAR binds to an epitope of the antigen nfP2X?. In such embodiments, the binding polypeptide comprises the amino acid sequence of the CDRs of the VH and / or VL chain of an antibody described in any one of: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531 ,171 , US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2007 / 001540 (or in corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in corresponding US publication US 2010-0036101 ), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181 ,320, US 9,944,701 or US 10,597,451 ), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 201 1 / 020155, US 9,127,059, US 9,688,771 , or US 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609) the entire contents of which are hereby incorporated by reference. Preferably the binding polypeptide comprises the amino acid sequence of the CDRs of the VH and / or VL chain of antibody 2-2-1 described in PCT / AU2010 / 001070 (or in any one of the corresponding US patents US 9,127,059, US 9,688,771 , or US 10,053,508) or BPM09 described in PCT / AU2007 / 001541 (or in corresponding US publication US 2010- 0036101 ) and produced by the hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under Accession no. 06080101 , W02013185010A1 or WO201 9056023. Alternatively, the binding polypeptide of the CAR may comprise theamino acid sequences of the CDRs of the antibody sdAbs 2-2-3, 2-472-2, or 2-2-12 described in WO 2017 / 041 143 (also published as US 2019 / 0365805), and WO 2019 / 222796 (corresponding to US application 17 / 057,060), incorporated herein by reference.

[0178] The binding polypeptide of the CAR may comprise the amino acid sequence of the VH and / or VL chains of an antibody described in any one of: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531 ,171 , US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2007 / 001540 (or in corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in corresponding US publication US 2010-0036101 ), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181 ,320, US 9,944,701 or US 10,597,451 ), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 201 1 / 020155, US 9,127,059, US 9,688,771 , or US 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609) the entire contents of which are hereby incorporated by reference. Preferably the binding polypeptide comprises the amino acid sequence of the VH and / or VL chains of the antibody 2-2-1 described in PCT / AU2010 / 001070 (or in any one of the corresponding US patents US 9,127,059, US 9,688,771 , or US 10,053,508) or BPM09 described in PCT / AU2007 / 001541 (or in corresponding US publication US 2010- 0036101 ) and produced by the hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under Accession no. 06080101 , W02013185010A1 or WO201 9056023. Alternatively, the binding polypeptide of the CAR may comprise the amino acid sequences of the VH and / or VL chains of the antibody sdAbs 2-2-3, 2-472-2, or 2-2-12 described in WO 2017 / 041 143 (also published as US 2019 / 0365805), and WO 2019 / 222796 (corresponding to US application 17 / 057,060), incorporated herein by reference.

[0179] The binding polypeptide of the CAR may comprise the amino acid sequence of an antibody or fragment thereof described in any one of: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531 ,171 , US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, orUS 10,450,380), PCT / AU2007 / 001540 (or in corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in corresponding US publication US 2010-0036101 ), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181 ,320, US 9,944,701 or US 10,597,451 ), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 201 1 / 020155, US 9,127,059, US 9,688,771 , or US 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609) the entire contents of which are hereby incorporated by reference. Preferably the binding polypeptide comprises the amino acid sequence of sdAb 2-2-1 described in PCT / AU2010 / 001070 (or in any one of the corresponding US patents US 9,127,059, US 9,688,771 , or US 10,053,508) or antibody BPM09 described in PCT / AU2007 / 001541 (or in corresponding US publication US 2010-0036101 ) and produced by the hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under Accession no. 06080101 , W02013185010A1 or WO2019056023. Alternatively, the binding polypeptide may comprise the amino acid sequences of sdAbs 2-2-3, 2-472-2, or 2-2-12 described in WO 2017 / 041 143 (also published as US 2019 / 0365805), and WO 2019 / 222796 (corresponding to US application 17 / 057,060), incorporated herein by reference.Universal CAR systems

[0180] It will be appreciated that the methods of the present invention also find utility in the context of immune cells comprising so-called universal CARs or “switch CARs”. Such “CARs” are intended for similar use as conventional CARs (eg: for expression on immune cells and for killing of target cells), but do not comprise antigen-binding domains for directly binding to antigens on target cells. Rather, these exogenous receptors may comprise binding domains for binding to an intermediate (tag or switch) or neo-epitope which is bound and thereby indirectly enables direction (or redirection) of the immune cells expressing the receptor, to a target cell.

[0181] In certain embodiments, the receptor is capable of binding to a neo-epitope or exogenous epitope or peptide “tag”, such as a dye (eg fluroscein isothiocyanate, FITC), biotin moiety, “AZip” tag (or Leucine zipper moiety), “Latch with Blm peptide”, “SpyTag”, the 5B9 tag (ie comprising 10 amino acids derived from the human nuclear autoantigenLa / SS-B) and the PNE tag (ie a 14 amino acid peptide neo-epitope derived from a yeast transcription factor) and D-Domain based Universal CAR T (eg ARC-SparX). In the art, such neo-epitopes or tags may also be referred to as “switch elements” in the context of so-called “Universal CAR” systems. The skilled person will appreciate that such neoepitopes are not typically epitopes associated with cancer antigens.Other portions of CARs and Universal CARs

[0182] A sequence encoding a CAR may also encode a "signal peptide" which refers to a peptide sequence that directs the transport and localisation of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0183] "Spacer" or "hinge" as used herein refers to the hydrophilic region that is between the antigen binding domain and the transmembrane domain. The CARs of the invention may comprise an extracellular spacer domain but it is also possible to leave out such a spacer. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge or CD28 hinge.

[0184] The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such a domain. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived for example from CD8alpha or CD28. When the key signalling and antigen recognition modules (domains) are on two (or even more) polypeptides, then the CAR may have two (or more) transmembrane domains. The splitting of key signalling and antigen recognition modules enables small moleculedependent, titratable and reversible control over CAR cell expression (Wu et al, 2015, Science 350: 293-303) due to small molecule-dependent heterodimerising domains in each polypeptide of the CAR.

[0185] The cytoplasmic domain (or the intracellular signaling domain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" means a specialised function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper cell activityincluding the secretion of cytokines. The intracellular signalling domain refers to the part of a protein that transduces the effector function signal and directs the cell expressing the CAR to perform a specialised function. The intracellular signalling domain may include any complete, mutated or truncated part of the intracellular signalling domain of a given protein sufficient to transduce a signal that initiates or blocks immune cell effector functions.

[0186] The function of the intracellular domains may be pro- or anti-inflammatory and / or immunomodulatory, or a combination of such.

[0187] Prominent examples of intracellular signalling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and coreceptors that initiate signal transduction following antigen receptor engagement.

[0188] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signalling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signalling sequences) and secondly those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signalling sequences, co-stimulatory signalling domain). Therefore, an intracellular signalling domain of a CAR may comprise one or more primary cytoplasmic signalling domains and / or one or more secondary cytoplasmic signalling domains.

[0189] Primary cytoplasmic signalling sequences that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs) signalling motifs.

[0190] Examples of ITAM containing primary cytoplasmic signalling sequences often used in CARs are those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. Most prominent is the sequence derived from CD3 zeta.

[0191] The cytoplasmic domain of the CAR may be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a co- stimulatory signalling region. The co-stimulatory signalling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that isrequired for an efficient response of lymphocytes to an antigen. Examples for a costimulatory molecule are CD27, CD28, 4-1 BB (CD137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function-associated antigen- 1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C and B7- H3.

[0192] The cytoplasmic signalling sequences within the cytoplasmic signalling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo-or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

[0193] As an example, the cytoplasmic domain may comprise the signalling domain of CD3-zeta and the signalling domain of CD28. In another example the cytoplasmic domain may comprise the signalling domain of CD3-zeta and the signalling domain of CD27. In a further example, the cytoplasmic domain may comprise the signalling domain of CD3- zeta, the signalling domain of CD28, and the signalling domain of CD27.

[0194] As aforementioned, either the extracellular part or the transmembrane domain or the cytoplasmic domain of a CAR may also comprise a heterodimerising domain for the aim of splitting key signalling and antigen recognition modules of the CAR.

[0195] Non-limiting examples of CARs that may be used in accordance with the present invention are provided in WO 2017 / 041 143 (also published as US 2019 / 0365805), and WO 2019 / 222796 (corresponding to US application 17 / 057,060), and WO 2022 / 187906, incorporated herein by reference.

[0196] A CAR for use in accordance with the present invention, i.e. a CAR comprising an nfP2X? E200 binding domain, may be designed to comprise any portion or part of the above-mentioned domains as described herein in any order and / or combination resulting in a functional CAR.

[0197] The CARs as disclosed herein, or polypeptide(s) derived therefrom, nucleic acid molecule(s) or recombinant expression vectors cells encoding said CARs, or populations of cells expressing said CARs, may be isolated and / or purified. The term "isolated" means altered or removed from the natural state. For example, an isolated population of cells means an enrichment of such cells and separation from other cells that are normally associated in their naturally occurring state with said isolated cells. An isolated population of cells means a population of substantially purified cells that are a more homogenouspopulation of cells than found in nature. Preferably, the enriched cell population comprises at least about 90% of the selected cell type. In particular aspects, the cell population comprises at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of the selected cell type.

[0198] The affinity at which the P2X? receptor binding domain of the CAR binds to the E200 moiety of the bridging molecule can vary, but generally the binding affinity may be in the range of 100 pM, 1 nM, 10 nM, or 100 nM, preferably at least about 1 pM or 10 pM, even more preferably at least about 100 pM.

[0199] CAR T cells targeted to EGFRvlll may be used to treat solid cancers. EGFRvlll is a frequent splice variant of EGFR skipping exons 2-7. EGFRvlll is relatively tumour specific and does not occur in healthy cells as EGFR is tightly regulated in normal cells. EGFRvlll is commonly expressed in glioblastoma but also in breast cancer and head and neck cancer. The EGFRvlll-CAR T may be targeted to the epitope resulting out of the fusion of the amino acid sequence starting at position 25-29 LEEKK (SEQ ID NO: 280), followed by the insertion of G and the subsequent amino acid sequence 298-304 NYVVTDH (SEQ ID NO: 281 ) the total epitope comprises or consists of the sequence LEEKKGNYVVTDH (SEQ ID NO: 134). The complete EGFR sequence is found at UniProtKB - P00533 (EGFR_HUMAN) and the complete protein counts 1210 amino acids in isoform 1 . An exemplary anti-EGFRvlll-CAR sequence is set forth in SEQ ID NO: 285.

[0200] EGFRvlll targeted CAR T cells may be used to treat glioblastoma in a conventional way to target EGFRvlll on cancer cells, but may also be redirected to other cancer-associated target antigens via the bridging molecules described herein if the EGFRvlll epitope moiety is integrated into the sequence of bridging molecules. The EGFRvlll CAR T cells then can be used in the same manner as outlined for the nfP2X? CAR targeted approach described herein. The peptide tag may be the 13-mer peptide LEEKKGNYVVTDH or a shortened or extended natural or artificial variant thereof, of SEQ ID NO: 134.

[0201] The amino acid sequence of EGFRvlll CAR compatible bridging molecules targeted to CD19 and CD33 are described in Table 2 as SEQ ID NO: 135 and 136; 137 and 138; and SEQ ID NOs: 139 and 140; 141 and 142, respectively.

[0202] CLDN6 targeted CAR T cells may be used to treat solid cancers e.g. ovarian cancer. The CLDN6-CAR T may be targeted to the second extracellular domain of CLDN6 [UniProtKB - P56747 (CLD6_HUMAN] cells directly via the amino acid sequence [ECD2, >sp|P56747|138-160 WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 143)] but may also be redirected to other cancer-associated target antigens, e.g. CD33 or CD19 via the bridging molecules described herein, if the CLDN6 epitope moiety is integrated into the sequence of bridging molecules. The CLDN6 CAR T cells then can be used in the same manner as outlined for the nfP2X7 CAR targeted approach described herein. The peptide tag may be the 23-mer peptide WTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 143) or a shortened or extended natural or artificial variant thereof. An exemplary anti-CLDN-CAR sequence is set forth in SEQ ID NO: 284.

