Tumour-transforming multispecific proteins

Abstract

The invention relates to a soluble multispecific protein for recruiting an immune cell to a target cell presenting a target peptide MHC complex (pMHC complex); wherein the protein comprises a half-lif

Description

Field of the Invention The present invention relates to multispecific molecules for bridging immune synapses between immune cells and target cells in a patient, to treat diseases such as cancer, which combine high potency with extended in vivo half life. Background T cells express variable T cell receptors (TCRs) complexed with CD3 polypeptides. The TCR is activated via binding to its cognate peptide-major histocompatibility complex (pMHC), a cell surface component which is found on most mammalian cells and which presents a sample of peptides from within the cell via the antigen processing and presentation pathway. MHO class I molecules on the surface of most mammalian cells present 8 to 11 mer peptides derived from the cell’s own cytosolic proteins, and are recognised by CD8+ T cells in which the TCR-CD3 complex and co-receptor CD8 bind to the pMHC (Figure 1). MHO class II molecules are found on specialised antigen presenting cells (APC) and present 12 to 20 mer peptides derived from proteins taken up by the cell from its extracellular environment, and are recognised by CD4+ T cells in which the TCR-CD3 complex and co-receptor CD4 bind to the pMHC. A repertoire of TCRs with diverse sequences are generated by somatic rearrangement of genomic DNA within a population of T cells in vivo, each T cell expressing a single (monoclonal) TCR containing random sequence elements. Positive and negative selection during T cell development permits survival of T cells expressing TCR which recognise pMHC with a threshold affinity and deletes self-reactive cells that exhibit high affinity binding, thereby producing a population of T cells which do not recognise normal “self pMHC but which may bind previously unencountered pMHC. When a TCR of a CD8+ cytotoxic T lymphocyte (CTL) recognises a peptide displayed on MHC class I, the binding of TCR to the pMHC initiates intracellular signalling via the CD3 component of the TCR complex, activating the CTL to release cytolytic molecules and immune cell-activating cytokines. Cells expressing proteins with neoepitopes (e.g., infected cells producing viral protein, and cells expressing mutated oncogenes) can thus be eliminated by the cellular adaptive immune system to maintain health. CTL activity has been harnessed and enhanced in a therapeutic setting to destroy tumour cells, using T cell redirecting bispecifics which bind CD3 and a tumour specific cell surface antigen. Binding of the antibody to the CD3 complex over-rides the natural specificity of the T cell for its cognate pMHC, activating polyclonal CTL to kill the target cell. Scientists at Immunocore fused a truncated extracellular region of a TCR (including variable and constant domains of the alpha and beta chains) to an anti-CD3 scFvto create a polypeptide “ImmTAC" for redirecting CTL to tumour cells presenting target pMHC (Boulter &Jakobsen Clin Exper Immunol 142:454-460 2005; Liddy et al, Nat Med 18(6):980-987 2012, Oates &Jakobsen, Oncolmmunology 2(2):e22891 2013). Tebentafusp, an ImmTAC containing a TOR specific for the glycoprotein 100 (gp100) peptide fragment YLEPGPVTA presented by human leukocyte antigen (HLA)-A*02:01, has been approved for treating HLA-A*02:01-positive uveal melanoma patients with metastatic cancer. Bispecifics have been described that recruit various types of immune cells (e.g., regulatory T cells, NK cells, as well as T effector cells) via other immune receptors (e.g., CD28, and checkpoint molecules such as PD-1). Bispecifics for engaging T cells and other immune effector cells were reviewed by Wu &Cheung, Pharmacol Ther 182:161-175 2018 and Fuca et al, ESMO 6(1) 2021. Engagement between an immune cell and a target cell presenting pMHC occurs at a receptor-dense membrane interface termed the immune synapse. It is desirable to provide a therapeutic molecule that is able to promote formation of a strong immune synapse between a target cell and an immune cell in a patient, and which has an extended in vivo half life. However, there is a design challenge in finding a multispecific molecular format that combines the advantages of (1) potency for triggering signalling at the immune synapse with (2) acceptable circulatory half life in vivo to limit dosing frequency of the drug product. Strategies for extending half life tend to involve the addition of bulky molecules that reduce clearance of the drug from the circulation. Examples of half life extenders are hydrophilic polymers such as PEG to increase hydrodynamic radius, antibody Fc regions that allow capture and recycling via FcRn, and serum albumin binders or serum albumin itself, which has a long circulatory half life as it also binds to FcRn. However, attachment of such half life extending regions may strain the spatial constraints for formation of tight immune synapses. Proximity between the plasma membranes of the two cells at the immune synapse contributes to effective signalling within the immune cell, and triggering of downstream responses. Bulky molecules may sterically hinder the immune synapse, leading to lower activation of immune receptor signalling, especially at low antigen densities. The tyrosine phosphatase CD45 restrains immune cell receptor signalling, and its exclusion from the immune synapse creates a phosphatase-free zone that is conducive to immune receptor signalling and subsequent immune cell activation (e.g., from CD3 signalling) or suppression (e.g., from PD-1 signalling). As CD45 has a large extracellular region, a short (~15 nM) inter-membrane distance between target cell and