[0203] The amino acid sequence of CLDN6 CAR compatible bridging molecules targeted to CD19 and CD33 are described in Table 2 as SEQ ID NO: 144 and 145; and SEQ ID NOs: 148 and 149; 150 and 151 , respectively.Bridging molecule (heterodimeric molecule)

[0204] It will be appreciated that the bridging molecule of the invention is typically in the form of an antibody (eg two “Fab” arms” and optionally one or more Fc regions from an antibody), and wherein the moieties capable of being bound by an exogenous immune receptor are present on any region of a single Fab arm. Examples of suitable architectures are depicted in Figure 3.

[0205] Any suitable linker may be used for linking the moieties for binding by the immune cell receptor, to the Fab arm. The linker may comprise a polypeptide, a peptide or a chemical group.

[0206] A 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, e.g., a peptide bond, formed between two moieties. Accordingly, in the context of the present invention the linker may have a length of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. For example, the herein provided bridging molecule may comprise a linker between each moiety and the Fab arm. Such linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein fold independently and behave as expected.

[0207] The skilled person will be familiar with the design and use of various peptide linkers comprised of various amino acids, and of various lengths, which would be suitable for use as linkers in accordance with the present invention. The linker may comprise various combinations of repeated amino acid sequences. The linker may be a flexible linker (such as those comprising repeats of glycine and serine residues), a rigid linker (such as those comprising glutamic acid and lysine residues, flanking alanine repeats) and / or a cleavable linker (such as sequences that are susceptible by protease cleavage).

[0208] The peptide linker may be any one or more repeats of Gly-Ser (GS), Gly-Gly- Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 315) or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 316) or variations thereof. In any embodiment, the linker may comprise or consist of the sequence GGGGSGGGGSGGGGS, i.e. (G4S)3 (SEQ ID NO: 146).

[0209] In any embodiment, the peptide linker can include the amino acid sequence GGGGGS (a linker of 6 amino acids in length) or even longer. The linker may 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 may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 317) or longer (GS)n or longer. It will be appreciated that n can be any number including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more.

[0210] In further embodiments, the linker may comprise inclusion of an amino acid that provides rigidity, such as lysine. For example, in certain embodiments, the linker region may also comprise the sequence GSGK (SEQ ID NO: 318).

[0211] The peptide linker may consist of a series of repeats of Thr-Pro (TP) comprising one or more additional amino acids N and C terminal to the repeat sequence. For example, the linker may comprise or consist of the sequence GTPTPTPTPTGEF (also known as the TP5 linker; SEQ ID NO: 147). In further aspects, the linker may be a short and / or alpha-helical rigid linker (e.g. A(EAAAK)3A, SEQ ID NO: 319; PAPAP, SEQ ID NO: 320; or a dipeptide such as LE or CC).

[0212] In further embodiments, as an alternative or in addition to a glycine-serine- based linker region as described above, the targeting moiety may be in the form of a fusion protein in which the targeting moiety is linked to the tumour-specific or tumour- associated antigen epitope moiety, preferably dysfunctional P2X? receptor epitope moiety, via a hinge region. The linking between the targeting moiety and tumour-specificor tumour-associated antigen epitope moiety may comprise a combination of hinge region and linker regions.

[0213] Examples of suitable hinge regions include hinge regions derived from immunoglobulins. The hinge region may be derived from an lgG1 , lgG2, lgG3 or lgG4, and may comprise one or more amino acid substitutions, (for example to prevent or reduce the likelihood of disulphide bridge formation). Alternative hinge sequences may be derived from alternative immunoglobulin domains, CD8A, CD8B, CD4 or CD28, TRAC, TRBC, TRGC, TRDC.

[0214] Table 5 below provides non-limiting examples of suitable hinge regions for use in joining the targeting moiety and tumour-specific / tumour-associated antigen epitope moiety, in the bridging molecules of the invention.

[0215] It will be appreciated that the targeting moiety may be joined to the tumour- specific / tumour-associated antigen epitope moiety by more than one linker and / or more than one hinge region. For example, the fusion protein may comprise (N to C terminus), the tumour-specific / tumour-associated antigen epitope moiety, conjugated directly to the targeting moiety. Alternatively, the fusion protein may comprise the tumour-specific / tumour-associated antigen epitope moiety, followed by a linker region, then the targeting moiety. Further still, the fusion protein may comprise the tumour-specific / tumour- associated antigen epitope moiety, followed by a linker region, then a hinge region, and then the targeting moiety. In a further embodiment still, the fusion protein may comprise the tumour-specific antigen epitope moiety, followed by a linker region, then a hinge region, a further linker region, then the targeting moiety.

[0216] Table 5: exemplary hinge regionsFab regions of the bridging molecule

[0217] The Fab regions of the bridging molecule of the invention preferably bind to a cell surface molecule on a target cell. The cell surface molecule may comprise 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-bound peptide, or a combination thereof. The cell surface molecule may comprise parts (e.g., coats, capsules, cell walls, flagella, fimbriae, and toxins) of bacteria, viruses, and other microorganisms. The cell surface molecule may be expressed by the target cell. 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 that is not the target cell and that is bound to the target cell or a cell surface molecule of the target cell. Also, by non-limiting example, the cell surface molecule may be a toxin, exogenous molecule or viral protein that is bound to a cell surface or cell surface receptor of the target cell.

[0218] In preferred embodiments, the Fab regions of the bridging molecules of the invention are for binding to antigens on the surface of cancer cells.

[0219] The bridging molecules may interact with a plurality of 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 come from an invading organism (e.g. yeast, worm, bacteria, fungus). Further disclosed herein are bridging molecules that interact with a molecule on a non-cell target. The non-cell target may be a virus or aportion thereof. The non-cell target may be a fragment of a cell. The non-cell target may be an extracellular matrix component or protein.

[0220] The target cell may be derived from a tissue. The tissue may be selected from brain, oesophagus, breast, gut, intestine, colon, lung, glia, ovary, uterus, testes, prostate, gastrointestinal tract, bladder, liver, spleen, thymus, bone, fat and skin. The target cell may be derived from one or more endocrine glands. Alternatively, or additionally, the target cell may be derived from one or more endocrine glands. The endocrine gland may be a lymph gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad or pineal gland.

[0221] 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.

[0222] 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, a brain, an oesophagus, a breast, a colon, a lung, a glia, an ovary, a uterus, a testicle, a prostate, a gastrointestinal tract, a bladder, a liver, a 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 a B cell, a T cell, a monocyte, a thrombocyte, a leukocyte, a neutrophil, an eosinophil, a basophil, a lymphocyte, a hematopoietic stem cell or an endothelial cell progenitor. The cancer cell may be derived from a CD19-positive B lymphocyte. 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 gland may be a lymph gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad or pineal gland.

[0223] The cell surface molecule of the target cell may be a receptor. The receptor may be an extracellular receptor. The receptor may be a cell surface receptor. By way of nonlimiting example, the receptor may bind a hormone, a neurotransmitter, a cytokine, a growth factor or a cell recognition molecule. The receptor may be a transmembrane receptor. The receptor may be an enzyme-linked receptor. The receptor may be a G- protein couple receptor (GPCR). The receptor may be a growth factor receptor. By way of non-limiting example, the growth factor receptor may be selected from an epidermal growth factor receptor, a fibroblast growth factor receptor, a platelet derived growth factorreceptor, a nerve growth factor receptor, a transforming growth factor receptor, a bone morphogenic protein growth factor receptor, a hepatocyte growth factor receptor, a vascular endothelial growth factor receptor, a stem cell factor receptor, an insulin growth factor receptor, a somatomedin receptor, an erythropoietin receptor and homologs and fragments thereof. The receptor may be a hormone receptor. The receptor may be an insulin receptor. By way of non-limiting example, the receptor may be selected from an eicosanoid receptor, a prostaglandin receptor, an oestrogen receptor, a follicle stimulating hormone receptor, a progesterone receptor, a growth hormone receptor, a gonadotropinreleasing hormone receptor, 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 luteinising hormone receptor. The cell surface molecule may be at least about 50% homologous to a luteinising hormone receptor. The receptor may be an immune receptor. By way of non-limiting example, the immune receptor may be selected from a pattern recognition receptor, a toll-like receptor, a NOD-like receptor, a killer-activated receptor, a killer inhibitor receptor, an Fc receptor, a B cell receptor, a complement receptor, a chemokine receptor and a cytokine receptor. By way of non- limiting example, the cytokine receptor may be selected from an interleukin receptor, an interferon receptor, a transforming growth factor receptor, a tumour necrosis factor receptor, a colony stimulating factor receptor, 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. By way of non-limiting example, the receptor kinase may activate a signalling protein selected from a Ras, a Raf, a PI3K, a protein kinase A, a protein kinase B, a protein kinase C, an AKT, an AMPK, a phospholipase, homo logs thereof and fragments thereof. The receptor kinase may activate a MAPK / ERK signalling pathway. The receptor kinase may activate Jak, Stat or Smad.

[0224] The cell surface molecule may be a non-receptor cell surface protein. The cell surface molecule may be a cluster of differentiation proteins. By way of non- limiting example, the cell surface molecule may be selected from CD3, CD4, CD8, CD1 1 a, CD1 1 b, CD13, CD14, CD15, CD16, CD22, CD24, CD25, CD30, CD31 , CD33, CD34, CD38, CD45, CD56, CD61 , CD91 , CD1 14, CD1 17, CD182, CD200, fragments thereof, and homologs thereof.

[0225] The cell surface molecule of the target cell may be a molecule that does not comprise a peptide. The cell surface molecule may comprise a lipid. The cell surface molecule may comprise a lipid moiety or a 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.

[0226] The cell surface molecule of the target cell may be an antigen. The antigen may be at least a portion of a surface antigen or a cell surface marker on a cell. The antigen may be a receptor or a co-receptor on a cell. The antigen may refer to a molecule or molecular fragment that may be bound by a major histocompatibility complex (MHC) and presented to a T-cell receptor. The term "antigen" may also refer to an immunogen. The immunogen may provoke an adaptive immune response if injected on its own into a subject. The immunogen may induce an immune response by itself. The antigen may be a superantigen, T-dependent antigen or a T-independent antigen. The antigen may be an exogenous antigen. Exogenous antigens are typically antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. Some antigens may start out as exogenous antigens, and later become endogenous (for example, intracellular viruses). The antigen may be an endogenous antigen. The endogenous antigen may be an antigen that has been generated within cells as a result of normal cell metabolism, or because of pathogenic infections (e.g., viral, bacterial, fungal, parasitic). The antigen may be an autoantigen. The autoantigen may be a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognised by the immune system of patients suffering from a specific autoimmune disease. These antigens should, under normal conditions, not be the target of the immune system, but, due to genetic and / or environmental factors, the normal immunological tolerance for such an antigen is not present in these patients. The antigen may be present or over-expressed due to a condition or disease. The condition or disease may be a cancer or a leukaemia. 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.

[0227] The present invention also may find application for the treatment of specific B- or T-lineage associated autoimmune diseases, for example using anti-idiotypic antibodies or fragments thereof or ligands thereof for targeting the B cell receptor and / or the T cell receptor. Such diseases include myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), solid organ transplant hyperacute,acute, chronic or mix-type rejection, bone marrow or stem cell transplant rejection, and graft versus host disease.

[0228] The cell surface molecule of the target cell may be an antigen that has been designated as a tumour antigen. Tumour antigens or neo-antigens may be antigens that are presented by MHC I or MHC II molecules on the surface of tumour cells. These antigens may sometimes be presented by tumour cells and never by the normal ones. In this case, they are called tumour-specific antigens (TSAs) and, in general, result from a tumour-specific mutation. More common are antigens that are presented by tumour cells and normal cells, and they are called tumour-associated antigens (TAAs). A TAA associated antigen is not unique to a tumour cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumour may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during foetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumour cells. Cytotoxic T lymphocytes that recognise these antigens may be able to destroy the tumour cells before they proliferate or metastasise. Tumour antigens may also be on the surface of the tumour in the form of, for example, a mutated receptor, in which case they may be recognised by B cells.

[0229] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1 , TRP-2 and tumour-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumour-suppressor genes such as p53, Ras, HER-2 / neu; unique tumour antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the 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-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1 , NuMa, K-ras, beta-Catenin, CDK4, Mum-1 , p 15, p 16, 43-9F, 5T4, 791 Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, 20 CD68\PI, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1 , RCASI, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0230] The cell surface molecule of the target cell may be an antigen selected from the group consisting of any surface expressed antigens. Exemplary target antigens may comprise but are not limited to: CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1 ), LeY, FR|3, MICA / B, CD305 (LAIR-1 ), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1 ), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD1 12 (Nectin2), CD1 17 (c-Kit), CD133, CD146 (MCAM), CD155 (PVR), CD171 (L1 CAM), 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 the target cell.

[0231] In preferred embodiments, the target cell is a cancer cell and the cell surface molecule of the cancer cell is an antigen that is associated with the cancer. The antigen may be a tumour-specific antigen or a tumour-associated antigen. The antigen may be one which is associated with a particular type of cancer. For example, overexpression of the antigen may be associated with a specific cancer or specific class of cancer. For example, where the cancer is breast cancer, the antigen may be associated with breast cancer but not with another form of cancer. Alternatively, the antigen may be associated with a class of cancers such as solid tumours, but not associated with haematological (ie “liquid”) tumours, or vice versa. The antigen may be associated with cancers of a particular lineage but not with others. For example the antigen may be associated with sarcomas but not lymphomas or carcinoma. As used herein the term “associated with” in relation to cancer, will be understood to mean that the antigen’s expression (whether increased or decreased) is considered a marker of the cancer. It will be appreciated that there may be low levels of expression of an antigen but this does not equate to the antigen being “associated” with a given cancer.

[0232] Suitable cancer antigens which may be bound by the Fab regions of the bridging molecule (or indeed by the antigen recognition domain of a CAR) 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 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate-binding protein (FBP), foetal acetylcholine receptor (AChR), folate receptor-a and [3 (FRa and [3), Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2 / ERB2), HER3, Epidermal Growth Factor Receptor vlll (EGFRvlll), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1 CAM), melanoma-associated antigen 1 (melanoma antigen family A1 , MAGE-A1 ), Mucin 16 (Muc-16), Mucin 1 (Muc- 1 ), NKG2D ligands, cancer-testis antigen NY-ESO-1 , oncofoetal antigen (h5T4), tumour- associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms’ tumour protein (WT-1 ), type 1 tyrosine-protein kinase transmembrane receptor (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-1 H, HERK-V, IL-1 1 Ra, 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 utilised for targeting by a bridging molecule in the present invention.

[0233] The bridging molecule may be in the form of an immunoglobulin selected from an IgG, an IgA, an IgD, an IgE, an IgM, a fragment thereof or a modification thereof. The immunoglobulin may be IgG. The IgG may be IgG 1 . The IgG may be lgG2. The IgG may be lgG3. The IgG may be lgG4. The IgG may have one or more Fc mutations for modulating endogenous T cell FcR binding to the bridging molecule. The IgG may have one or more Fc mutations for removing the Fc binding capacity to the FcR of FcR-positive cells. The one or more Fc mutations may remove a glycosylation site. The one or more Fc mutations may be selected from E233P, L234V, L235A, delG236, A327G, A330S, P331 S, N297Q and any combination thereof. The one or more Fc mutations may be in lgG1. The one or more Fc mutations in the lgG1 may be L234A, L235A, or both. Alternatively, or additionally, the one or more Fc mutations in the lgG1 may be L234A, L235E, or both. Alternatively, or additionally, the one or more Fc mutations in the lgG1 may be N297A. Alternatively, or additionally, the one or more mutations may be in lgG2. The one or more Fc mutations in the lgG2 may be V234A, V237A, or both.

[0234] The bridging molecule may be an Fc null immunoglobulin or a fragment thereof.

[0235] As used herein, the term "antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered. 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 specifically noted otherwise, statements and claims that use the term "antibody" or "antibodies" may specifically include "antibody fragment" and "antibody fragments."

[0236] The bridging molecule may be human, fully human, humanised, human engineered, non-human, and / or chimeric antibody. The non-human antibody may be humanised to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Chimeric antibodies may refer to antibodies created through the joining of two or more antibody genes that originally encoded for separate antibodies. A chimeric antibody may comprise at least one amino acid from a first antibody and at least one amino acid from a second antibody, wherein 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 antibody may be from cynomolgus monkey.

[0237] The bridging molecule 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, cetaceans, odd-toed ungulates and even-toed ungulates. The mammal may be a human, non-human primate, mouse, sheep, cat, dog, cow, horse, goat, or pig.

[0238] The bridging molecule may recognise or bind an antigen selected from, by nonlimiting example, CD19, Her2, CLL-1 , CD33, EGFRvlll, CD20, CD22, BCMA or a fragment thereof. The antigen may comprise a wild-type antigen. The antigen may comprise one or more mutations.

[0239] A Fab region of the bridging molecule may be an anti-CD19 antibody or a fragment thereof. The bridging molecule may be an anti-CD22 antibody. A Fab region of the bridging molecule may be an anti-BCMA antibody or a fragment thereof. A Fab region of the bridging molecule may be an anti-EGFRvlll antibody or a fragment thereof. A Fab region of the bridging molecule may be an anti-Her2 antibody or a fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD20 antibody or antibody fragment. The bridging molecule may comprise rituximab. A Fab region of the bridging molecule may comprise an anti-EGFR antibody or antibody fragment. A Fab region of the bridging molecule may comprise an anti-CEA antibody or antibody fragment. A Fab region of the bridging molecule may comprise an anti-CLL-1 antibody or antibody fragment. A Fab region of the bridging molecule may comprise an anti-CD33 antibody or antibody fragment. A Fab region of the bridging molecule may comprise an anti-EpCAM antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD30 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD79B antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD37 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD38 antibody or fragment thereof. A Fab region of the bridging molecule le may comprise an anti-CD70 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD276 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-GD2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD371 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD135 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD105 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD123 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-ROR-1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-PD-L1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MET-R antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-PDGFRalpha antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-Her3 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-FRalpha antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-GPC3 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-SLAMf7 antibody or fragment thereof. A Fabregion of the bridging molecule may comprise an anti-TNFRSF1 OB antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-GPNMB antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-VEGFR2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-alpha4beta7 / alphaEbeta7 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CSPG4 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD80 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CCR4 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD1 15 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-ENOX-2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD56 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-huVH1 -69 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD1 17 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD133 antibody or fragment thereof. A Fab region of the bridging molecule comprise an anti-MUC1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MSLN antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-ROR-2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-IL13Ra2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-EPHA2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-EGFRvlll antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-PSMA antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CEA antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-Lewis Y antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-PSCA antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MUC1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD171 L1 CAM antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-EpCAM antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-ALK antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-IGF-1 R CD221 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-Nectin 4 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-FAP antibody or fragment thereof.A Fab region of the bridging molecule may comprise an anti-AXL antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD138 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CLDN6 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-Her4 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-Claudin 18.2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-O-acetylated GD2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-GD3 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-GM2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-TM4SF1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD147 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CEACAM5 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-VEGFR-1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-PDPN antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-WT 1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-GPC2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-FGFR4 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-EphB4 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-STEAP-1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-STEAP-2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MUC1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-chlorotoxin antibody or fragment thereof. A Fab region of the bridging molecule comprise an anti-206 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-ILRAP1 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MICA antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MAGE-A1 scTcR antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MAGE- A1 sTCR antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MICA antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-TRBC1 / 2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-B7-H7 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD34 antibody or fragment thereof.A Fab region of the bridging molecule may comprise an anti-CD7 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-TIM3 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD191 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD66b antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-CD1 1 b antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-EMR2 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-MUC16 antibody or fragment thereof. A Fab region of the bridging molecule may comprise an anti-NYESO-1 HLA-A2 antibody or fragment thereof or a soluble TcR or variant thereof. A Fab region of the bridging molecule may comprise an anti-SURVIVIN HLA-A2 antibody or fragment thereof or a soluble TcR or variant thereof. A Fab region of the bridging molecule may comprise an anti-CD200 antibody or fragment thereof.

[0240] The bridging molecule may comprise or may be derived from any commercially available antibody, such as: from trastuzumab (for binding to Her2), alemtuzumab (for binding CD52), bevacizumab (for binding VEGF-A), brentuximab (for binding CD30, gemtuzumab (for binding CD33), ipilimumab (for binding VTLA-4), ibritumomab (for binding CD20), panitumumab (for binding EGFR), cetuximab (for binding EGFR), rituximab (for binding CD20), and fragments thereof.

[0241] The bridging molecule may be any referred to in Table 1 or 2 or an antigen binding fragment 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.

[0242] It will be well within the purview of the skilled person to be able to determine the appropriate design of a bridging molecule as described herein, including selection of the Fab regions (antigen binding domains) of the bridging molecule and moieties for being bound by the exogenous immune cell receptor.

[0243] More specifically, the skilled person, having knowledge of the CAR for use in the methods (including “redirection” as described herein) would be able to select an alternative targeting moiety to which the CAR should be directed, to facilitate treatment of the cancer or disease requiring treatment.

[0244] As described herein, the Fab region of the bridging molecule may be for binding to any cell surface molecule on a target cell (such as a tumour-associated antigen expressed on a cancer cell). The skilled person can select an appropriate cell surface molecule depending on the disease (eg cancer) requiring treatment. For example, if the disease is a Her2+ cancer, then the skilled person may select a Fab that is capable of specifically binding to Her2 on the cancer cell. Similarly, if the disease is a CD19+ cancer cell, then the skilled person may select a Fab that is capable of specifically binding to CD19 on the cancer. The skilled person will be familiar with methods for determining the antigen expression profile of the cancer or condition to be treated in order to identify suitable surface molecules expressed on the target cell.

[0245] Having identified a suitable cell surface molecule (target antigen) on the target cell (eg: CD19, Her2, CLL-1 , CD33, EGFRvlll, CD20, CD22, BCMA or any other tumour- associated antigen described herein or that may be selected for targeting), the skilled person can readily determine the sequence of a Fab binding domain that can bind to said target antigen, including by reference to commercially available antibodies or to published literature describing antigen binding domains of known antibodies that bind to the antigen. The skilled person can then formulate the structure of a bridging molecule (fusion protein) as described herein, comprising a Fab that binds to the cell surface molecule on a target cell. The composition of the remaining components of the bridging molecule (eg: the tumour-specific antigen epitope moiety) are described further herein.

[0246] Finally, the skilled person, having identified a suitable cell surface molecule (target antigen) on the target cell, and tumour-specific antigen epitope moiety in order to arrive at a bridging molecule of the invention, can readily determine, using routine techniques, whether the bridging molecule: a) binds to the target cell, b) binds to the CAR T cell and c) can successfully redirect the CAR T cell to the target cells to induce cell killing. Methods of determining binding to target antigens, and cytotoxicity are well known in the art. Non-limiting methods and various experimental protocols for determining binding to target cell, CAR T cell and induction of cell killing are described in detail herein in the Examples.P2X? receptor epitope moiety

[0247] The present invention contemplates the use of an antigen epitope moiety in the form of an epitope from dysfunctional P2X? receptor.

[0248] A dysfunctional P2X? receptor epitope moiety may be provided in the form of a dysfunctional P2X? receptor, or a fragment of a dysfunctional P2X? receptor, that has at least one of the three ATP binding sites that are formed at the interface between adjacent correctly packed monomers that are unable to bind ATP. Such receptors are unable to extend the opening of the non-selective calcium channels to apoptotic pores.

[0249] A range of peptide fragments of a dysfunctional P2X? receptor are known and discussed in PCT / AU2002 / 000061 (and in corresponding publications WO 2002 / 057306 and US 7,326,415, US 7,888,473, US 7,531 ,171 , US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2008 / 001364 (and in corresponding publications WO 2009 / 033233 and US 8,440,186, US 9,181 ,320, US 9,944,701 or US 10,597,45) and PCT / AU2009 / 000869 (and in corresponding publications WO 2010 / 000041 and US 8,597,643, US 9,328,155 or US 10,238,716) the contents of all of which are incorporated in entirety. Exemplary peptides within these specifications that include epitopes contemplated for use in this invention are described below.PCT publication Peptide sequenceWO 2002 / 057306 GHNYTTRNILPGLNITC (SEQ ID NO: 153) (also referred to herein as the “E200” epitope)WO 2009 / 033233 KYYKENNVEKRTLIK (SEQ ID NO: 163) (also referred to herein as the “E300” epitope)WO 2010 / 000041 GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 165) (also referred to herein as the “E200 / E300” or “composite” epitope)

[0250] In any embodiment, the amino acid sequence of the P2X? receptor epitope moiety of any bridging molecule described herein, comprises or consists of a sequence as set forth in any of SEQ ID Nos: 155 to 218, or sequences 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 P2X? receptor epitope moiety comprises at least the sequence of SEQ ID NO: 162.

[0251] The P2X? receptor epitope moiety may have any functional chemical group such as a carboxyl group, an active ester, an acetamide or maleimide capable of coupling to a targeting moiety as disclosed herein, for example an antibody or fragment thereof using NH2 or SH groups for coupling thereto.

[0252] Various examples of P2X? receptor epitope moieties are described herein, particularly in Table 3. As is clear from Table 3, the minimum E200 sequence comprises at least the sequence of SEQ ID NO: 162. In preferred embodiments, the minimum E200 sequence comprises at least the sequence of SEQ ID NO: 155, or a variant thereof.

[0253] In certain examples, the E200 epitope for inclusion in the bridging molecules of the invention, may comprise extensions in the N-terminal and / or C-terminal region. The extensions may be derived from the naturally occurring nfP2X7 receptor sequence, and may comprise extensions of at least 1 amino acid residue, at least 2 amino acid residues, at least 3, at least 4, at least 5 or more amino acid residues. For example, the E200 epitope defined in SEQ ID NO: 155 may comprise an N-terminal extension of the sequence DFP, which corresponds to the three amino acid residues immediately N terminal to the E200 sequence in SEQ ID NO: 1. Further, the E200 epitope defined in SEQ ID NO: 155 may comprise a C terminal extension, as depicted, for example in SEQ ID NO: 155 and 156. It will be appreciated that such N and C terminal extensions may serve to improve the efficacy of binding of the CAR-T cell to the epitope on the bridging molecule.

[0254] In any embodiment, the P2X? receptor epitope moiety is conjugated to the Fab region of the bridging molecule so as to enable sufficient accessibility for binding by an nfP2X? receptor CAR. Thus, while the minimum dysfunctional P2X? receptor epitope moiety sequence comprises the minimum E200 sequence at least the sequence of SEQ ID NO: 162, in preferred embodiments, the minimum E200 sequence comprises at least the sequence of SEQ ID NO: 154, or a variant thereof, in addition to one or more linker or hinge regions for enabling binding by an nfP2X? receptor CAR. In certain embodiments, the total length of the dysfunctional P2X? receptor epitope moiety (including linker and hinge region) 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, 45, 47, 48, 49, or 50 amino acids in length. Preferably the dysfunctional P2X? receptor epitope moiety is no more than about 100 amino acids in length, more preferably, no more than 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 in length.

[0255] In the context of a bridging molecule for targeting CD19, preferably the dysfunctional P2X? receptor epitope moiety is no more than about 35 amino acids in length and at least 20 amino acids in length, preferably at least 21 , 22, 23, 24, 25, or 26 amino acids in length.

[0256] In the context of a bridging molecule for targeting CD33, preferably the dysfunctional P2X? receptor epitope moiety is no more than about 45 amino acids in length and at least 20 amino acids in length, preferably at least 21 , 22, 23, 24, 25, or 26 amino acids in length.PNE epitope moiety

[0257] In any embodiment, the amino acid sequence of the PNE neo-epitope moiety of any bridging molecule described herein, comprises or consists of a sequence as set forth in any of SEQ ID Nos: 125, or sequences 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.EGFRvlll epitope moiety

[0258] In any embodiment, the amino acid sequence of the EGFRvlll epitope moiety of any bridging molecule described herein, comprises or consists of a sequence as set forth in any of SEQ ID Nos: 267, or sequences 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 EGFRvlll epitope moiety comprises at least the sequence of SEQ ID NO: 134.CLDN6 epitope moiety

[0259] In any embodiment, the amino acid sequence of the CLDN6 epitope moiety of any bridging molecule described herein, comprises or consists of a sequence as set forth in any of SEQ ID Nos: 143, 282 or 283, or sequences 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 moiety comprises at least the sequence of SEQ ID NO: 143.Exemplary bridging molecules

[0260] The present specification provides various non-limiting examples of tumourspecific antigen epitope moiety (e.g. P2X? receptor epitope, CLDN6 or EGFRIII moieties) I Fab region pairs.

[0261] Exemplary bridging molecules of the invention are described in Tables 1 and 2. For those bridging molecules that are described in Table 1 that include a nfP2X? epitope moiety, the specification includes those bridging molecules but with the nfP2X? epitope moiety substituted for a PNE, EGFRvlll or CLDN6 epitope moiety (in Table 2).

[0262] In examples where the bridging molecules comprise a Fab region for binding to CD19, the bridging molecule may comprise or consist of a heavy and paired light variable chain combination as set forth in Tables 1 or 2 (heavy and light chain, respectively; or sequences 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.

[0263] In any embodiment, the bridging molecule of the invention may comprise the amino acid sequences of SEQ ID NOs: 1 , 9 and 10, or of SEQ ID NOs: 2, 8 and 10, or of SEQ ID NO: 287, 8 and 10, or of SEQ ID NOs: 3 and 9, or of SEQ ID NOs: 4, 1 1 and 12, or of SEQ ID Nos: 5, 11 and 12, or of SEQ ID NOs: 6, 13 and 14, or of SEQ ID NOs: 7, 13 and 14, or of SEQ ID NOs: 1 , 2 and 288, or of SEQ ID Nos 4 and 1 1 in combination with SEQ ID NOs: 8 and 287, 1 and 288, 2 and 8, 1 and 10 or 287 and 288; or sequences 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.Nucleic acidsIll

[0264] In a further aspect, the present invention provides a nucleic acid molecule encoding a bridging molecule of the invention, or part thereof.

[0265] The nucleic acid molecule may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified, or modified, RNA or DNA. For example, the nucleic acid molecule may include single- and / or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, doublestranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecule may comprise triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecule may also comprise one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. A variety of modifications can be made to DNA and RNA; thus the term "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms.

[0266] In some embodiments of the second aspect of the invention, the nucleic acid molecule comprises a nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 151. Preferably, the nucleic acid comprises a nucleotide sequence encoding the heavy chain and light chain pairs described above.

[0267] Further, the present invention provides a nucleic acid construct including a nucleic acid molecule encoding a bridging molecule of the invention, or part 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 that is active in one or more hosts; and / or one or more transcriptional control sequences.

[0268] As used herein, the term “selectable marker gene” includes any gene that confers a phenotype on a cell in which it is expressed, to facilitate the identification and / or selection of cells that are transfected or transformed with the construct.

[0269] “Selectable marker genes” include any nucleotide sequences which, when expressed by a cell transformed with the construct, confer a phenotype on the cell that facilitates the identification and / or selection of these transformed cells. A range of nucleotide sequences encoding suitable selectable markers are known in the art (for example Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5).Exemplary nucleotide sequences that encode selectable markers include: Adenosine deaminase (ADA) gene; Cytosine deaminase (CDA) gene; Dihydrofolate reductase (DHFR) gene; Histidinol dehydrogenase (hisD) gene; Puromycin-N-acetyl transferase (PAC) gene; Thymidine kinase (TK) gene; Xanthine-guanine phosphoribosyltransferase (XGPRT) gene or antibiotic resistance genes such as ampicillin-resistance genes, puromycin-resistance genes, Bleomycin-resistance genes, hygromycin-resistance genes, kanamycin-resistance genes and ampicillin-resistance genes; fluorescent reporter genes such as the green, red, yellow or blue fluorescent protein-encoding genes; and luminescence-based reporter genes such as the luciferase gene, amongst others which permit optical selection of cells using techniques such as Fluorescence-Activated Cell Sorting (FACS).

[0270] Furthermore, it should be noted that the selectable marker gene may be a distinct open reading frame in the construct or may be expressed as a fusion protein with another polypeptide (e.g. the CAR).

[0271] As set out above, the nucleic acid construct may also comprise one or more transcriptional control sequences. The term “transcriptional control sequence” should be understood to include any nucleic acid sequence that effects the transcription of an operably connected nucleic acid. A transcriptional control sequence may include, for example, a leader, polyadenylation sequence, promoter, enhancer or upstream activating sequence, and transcription terminator. Typically, a transcriptional control sequence at least includes a promoter. The term “promoter” as used herein, describes any nucleic acid that confers, activates or enhances expression of a nucleic acid in a cell.

[0272] In some embodiments, at least one transcriptional control sequence is operably connected to the nucleic acid molecule of the second aspect of the invention. For the purposes of the present specification, a transcriptional control sequence is regarded as “operably connected” to a given nucleic acid molecule when the transcriptional control sequence is able to promote, inhibit or otherwise modulate the transcription of the nucleic acid molecule. Therefore, 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.

[0273] The "nucleic acid construct" may be in any suitable form, such as in the form of a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which is capable ofreplication when associated with the proper control elements and which can transfer gene 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.

[0274] A promoter may regulate the expression of an operably connected nucleic acid molecule constitutively, or differentially, with respect to the cell, tissue, or organ at which expression occurs. As such, the promoter may 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 specific environmental or physiological conditions. The present invention contemplates the use of any promoter that is active in a cell of interest. As such, a wide array of promoters would be readily ascertained by one of ordinary skill in the art.

[0275] Mammalian constitutive promoters may include, but are not limited to, Simian virus 40 (SV40), cytomegalovirus (GMV), P-actin, Ubiquitin C (UBC), elongation factor-1 alpha (EF1 A), phosphoglycerate kinase (PGK) and CMV early enhancer / chicken [3 actin (CAGG).

[0276] Inducible promoters may include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters that have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: tetracycline regulated promoters (e.g. see US Patent 5,851 ,796 and US Patent 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (e.g. see US Patent 5,512,483), ecdysone receptor promoters (e.g. see US Patent 6,379,945) and the like; and metal-responsive promoters such as metallothionein promoters (e.g. see US Patent 4,940,661 , US Patent 4,579,821 and US 4,601 ,978) amongst others.

[0277] In the context of the present invention, it will be appreciated that it may be desirable in certain circumstances for the expression of the bridging molecule to be under the control of an inducible promoter. This enables a switching on and switching off of the expression of the nucleic acid encoding the bridging molecule.

[0278] In certain embodiment, and in the case of an inducible expression construct, an immune cell expressing a CAR can be genetically modified with a) a nucleic acid encoding an antigen binding receptor and b) an inducible expression construct encoding the bridging molecule. Upon binding of dysfunctional P2X? receptor, the immune cell induces expression of the gene encoding the bridging molecule. In certain embodiments, expression of such gene facilitates and / or improves treatment of cancer.

[0279] As mentioned above, the control sequences may also include a terminator. The term “terminator” refers to a DNA sequence at the end of a transcriptional unit that signals termination of transcription. Terminators are 3'-non-translated DNA sequences generally containing a polyadenylation signal, which facilitate the addition of polyadenylate sequences to the 3'-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence that is operable in the cells, tissues or organs in which it is intended to be used. Suitable terminators would be known to a person skilled in the art.

[0280] As will be understood, the nucleic acid constructs of the invention can further include additional sequences, for example sequences that permit enhanced expression, cytoplasmic or membrane transportation, and location signals. Specific non-limiting examples include an Internal Ribosome Entry Site (IRES) or cleavage site (e.g. P2A, T2A).

[0281] The present invention extends to all genetic constructs essentially as described herein. These constructs may further include nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotes and / or the integration of the genetic construct or a part thereof into the genome of a eukaryotic cell.

[0282] Methods are known in the art for the deliberate introduction (transfection / transduction) of exogenous genetic material, such as the nucleic acid construct of the third aspect of the present invention, into eukaryotic cells. As will be understood, the method best suited for introducing the nucleic acid construct into the desired host cell is dependent on many factors, such as the size of the nucleic acid construct, the type of host cell, the desired rate of efficiency of the 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 structuressuch 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.

[0283] The nucleic acid construct will be 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 for introducing the nucleic acid construct into a host cell is viral transduction. Methods are known in the art for utilising viral transduction to elicit expression of a CAR in a PBMC (Parker, LL. et al. Hum Gene Ther. 2000;1 1 : 2377-87) and more generally utilising 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, a cosmid, an artificial chromosome or the like, and can be transfected into the cell by any suitable method known in the art.Modified cells

[0284] As described herein, the invention includes the use of a cell expressing a chimeric antigen receptor comprising a binding domain and signalling domain. The cell may be an "engineered cell", "genetically modified cell", “immune cell” or “immune effector cell” as described herein. Further, the cell may be capable of differentiating into an immune cell. A cell that is capable of differentiating into an immune cell may be a stem cell, multi-lineage progenitor cell or induced pluripotent stem.

[0285] In any embodiment, the cell may be a T cell, wherein optionally said T cell does not express TcRap, PD1 , CD3 or CD96 (e.g. by way of knocking down or knocking out one of these genes on a genetic level or functional level).

[0286] In any embodiment, the cell may be an immune cell, wherein optionally said cell does not express accessory molecules that can be checkpoint, exhaustion or apoptosis- associated signalling receptors as well as ligands such as PD-1 , LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (e.g. by way of knocking out one of these genes on a genetic level or functional level).

[0287] In some embodiments, the genetically modified cell includes two or more different CARs.

[0288] In some embodiments of the invention, the genetically modified cell includes a nucleic acid molecule, or a nucleic acid construct, that encodes for two or more different CARs. In some embodiments of the invention, the genetically modified cell includes two or more nucleic acid molecules, or two or more nucleic acid constructs, each of which encodes for a different CAR.

[0289] As referred to herein, a "genetically modified cell" includes any cell comprising a non-naturally occurring 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 in the cell as a discreet DNA molecule, or it may be integrated into the genomic DNA of the cell.

[0290] Genomic DNA of a cell should be understood in its broadest context to include any and all endogenous DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA and the like. As such, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, and the like. The "genomically integrated form" of the construct may be all or part of the construct. However, in some embodiments the genomically integrated form of the construct at least includes the nucleic acid molecule of the second aspect of the invention.

[0291] As used herein, the term “different CARs” or “different chimeric antigen receptors” refers to any two or more CARs that have either non-identical antigenrecognition and / or non-identical signalling domains. In one example, “different CARs” includes two CARs with the same antigen-recognition domains (e.g. both CARs may recognise a dysfunctional P2X? receptor), but have different signalling domains, such as one CAR having a signalling domain with a portion of an activation receptor and the other CAR having a signalling domain with a portion of an co-stimulatory receptor. As will be understood, at least one of the two or more CARs within this embodiment will have an antigen-recognition domain that recognises the dysfunctional P2X? receptor and the other CAR(s) may take any suitable form and may be directed against any suitable antigen.

[0292] Accordingly, in some embodiments of the invention the two or more different CARs have different signalling domains, and may have identical, or different, antigenrecognition domains. Specifically, the genetically modified cell of the invention may include a first chimeric antigen receptor with a signalling domain that includes a portionderived from an activation receptor and a second chimeric antigen receptor with a signalling domain including a portion derived from a co-stimulatory receptor.

[0293] In some embodiments, the activation receptor (from which a portion of signalling domain is derived) is the CD3 co-receptor complex or is an Fc receptor.

[0294] In some embodiments, the co-stimulatory receptor (from which a portion of signalling domain is derived) is selected from the group consisting of CD27, CD28, CD- 30, CD40, DAP10, 0X40, 4-1 BB (CD137) and ICOS.

[0295] In some embodiments, the co-stimulatory receptor (from which a portion of signalling domain is derived) is selected from the group consisting of CD28, 0X40 or 4- 1 BB.

[0296] In some embodiments, the genetically modified cell is further modified to constitutively express co-stimulatory receptors.

[0297] As described above, a cellular immune response is typically only induced when an activation signal (typically in response to an antigen) and a co-stimulation signal are simultaneously experienced. Therefore, by having a genetically modified cell in accordance with some of the above embodiments, which includes two or more CARs that in combination provide both an intracellular activation signal and an intracellular costimulation signal, ensures that a sufficient immune response can be induce in response to the recognition by the CAR(s) of their cognate antigen. Alternatively, the genetically modified cell may include only one CAR, which has an antigen-recognition domain that recognises a dysfunctional P2X? receptor, and may constitutively express co-stimulatory receptors, thereby increasing the likelihood of co-stimulation being provided simultaneously when the CAR is activated. Alternatively, the genetically modified cell may be further modified to constitutively express both co-stimulatory receptor(s) and its / their ligand(s). In this way the cell is continuously experiencing co-stimulation and only needs the activation of a CAR, with a signalling domain including a portion from an activation receptor, for immune activation of the cell.

[0298] Therefore, in some embodiments, the genetically modified cell expressing the CAR is further modified so as to constitutively express co-stimulatory receptors. In further embodiments, the genetically modified cell is further modified so as to express ligands for the co-stimulatory receptors, thereby facilitating auto-stimulation of the cell. Examples ofCAR-expressing T cells that also express both co-stimulatory receptors and their cognate ligands (so as to induce auto-stimulation) are known in the art and include, inter alia, those disclosed in Stephan MT. et al. Nat Med, 2007; 13: 1440-9.

[0299] The potency of a genetically modified cell including a CAR can be enhanced by further modifying the cell so as to secrete cytokines, preferably pro-inflammatory or pro- proliferative cytokines. This secretion of cytokines provide both autocrine support for the cell expressing the CAR, and alters the local environment surrounding the CAR- expressing cell such that other cells of the immune system are recruited and activated. Consequently, in some embodiments of the fourth or fifth aspects of the invention the genetically modified cell is further modified to secret cytokines. This secretion may be constitutive, or may be inducible upon recognition of a CAR of its cognate antigen of ligand.

[0300] Whilst any one or more cytokines can be selected depending on the desired immune response, preferable cytokines and / or chemokines include IL-2, IL-7, IL-12, IL- 15, IL-17, IL-18 and IL-21 , CCL19, CCL21 or a combination thereof.

[0301] The immune cell of the invention can be any suitable immune cell, or progenitor cell thereof, or can be a homogeneous or a heterogeneous cell population. In some embodiments, the cell is a leukocyte, a Peripheral Blood Mononuclear Cell (PBMC), a lymphocyte, a T cell, a CD4+ T cell, a CD8+ T cell, a natural killer cell, a natural killer T cell, or a yb T cell.

[0302] The immune cell may be a T cell, wherein optionally said T cell does not express TcRap, PD1 , CD3 or CD96 (e.g. by way of knocking down or knocking out one of these genes on a genetic level or functional level).

[0303] The immune cell may not express accessory molecules that can be checkpoint, exhaustion or apoptosis-associated signalling receptors as well as ligands such as PD-1 , LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (e.g. by way of knocking out, or knocking down, one of these genes on a genetic level or functional level).Methods of treatment and administration

[0304] As discussed further in this document, the present invention finds application in the treatment of a variety of conditions, although preferably in the treatment of cancers.

[0305] The present invention also contemplates various scenarios for the use of the two components of the therapeutics described herein.

[0306] In one scenario, the individual requiring treatment is administered a single composition comprising both the CAR T cells and the bridging molecule.

[0307] In further scenarios, the individual requiring treatment is administered a population of CAR T cells, which cells comprise an expression vector encoding the bridging molecule. The expression vector may facilitate constitutive or inducible expression of the nucleic acid sequence encoding the bridging molecule.

[0308] Further still, the individual requiring treatment may be administered the CAR T cells, and at a later date, be administered a composition comprising the bridging molecule (e.g., via infusion), or a nucleic acid sequence encoding the bridging molecule. Such a scenario may be appropriate in circumstances where the individual is first treated with the CAR T cells for targeted treatment of cancers that are positive for dysfunctional P2X? receptor and wherein the subsequent administration of the bridging molecule is for the purposes of redirecting the CARs to alternative cancer antigens, or to peptides derived from an infectious agent and which are presented on MHC I or II molecules of cells.

[0309] Thus the bridging molecule may be administered prior to, at the same time as, or after the subject receives treatment with the CAR T cell.

[0310] Where the bridging molecule and CAR T cells are administered to the subject at the same time, they can be administered via the same route of administration (including in a single composition), or alternatively via different routes of administration. For example, the CAR T cells may be administered by injection into the blood stream of the subject, while the bridging molecule may be administered via another route of administration such as intramuscularly, intradermally, subcutaneously or intraperitoneally.

[0311] A bridging molecule may be produced or expressed inside the body by genetically engineered cells secreting bridging molecules spontaneously or upon stimulation via a stimulating agent e.g. a small molecule. Alternatively, cells may continuously secrete bridging molecules and will stop secreting them upon application of a stimulating agent, e.g. a small molecule.

[0312] It will be clearly understood that, although this specification refers specifically to applications in humans, the invention is also useful for veterinary purposes. Thus in all aspects the invention is useful for domestic animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term "subject” or “subject to be I being treated" is understood to include all animals (such as humans, apes, dogs, cats, horses, and cows).

[0313] The term "administered" means administration of a therapeutically effective dose of the aforementioned composition including the respective cells to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localised delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0314] Subjects requiring treatment include those already having a benign, pre- cancerous, or non-metastatic tumour as well as those in which the occurrence or recurrence of cancer is to be prevented. Subjects may have metastatic cells, including metastatic cells present in the ascites fluid and / or lymph node.

[0315] The objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the disorder.

[0316] Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and / or quality of life.

[0317] The method is particularly useful for extending time to disease progression.

[0318] The method is particularly useful for extending survival of the human, including overall survival as well as progression free survival.

[0319] The method is particularly useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured.

[0320] The method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumours or lesions, or in the extent of cancer in the body, in response to treatment.

[0321] The objective or outcome of treatment may be any one or more of the following: to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; relieve to some extent one or more of the symptoms associated with the disorder.

[0322] In one embodiment, subjects requiring treatment include those having a benign, pre-cancerous, non-metastatic tumour.

[0323] In one embodiment, the cancer is pre-cancerous or pre-neoplastic.

[0324] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher haemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain. The secondary cancer may be detected in the ascites fluid and / or lymph nodes.

[0325] In one embodiment, the cancer may be substantially undetectable.

[0326] “Pre-cancerous" or “preneoplasia” generally refers to a condition or a growth that typically precedes or develops into a cancer. A "pre-cancerous" growth may have cells that are characterised by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle.

[0327] The cancer may be a solid or a “liquid” tumour. In other words, the cancer may be growth in a tissue (carcinoma, sarcoma, adenomas etc) or it may be a cancer present in bodily fluid such as in blood or bone marrow (e.g., lymphomas and leukaemias).

[0328] In certain preferred embodiments, the cancer requiring treatment may be a cancer characterised by low levels of expression of dysfunctional P2X? receptor. Examples of such cancers include Burkitt’s lymphoma. However, immunohistochemical analyses of surface expression of the dysfunctional P2X? (nfP2X?) receptor on patient tumour biopsies reveals a range from 1 + to 3+ in IHC score. Samples with low expression may therefore be found in a wide range of tumour types. Examples are found in solid tumours of various types, including but not limited to neuroblastoma, colorectal cancers, lung cancers, kidney cancers, skin cancers, breast cancers, brain cancers and prostate cancer. Such differences in expression level in different tissues may be due to the formation of tumours from cells that are at an earlier state of transformation (the tissues with the highest receptor expression may be those undergoing the highest rate of proliferation).

[0329] Other examples of cancers that can be treated in accordance with the methods of the present invention include blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SCKC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including 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, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumours of the biliary tract, as well as head and neck cancer.

[0330] In further examples, the methods of treatment contemplated within the scope of the present invention, include methods for treating or preventing an infectious disease. Thus, the bridging molecules of the invention can be utilised to redirect the CAR T cellstowards an additional surface accessible antigen, for example wherein the antigen is a non-cancer associated pathogenic antigen presented on an MHC I or MHC II molecule as further described herein.

[0331] The subject requiring treatment for an infectious disease may be at risk or have been diagnosed with the disease. Subjects at risk include those who are immunocompromised. Thus, the methods of the present invention also allow for the prevention of onset of infectious disease in individuals receiving therapy (such as for treating cancer) that renders them immunocompromised and therefore susceptible to infection.

[0332] Examples of intracellular pathogens from which peptides are presented on MHC I or MHC II molecules include: viral infections, intracellular bacterial infections, protozoan infections, and intracellular fungal infections.

[0333] Examples of viral infections that may be treated using the methods of the present invention include: HIV, hepatitis (e.g., Hepatitis A, B or C), a coronavirus (e.g. SARS-CoV-2), an influenza virus, varicella zoster virus, mumps virus.

[0334] Examples of intracellular bacterial infections which may be treated using the methods of the present 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.

[0335] Examples of intracellular infections caused by fungal pathogens: Histoplasma capsulatum, Cryptococcus neoformans, and Pneumocystitis jirovecii.

[0336] Examples of obligate intracellular protozoan pathogens include: Apicomplexans (Plasmodium spp., Toxoplasma gondii and Cryptosporidium parvum), and Trypanosomatids (Leishmania spp. and Trypanosoma cruzi).

[0337] Immune cells that may be targeted to modulate the immune system in the context of cancer and / or autoimmune disease may be B cells (CD19, CD20, CD22), plasma cells (BCMA, CD38, CD138), T cell subsets via (TRBC1 or TRBC2, a4[37 & aE|37, CD7), macrophages and TAMs (CD163 and CD206). In the context of allogeneic stemcell transplantation, immune-based conditioning may be undertaken by targeting (CD34, CD1 17, CD133, CD33 and CD38) especially in case of non-malignant diseases e.g. thalassaemia major or sickle cell anaemia and / or in case of DNA-repair defects like Fanconi anaemia.

[0338] Targeting senescent tumour cells via the marker (uPAR) will help to eliminate tumour cells in a resting state and which are likely to expand at later time points and promote even faster proliferation of cancer cells in the latter by secreting tumour promoting cytokines and shaping a tumour-suppressive environment protecting new cancerous subclones.

[0339] CAR T cells may be constructed in a way that they are able to immunosuppress other immune cells, e.g. TREG CAR T cells or by secreting immunosuppressive cytokines (TGFbeta, IL10) and chemokines by introducing the corresponding inducible expression cassette [NFAT-dependent cytokine secretion] and the signalling in the construct.

[0340] The bridging molecules of the invention may be formulated for administration to a subject using techniques known to the skilled artisan. Formulations of the bridging molecules may include pharmaceutically acceptable excipient(s) (carriers or diluents). Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-injection, glycerol, ethanol, and combinations thereof, stabilising agents, solubilising agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0341] A formulation of bridging molecules may include one type of bridging molecule, or more than one type of bridging molecule (i.e., wherein the bridging molecules may have the same or different targeting and / or dysfunctional P2X? receptor epitope moieties).

[0342] The bridging molecules may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous, intraperitoneal, and intratumoural injection. Other modes include, without limitation, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intraarterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).

[0343] Formulations comprising the bridging molecule are administered to a subject in an amount that is effective for treating the specific indication or disorder. In general,formulations comprising at least about 0.01 pg / kg to about 100 mg / kg body weight of the bridging molecule may be administered to a subject in need of treatment. In most cases, the dosage may be from about 100 pg / kg to about 10 mg / kg body weight of the bridging molecules daily, taking into account the routes of administration, symptoms, etc. However, the amount of bridging molecules in formulations administered to a subject may vary between wide limits, depending upon the location, source, identity, extent and severity of the disorder, the age and condition of the individual to be treated, etc. A physician may ultimately determine appropriate dosages to be used. The bridging molecules may be administered as a continuous infusion or a bolus application.

[0344] The timing between the administration of the CAR T cell and the bridging molecule formulation may range widely depending on factors that include the type of (immune) cells being used, the binding specificity of the CAR, the identity of the targeting moiety and the identity of the target cell, e.g. cancer cell to be treated, the location of the target cell in the subject, the means used to administer the formulations to the subject, and the health, age and weight of the subject being treated. Indeed, the TCBM formulation may be administered prior to, simultaneous with, or after the genetically engineered (immune) cell formulation.

[0345] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.Examples

[0346] Bivalent bridging molecules of various formats were tested for their ability to stimulate cell killing by CAR T cells. In these examples, the CAR T cells tested all comprised antigen binding domains for binding to the E200 epitope of P2X? receptor (eg comprising an antigen binding domain as set forth in SEQ ID NO: 286, such as described in WO 2022 / 187906).Example 1 : dual-tagged bridging molecules can cause unwanted cytotoxicity

[0347] The inventor identified that bridging molecules have two tags present on each “side” of a bridging molecule can result in unwanted cytotoxic events.

[0348] More specifically, it was found that a symmetric antibody architecture of bridging molecules induced a significantly higher expression of the activation markers CD25+CD69+ as well as the exhaustion marker PD-1 - compared to the XmAb1and XmAb2bridging molecule formats.

[0349] Figure 9A shows fold change in CD25+CD69+ expression, and Figure 9B shows fold change PD-1 expression. This experiment was done using 72h cytokine deprived adapter CAR T cells being incubated at a range of bridging molecule concentrations. The reference to calculate the fold change is the condition of cytokine deprived adapter CAR T cells without any addition of bridging molecule (0 ng / mL). In the other conditions, the various bridging molecule formats as depicted were added at the indicated concentrations and the CD25 and CD69 positive fraction (A) or PD-1 (B) was quantified and divided by the condition without bridging molecule (0 ng / mL).

[0350] The XmAb1and XmAb2format bridging molecules demonstrated significantly lower potential to induce unwanted target-independent activation than their symmetric counterparts. In conclusion, the XmAb bridging molecules facilitate an improved anticancer potency while reducing unwanted activation, exhaustion and induction of Activation Induced Cell Death (AICD) of CAR T cells.Example 2: design of alternative bridge molecule format

[0351] The inventors designed bivalent bridging molecules in antibody (lgG1 ) format. The bridging molecules were generated using knobs-in-holes (KIH) format in combination with CROSS-mAb or DUET-Mab technologies to control heavy and light chain pairing. Bridging molecules further also comprised either a single tag for binding by anti-E200 CAR T cells or multiple tags.

[0352] Figure 3 shows various architectures for various mono- dual, triple- or tetratagged antibody-format bridging molecules.

[0353] The inventors determined that the optimum distance between tags on a single antibody-format bridging molecule was about 25 nm. This meant that location of the tags on the Fab portion of the antibodies was determined to be optimal (eg compared to location of the tag on the Fc portion of the antibody). Furthermore, the inventors determined that the optimal location of the tags on the Fab portion of the antibody wassuch that multiple tags should all be linked to the same Fab “arm” of the antibody so as to prevent cytotoxic events as described in example 1 .Example 3: Cell killing by anti-E200 CAR T cells in the presence of CD33 targeting antibody format bridging molecules

[0354] Figure 4 shows the results of cell-killing experiments of U937 cells (expressingCD33)

[0355] In these experiments, the anti-E200 CAR comprised the VH BIL03 2-2-1 and the VL WTB1 (sequences disclosed in WO 2023 / 028653), the hinge region of CD8A, the transmembrane domain of CD28, and the co-stimulatory / signalling domain of CD28, 41 BB and CD3zeta.

[0356] The results shown in Figure 4 demonstrate that dual-tagged antibody-format bridging molecules facilitate greater cell killing by the anti-E200 CAR T cells comparted to mono-tagged antibody format bridging molecules.

[0357] A similar experiment was conducted using the same CAR T cells and bridging molecules but with Jeko-1 cells, which do not express CD33. The results (shown in Figure 5) demonstrate that there is no undirected cytolysis of the CAR T cells by the bridging molecules.Example 4: Cell killing by alternative anti-E200 CAR T cells in the presence of CD33 targeting antibody format bridging molecules

[0358] A further experiment was conducted using an alternative CAR format comprising the single domain antibody BIL03 (2-2-1 ) and the hinge region of CD8A, the transmembrane domain of CD28, and the co-stimulatory / signalling domains of CD28, 41 BB (CD137) and CD3zeta.

[0359] The results, shown in Figure 6, further demonstrate the utility of the dual-tagged anti-CD33 antibody-format bridging molecules to elicit cell-death of CD33 expressing cells.Example 5: Consideration of orthogonal tag presentation

[0360] The inventors further investigated optimal tag presentation to maximise engagement of cancer and T cells by bridging molecules of the invention. Figure 7 shows a schematic depicting optimal (A) and non-optimal (B) presentation of the tag.

[0361] Figure 8 shows the results of a series of experiments where the cell killing of either U937 or Jeko-1 cells by anti-E200 CAR T cells was elicited through engagement of anti-CD33 or ant-CD19 bridging molecules comprising one or two E200 tags.

[0362] Green binding domains refer to anti-CD33 binding regions of the bridging molecules. Red binding domains refer to anti-CD19 binding regions of the molecules.

[0363] As can be seen in Figure 8, the inventors compared the cell-killing enhancing capacity of mono- or dual-tagged bivalent monospecific bridging molecules and of mono- or dual-tagged bivalent bispecific bridging molecules.

[0364] In the context of U937 cells, there is no binding of the cells via the red coloured arm of the antibody (since U937 cells do not express CD19). Similarly, in the context of Jeko-1 cells, there is no binding of the cells via the green coloured arm of the antibody (since Jeko-1 cells do not express CD33).

[0365] The results indicate that superior results are obtained when using a bivalent, monospecific IgG with dual tags, where the binding domains of the IgG are for binding an antigen on the target cancer cell (eg CD33) and wherein the tags are located on the same Fab arm of the IgG.

[0366] The results also show that a dual-tagged bivalent, bispecific bridging molecule (eg as shown in orange bars) provides for greater cell killing of U937 (eg compared a monotagged version of the same molecule as shown in purple bars) even though there is no binding to the target cell via the anti-CD19 Fab portion of the antibody. This is also shown in the context of Jeko-1 cells whereby a dual-tagged, bivalent, bispecific bridging molecule as shown in peach bars provides for greater cell killing of Jeko-1 cells compared a monotagged version of the same molecule as shown in pink bars.

[0367] These results also demonstrate that in the context of bispecific bridging molecules, it is preferable for the tags to be located on a Fab portion of the antibody that is capable of binding to the target cell.Example 6: cytolysis of AML cell line using anti-E200 CAR and bridging molecules comprising E200 peptide tag

[0368] Cytolysis of the AML cell line U937 by an anti-E200 CAR (comprising an antigen binding domain comprising BIL03 sdAb) was measured using a luciferase-based kill assay at an ET ratio of 0.5:1 (CAR T to target ratio) in conjunction with and without the depicted bridging molecule variants (targeted to CD33, lintuzumab based) at indicated concentrations.

[0369] The bridging molecules tested were: right side of the antibody: HOLE, CROSS- MAb: SEQ ID NO: 4, 11 for all sequences. Left side of the antibody: KNOB, CROSS-mAb were:

[0370] Control experiments (data shown in Figure 10 Aii, Bii and Cii) makes use of a CAR T product that has no capability of binding to the tag.

[0371] The results are shown in Figure 10 Ai (cytolysis of AML cell line U937 at 24 hours) and Figure 10Bi (cytolysis of AML cells at 48 hours) and Figure 10Ci (cytolysis at 72 hours).

[0372] This control demonstrates that i) the CAR does not have an impact on cancer cell elimination if it cannot bind to the tag sequence present on the bridging molecule and ii) the bridging molecules alone (ie without a CAR T capable of recognising the tag sequence on the bridging molecules) do not impact on cancer cell growth and are immunologically inert .

[0373] In this assay, the inventors demonstrate that the XmAb2 variant was capable of eliminating the cancer cells with equal efficiency as more toxic counterparts in a symmetric mAb format. (The toxicity as measured by unwanted target-independent activation and exhaustion is illustrated in Figure 9).

[0374] Figure 1 1 further shows a comparison of cytolysis of AML cell line U937 by an anti-E200 CAR T cell at 1 ng / mL of the corresponding bridging molecule formats at 24 hours, 48 hours, and 72 hours.

[0375] Statistical analysis of the various bridging molecules demonstrated a superior function of the symmetric antibodies mAb2, mAb4, and XmAb2compared to XmAb1and Fab1. Thus, the XmAb2has a similar function to symmetric mAb2and mAb4, however, with an improved toxicity profile for the CAR T cells (reduced unspecific activation, AICD) as well as theoretically for the patient reducing target antigen unrelated “unspecific” activation of the CAR T cells which are induced via undirected secretion of cytokines which can trigger the cascade of severe life-threatening side effects, such as cytokine release syndrome (CRS), and / or immune effector cell associated neurotoxicity syndrome (ICANS), and / or macrophage activating syndrome (MAS).

[0376] In other words, the superior activity of symmetrically tagged antibodies comes at the price of unwanted target-independent toxicities and AICD. Thus, in the in vitro assay they may function better, but the overall performance is less due to their toxic potential on the CAR T cell product and they also induce unwanted toxic effects in patients due to target unrelated activation of the CAR T cells.Example 7: Efficacy of bridging molecules + CAR T cells in AML model

[0377] In the AML (MOLM-13 based) cell line derived xenograft model, 0.5x106MOLM- 13 cells were injected into the tail vein on day -3. On day 0 treatment was initiated after confirmation of leukaemic engraftment using bioluminescence imaging. MOLM-13 were equipped to express eGFP and firefly luciferase as reporter genes for cell line development and use for functional assessment including BLI (oxidation of D-luciferin potassium after in vivo distribution of chemical compound). No treatment (tumour only group), 25x106untransduced, TransAct activated and cytokine cultured (IL7 and IL15 at 10 ng / mL) and expanded T cells, or 5x106CD33-targeted CAR positive T cells (based on Lintuzumab), or 5x106E200-targeted CAR positive T cells (based on BIL03 2-2-1 ) were administered i.v.. Twice per week CD33-targeted XmAb2(based on Lintuzumab) was injected at 50 pg / mouse (2 x 50 pg / mouse = 100 pg / mouse per week) intraperitoneally in the E200-targeted CAR T cell group. Bioluminescence was performed twice per week over the course of treatment. Overall survival was calculated according to Kaplan-Meier.

[0378] Figure 12 shows a significantly reduced survival was detected in the tumour only group and T cell treated group compared to the CD33 CAR T cell group and E200- targeted CAR T plus CD33 XmAb2Lintuzumab treatment.Example 8: Demonstration of the efficacy of the XmAbs of the invention in a universal CAR (PNE) system

[0379] The ability of the bridging molecules of the invention to elicit cell death was tested in a system using a universal CAR system. The data shown in Figure 13 were generated using cryopreserved PNE CAR T cells, thawed on the day of the assay, and used after a reconstitution time of approximately 6 hours at an effector to target ratio of 1 :1 .

[0380] The antibody-based bridging molecules used are indicated at the top of Figure 13 as crossmab XmAb1(one tag on the N-TERMINAL light chain) and XmAb2(two tags on N-TERMINAL light chain and heavy chain). The last antibody or condition on the right of the figure (in brown) did not include a PNE tag but two E200 tags and thus was incompatible with the PNE CAR. This served as a negative control.

[0381] At the indicated concentrations there was no difference in performance. However, even though the cytotoxic potential of XmAb1and XmAb2is similar at 100 ng / mL to symmetrical antibodies (such as shown in purple, orange and black in the figure), XmAb1and XmAb2(red and green in the figure) will induce less unwanted targeted independent activation of the CAR T cells (similarly to the data shown in Figure 9), leading to ongoing activation and exhaustion as well as fratricide.Example 9: In vivo demonstration of the superiority of the asymmetrical bridging molecule design in an AML disease model

[0382] In this series of experiments, 1 x1 Oe6MOLM13 cells were injected into the tail vein of mice on day -3. On day 0 treatment was initiated after confirmation of leukemic engraftment using bioluminescence imaging. MOLM-13 cells were equipped to express eGFP and firefly luciferase as reporter genes for cell line development and use for functional assessment including BLI (oxidation of D-luciferin potassium after in vivo distribution of chemical compound).

[0383] No treatment (tumour only group), 5x1 Oe6CD33-targeted CAR positive T cells, or 10x10e6E200-targeted CAR positive T cells (comprising the BIL03 2-2-1 antigenbinding domains as describe elsewhere herein) were administered i.v.. Twice per week CD33-targeted XmAb1(one tag on the N-TERMINAL light chain; eg SEQ ID NO: 29, 77, 93, 109) XmAb2(two tags on N-TERMINAL light chain and heavy chain; eg SEQ ID NO: 61 , 29, 93, 109) or mAb2(two tags but one on each Fab arm; eg SEQ ID NO: 322 and [323 or 324]) were injected at 25 pg / mouse (2 x 25 pg / mouse = 50 pg / mouse per week) intraperitoneally in the E200-targeted CAR T cell group. Bioluminescence was performed twice per week over the course of treatment.

[0384] The results, shown in Figure 14A and B, show the bioluminescence (as measured by total flux) in the cells following CAR T cell injection into mice bearing the MOLM-13 cell xenograft. The level of bioluminescence is an indicator of the number of MOLM-13 cells remaining in the animal; with reduced bioluminescence being an indicator of cell killing by the CAR T cells. Figure 14A shows the individual animal bioluminescence readings during the treatment course. 3 out of 5 Xmabl -treated mice reached cancer clearance. Figure 14B shows the mean bioluminescence of all five treated animals.

[0385] The results show that although Xmab2 and mAb2 both comprise two E200 tags for binding to the CAR T cells, the asymmetrical design of Xmab2 demonstrated better efficacy than the symmetrical mAb2. As such, these results demonstrate that the asymmetric molecule design achieves greater cell tumour killing compared to the symmetrical bridging molecule design.

[0386] Figure 14C shows the flow cytometry results of analysis of the peripheral blood cell subpopulation in the xenograft animals once the experiment reached end-point. The blood cell subpopulations are defined as mouse blood cells (mouse CD45 positive cells), MOLM13 cancer cells (GFP positive), injected CAR T cells (mouse CD45 negative, human CD3 positive, CAR positive), and CAR negative human T cells (mouse CD45 negative, human CD3 positive, CAR negative). The percentage of each population to total blood cells was plotted. Similarly to the results shown in Figures 14A and B, although Xmab2 and mAb2 both comprise two E200 tags for binding to the CAR T cells, the asymmetrical design of Xmab2 provided for better CAR T persistence than the symmetrical design of mAb2. These results indicate that using an asymmetric design bridging molecules facilitates improved CAR T cell persistence in vivo.Example 10: Recognition of tumour antigens by bispecific bridging molecules of the invention

[0387] The wild type U937 cell line (AML; WT), which expresses both CD33 and CLL1 , was genetically modified using CRISPR-Cas9 technology to generate a U937-CD33 knock-out (KO) cell line. The U937 cell line does not express CD19.

[0388] The U937 WT and KO cells were incubated with 1 pg / mL purified CD33 monospecific, CLL1 monospecific, or CD33xCLL1 , CD33xCD19, CLL1 xCD19 bispecific bridging antibodies with an E200 tag on the left Fab arm. The binding of the bridging antibodies was detected by fluorochrome-conjugated BII03 (2-2-1 ) secondary antibody. The median fluorescence intensity ratio (MFIR) of the BII03 antibody binding was analyzed by flow cytometry.

[0389] The results are shown in the below table. Schematics of the bridging molecules used are shown in Figure 15.

[0390] The asymmetrical E200 tagging design of the monospecific CD33 and CLL1 antibodies and CD33xCLL1 bispecific antibodies enabled recognition of the target antigen on the cancer cells. This binding could be detected by E200-peptide targeted BII03 (2-2- 1 ) antibody. It was noticed that the control CD33xCD19 bispecific antibody did not bind to the U937 CD33 KO cells, but still could engage with the CD33 antigen on the U937 WT cells. This demonstrated that the single arm could form the immune synapse with the target antigen with specificity.Example 1 1 : / / ? vitro efficacy of bridge molecules of the invention in U937 cells

[0391] A series of in vitro efficacy experiments were conducted using a BilO3-CAR that comprised the single domain antibody BIL03 (2-2-1 ; SEQ ID NO: 286). Cancer target cells in this experiment were AML U937 cells, comprising U937 wild type (WT) , genomically engineered CD33 knocked out (CD33KO), and CLL1 knocked out (CLL1 KO) cells.

[0392] CD33 monospecific Crossmab, CLL1 monospecific Crossmab, and CD33xCLL1 bispecific Crossmab were added into the BilO3-CAR and cancer cell co-culture system at indicated concentration and / or combination. Effective CAR positive and target ratio was 1 :1 . Cancer cell viability was read at 24 hours.

[0393] Figure 16 show the percentage cell viability of wildtype U937 cells with increasing concentration of bridging molecules. The results demonstrate comparable efficacy of a CLL1 xCD33 bispecific Crossmab to 1 :1 mixed CD33 and CLL1 monospecific Cossmabs on U937 WT cells, which express CD33 and CLL1 .

[0394] A similar experiment was conducted using U937 CLL1 KO cells. For these cells, CLL1 x CD33 bispecific Crossmab with tag on CLL1 Fab could elicit cancer cell death however is less effective than CD33 monospecific Crossmab. These results (shown in Figure 18) demonstrate that in the context of bispecific bridging molecules, tags are preferably located on a Fab portion of the antibody that is capable of binding to the target (tumour) cell and facilitating orthogonal E200 tag presentation to CAR T cells.

[0395] A further experiment was conducted using U937 CD33 KO cells. For these cells, CLL1 x CD33 bispecific Crossmab with the tag on the CLL1 Fab arm could elicit cancer cell death effectively with comparable efficacy to CLL1 monospecific Crossmab. These results (shown in Figure 19) demonstrate that in the scenario of tumour CD33 antigen escape or low expression level, the CLL1 xCD33 bispecific Crossmab could mediate cancer cell cytolysis. These results demonstrate that a bispecific Crossmab molecule is useful for preventing target antigen escape.

[0396] In a final experiment, the WT, CD33KO and CLL1 KO U937 cells were mixed at 1 :1 :1 ratio as target cells. In this mixed U937 context, bispecific CD33xCLL1 crossmab elicited more cancer cell cytolysis than monospecific CD33 or CLL1 crossmab alone at the concentration of 5ng / mL or above. These results demonstrate that in the scenario ofa heterogenous cancer cell population, bispecific Crossmab molecules can achieve better tumour cell killing efficiency than monospecific CrossMabs.

[0397] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

CLAIMS1 . A molecule capable of simultaneously binding to a target cell and to an immune cell expressing an exogenous cell surface receptor comprising an intracellular signalling domain, the molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

2. A molecule capable of simultaneously binding to a target cell and to an immune cell expressing an exogenous cell surface receptor comprising an intracellular signalling domain, the molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least two moieties capable of being bound by the exogenous immune cell receptor, and wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor.

3. The molecule of claim 2, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

4. A two-component therapeutic comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm of an antibody and a second portion comprising a second Fab arm of an antibody, wherein the first and second Fab arms each comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

5. A composition comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising and an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell,wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

6. A kit comprising:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains.

7. The molecule of claim 1 to 3 or the bridging molecule defined in any one of claims 4 to 6, wherein the first and second portions comprise an Fc region of an antibody.

8. The molecule of claim 7 wherein the Fc region of an antibody is a CH2 region of an antibody.

9. The molecule of claim 7 wherein the Fc region of an antibody is a CH3 region of an antibody.

10. The molecule of claim 7 wherein the Fc region of an antibody comprises a CH2 and a CH3 region of an antibody.11 . The molecule of any one of claims 7 to 10 wherein the antigen binding domains of the first and second Fab arms of the heterodimeric molecule are identical in amino acid sequence.

12. The molecule of any one of claims 7 to 10, wherein the antigen binding domains are non-identical but bind to the same epitope of an antigen, or bind to different epitopes of the same antigen.

13. The molecule of any one of claims 7 to 10 wherein the antigen binding domains of the first and second Fab arms of the heterodimeric molecule are for binding to different antigens.

14. The molecule of any one of claims 7 to 13, wherein the first and second Fab arms are capable of specifically binding to an antigen on a cancer cell.

15. The molecule of any one of claims 7 to 14, wherein the first Fab arm of the heterodimeric molecule comprises 3 or 4 or 5.

16. The molecule of any one of claims 7 to 15, wherein the moieties capable of being bound by the exogenous immune cell receptor are selected from: a peptide, polysaccharide, dye, lipid, or small molecule.

17. The molecule of any one of claims 7 to 16, wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides.

18. The molecule of claim 17, wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising the amino acid sequence of a neo-epitope.

19. The molecule of claim 17, wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising the amino acid sequence of a tumour-specific or tumour-associated antigen.

20. The molecule of claim 19, wherein the antigen is selected from an antigen of P2X?, EGFRvlll or CLDN6 and the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising the amino acid sequence an epitope derived from P2X7, EGFRvlll or CLDN6.21 . The molecule of claim 20, wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising the amino acid sequence an epitope derived from P2X7receptor22. The molecule of claim 21 , wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising the amino acid sequence an epitope derived from the E200 or E300 epitopes of P2X7.

23. The molecule of claim22 wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 155 to 218.

24. The molecule of claim 22, wherein the moieties capable of being bound by the exogenous immune cell receptor are peptides comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 162.

25. The therapeutic of claim 4, the composition of claim 5, the kit of claim 6 or the molecule of any one of claims 1 to 3 and 7 to 24, wherein the exogenous immune cell receptor is a CAR comprising an antigen recognition domain and a signalling domain, wherein the antigen recognition domain is capable of binding to a tumour-specific or tumour-associated antigen.

26. The therapeutic of claim 4, the composition of claim 5, the kit of claim 6 or the molecule of any one of claims 1 to 3 and 7 to 24, wherein the exogenous immune cell receptor is a CAR comprising an antigen recognition domain for binding to an epitope of the P2X7receptor, preferably to a cancer-associated form of the receptor (such as dysfunctional or non-functional forms of the receptor).

27. The therapeutic, composition, kit or molecule of claim 26 wherein the binding domain of the exogenous receptor binds to an epitope associated with an adenosine triphosphate (ATP)-binding site of the P2X7receptor.

28. The therapeutic, composition, kit or molecule of claim 27 wherein the epitope of P2X? comprises or is a derivative of the E200 epitope as herein defined and preferably comprises at least the amino acid sequence as set forth in SEQ ID NO: 162.

29. The therapeutic, composition, kit or molecule of claim 26 or 27 wherein the antigen-recognition domain binds to an epitope that includes the proline at amino acid position 210 of the P2X? receptor.

30. The therapeutic, composition, kit or molecule of any one of claims 27 to 29, wherein the antigen-recognition domain binds to an epitope that includes one or more amino acid residues spanning from glycine at amino acid position 200 to cysteine at amino acid position 216, inclusive, of the P2X? receptor.

31. The therapeutic, composition, kit or molecule of any one of claims 27 to 30, wherein the antigen-recognition domain comprises a binding polypeptide that comprises the amino acid sequence of the CDRs of the VH and / or VL chain of an antibody, or the amino acid sequence of the VH and / or VL chains of an antibody, or the amino acid sequence of an antibody or fragment thereof, wherein the antibody or fragment thereof comprises the amino acid sequences of any antibody described in: PCT / AU2002 / 000061 or PCT / AU2002 / 001204 (or in any one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531 ,171 , US 8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380), PCT / AU2007 / 001540 (or in corresponding US patent US 8,067,550), PCT / AU2007 / 001541 (or in corresponding US publication US 2010- 0036101 ), PCT / AU2008 / 001364 (or in any one of the corresponding US patents US 8,440,186, US 9,181 ,320, US 9,944,701 or US 10,597,451 ), PCT / AU2008 / 001365 (or in any one of the corresponding US patents US 8,293,491 or US 8,658,385), PCT / AU2009 / 000869 (or in any one of the corresponding US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT / AU2010 / 001070 (or in any one of the corresponding publications WO / 2011 / 020155, US 9,127,059, US 9,688,771 , or US 10,053,508), and PCT / AU2010 / 001741 (or in any one of the corresponding publications WO 2011 / 075789 or US 8,835,609).

32. The therapeutic, composition, kit or molecule of any one of claims 27 to 31 , wherein the antigen recognition domain comprises the CDR amino acid sequences of the 2-2-1 antigen binding domain described in PCT / AU2010 / 001070 (or in any one of the corresponding US patents US 9,127,059, US 9,688,771 , or US 10,053,508) or BPM09described in PCT / AU2007 / 001541 (or in corresponding US publication US 2010- 0036101 ) and produced by the hybridoma AB253 deposited with the European Collection of Cultures (ECACC) under Accession no. 06080101 .

33. The therapeutic, composition, kit or molecule of any one of claims 27 to 32 wherein the antigen-recognition domain comprises the amino acid sequence of a binding domain as described in WO 2023 / 028653, optionally as defined in SEQ ID NO: 286.

34. The therapeutic, composition, kit or molecule of any one of claims 27 to 33 wherein the signalling domain of the immune cell receptor comprises a portion derived from an activation receptor; optionally wherein the activation receptor is a member of the CD3 co-receptor complex or is an Fc receptor, optionally wherein the portion derived from the CD3 co-receptor complex is CD3- ; optionally wherein the portion derived from the Fc receptor is FcsRI or FcyRI.

35. The therapeutic, composition, kit or molecule of any one of claims 27 to 34 wherein the signalling domain includes a portion derived from a co-stimulatory receptor. In some embodiments, the signalling domain includes a portion derived from an activation receptor and a portion derived from a co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, 0X40, 4-1 BB (CD137) and ICOS.

36. A nucleic acid comprising a nucleotide sequence encoding a molecule of any one of claims 1 to 3 and 7 to 24.

37. A method of treating a disease or disorder in a subject, the method comprising administering to the subject:(a) an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm,wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell associated with the disease or disorder, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, and wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, thereby treating the disease or disorder in the subject.

38. A method of treating a cancer in a subject, the method comprising administering to the subject:(a) an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a tumour cell, wherein the first Fab arm comprises at least two moieties capable of being bound by the exogenous immune cell receptor, and wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains,thereby treating the cancer in the subject.

39. A method of killing a target cell, the method comprising exposing or contacting the target cell with:(a) an immune cell or progenitor thereof, expressing an exogenous receptor comprising an intracellular signalling domain; and(b) a bridging molecule comprising: a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a tumour cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, and wherein the second the Fab arm portion does not comprise any moieties capable of being bound by the exogenous immune cell receptor, wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, thereby killing the target cell.

40. Use of a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, andwherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, in the manufacture of a medicament for treating a disorder, wherein preferably the disorder is cancer.41 . A pharmaceutical composition comprising a heterodimer consisting of a first portion comprising a first Fab arm and a second portion comprising a second Fab arm, wherein the first and second Fab arms comprise an antigen binding domain for binding to a cell surface molecule on a target cell, wherein the first Fab arm comprises at least one moiety capable of being bound by the exogenous immune cell receptor, wherein the second Fab arm does not comprise any moieties capable of being bound by the exogenous immune cell receptor, and wherein the first and second portions of the heterodimer comprise amino acid sequences for enabling heterodimerisation via pairing of distinct heavy chains and / or pairing of cognate light chains to the heavy chains, optionally comprising a pharmaceutically acceptable excipient.

42. The pharmaceutical composition of claim 41 , wherein the composition further comprise an immune cell or progenitor thereof expressing an exogenous receptor comprising an intracellular signalling domain and optionally further comprising an antigen recognition domain.

43. The method of any one of claims 37 to 39, or the use of claim 40, or the composition of claims 41 or 42, wherein the first and second portions of the bridging molecule comprise an Fc region of an antibody, such as a CH2 region of an antibody or a CH2 and a CH3 region of an antibody.

44. The method, use or composition of claim 43, wherein the first and second Fab regions are capable of specifically binding to an antigen on a cancer cell.

45. The method, use or composition of claim 43 or 44, wherein the moiety capable of being bound by the exogenous immune cell receptor is a peptide.

46. The method, use or composition of claim 45, wherein the moiety capable of being bound by the exogenous immune cell receptor is a peptide comprising the amino acid sequence of a tumour-specific or tumour-associated antigen.

47. The method, use or composition of claim 46, wherein the antigen is selected from P2X?, EGFRvlll or CLDN6 and the moiety capable of being bound by the exogenous immune cell receptor is a peptide comprising the amino acid sequence an epitope derived from the antigens P2X?, EGFRvlll or CLDN6.

48. The method, use or composition of claim 47, wherein the moiety capable of being bound by the exogenous immune cell receptor is a peptide comprising the amino acid sequence an epitope derived from the antigen P2X?.

49. The method, use or composition of claim 48, wherein the moiety capable of being bound by the exogenous immune cell receptor is a peptide comprising the amino acid sequence an epitope derived from the E200 or E300 epitopes of P2X?.

50. The method, use or composition of claim 49, wherein the moiety capable of being bound by the exogenous immune cell receptor is a peptide comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 155 to 218.51 . The method, use or composition of claim 50, wherein the moiety capable of being bound by the exogenous immune cell receptor is a peptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 162.

52. The method, use or composition of any one of claims claim 46 to 51 , wherein the heterodimeric bridging molecule is bivalent and bispecific.

53. The method, use or composition of claim 52, wherein the epitope moieties located on the molecule are located on a Fab portion which is capable of binding to the target cell.

54. The method, use or composition of any one of claims claim 46 to 51 , wherein the heterodimeric bridging molecule is bivalent and monospecific for binding to a target cell (such as a cancer cell).

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