immune cell effectively excludes CD45, which is pushed out to a distal ring around the synapse. At larger synapses (> ~15 nM), CD45 dampens intracellular signals from the immune cell receptor, although this may be overcome in the presence of a high copy number of target pMHC being presented to the immune cell, resulting in a high number of immune cell receptor engagments and consequent high volume of signalling induced within the immune cell, outweighing the negative infuence of tyrosine phosphatase. Under many physiological conditions, however, the density of target pMHC is very low, so a tight immune synapse is required for effective immune cell signalling. Copy number of some target pMHC on the tumour cell surface has been estimated to be between 1-10 (Liddy et al., Nat Med 18(6):981-987 2012). Spatial constraints are therefore important when designing multispecific molecules to bridge the immune synapse and promote effective immune receptor signalling. Scientists at Immatics designed a format for a TCR-immunoglobulin bispecific molecule including a diabody-type structure coupled to an Fc domain. As disclosed in WO2019 / 012138, the bispecific comprises a first polypeptide chain comprising a first TCR variable domain, a first antibody variable domain and a first Fc region, paired with a second polypeptide chain comprising a second antibody variable domain, a second TCR variable domain and a second Fc region. The assembled bispecific, termed “TCER”, is a heterodimer comprising paired TCR variable domains forming a pMHC binding site and paired antibody domains forming a CD3 binding site, and paired Fc regions. This format benefits from extended in vivo half life, but is less potent than the “ImmTAC” (scFv-TCR fusion, described above). Dickopf, Georges &Brinkmann (Computational and Structural Biotechnology Journal 18:1221-1227 2020) discussed the importance of geometry for multispecific molecules that elicit intercellular processes such as the formation of immunological synapses. They illustrated that molecular formats of the same size and same domain sequences, but different geometries, had different efficacies. A compact (5-6 nm) format termed “contorsbody” was reported, composed of three antibody Fab regions, in which a central Fab (anti-CD3) is oriented anti-parallel to two antigen-targeting Fabs, so that the CD3-binding and target antigen-binding sites face in opposing directions. The small barrel-like design of the molecule and close proximity of the dual antigen-binding sites was suggested to lead to denser TCR clustering, and reportedly showed higher potency compared with regular Y-shaped IgG bispecific molecules. Froning et al. (J. Immunother Cancer 10:e004281 2022) compared soluble TCR-anti-CD3 bispecific formats. TCR-scFv, TCR-Fab and TCR-IgG anti-CD3 bispecifics were compared in a panel of assays for antigen binding and T cell redirected cell killing. The tandem fusion formats (TCR-scFv, TCR-Fab) showed reduced binding to CD3 compared with the TCR-IgG, which was attributed to the N-terminal fusion hindering the kinetics of CD3 engagement in the tandem formats. Despite this, however, the TCR-Fab and TCR-scFv were more active than the TCR-IgG in cell killing assays. Two different TCR-IgG anti-CD3 bispecific molecules, with TCRs specific for two different pMHC, were both inactive in assays forT cell engaging activity despite being capable of binding their cognate pMHC complex and CD3 in separate binding assays. Efforts to increase avidity by including an additional TCR moiety in the various formats did not improve their cell killing potency, and in most cases actually reduced activity. The authors concluded that only small, tandem bispecifics were capable of inducing strong redirected cytolytic activity against tumours, while IgG-like or Fc-containing molecules demonstrated poor activity because the larger and less flexible TCR-IgG is less able to form a tight immune synapse. WO2020 / 157211 described linkage of an Fc region or albumin-binding domain to an ImmTAC. Although these half-life extended molecules retain some function and would have the advantage of less frequent dosing compared with the original ImmTAC format, their larger size renders them less suitable for mediating formation of a tight immune synapse for cell killing. Efforts have been made to limit the loss of potency in ImmTAC molecules that incorporate a half life extending region. For example, WO2024 / 038193 described an alternative format of Fc-linked ImmTAC with reportedly higher potency than that in WO2020 / 157211. WO2024 / 038198 described an Fc-linked ImmTAC in a coiled single chain arrangement, which was also reported to have higher potency than that in WO2020 / 157211. While the ability of a multispecific synaptic bridging molecule to mediate potent immune cell signalling is key to its therapeutic activity, its specificity for target cells versus non-target cells is another major factor for clinical success. Some target pMHC are presented by normal tissues as well as target cells, and may differ only in the level of expression (copy number), requiring a drug product that discriminates between higher and lower level expression of target pMHC in order to limit toxicity. In this situation a lower-potency molecule offers greater safety, but its therapeutic efficacy may be compromised. Another risk is cross-reactivity of the therapeutic molecule. TCRs naturally have a relatively low affinity for pMHC, compared with the affinity of antibody:antigen interaction. TCRs used in soluble drug molecules are typically engineered to improve affinity of binding, but raising affinity may undesirably reduce specificity, where changes result in higher affinity binding to non-target antigen as well as to the target antigen (Zhao et aL, J Immunol 179:5845-5854 2007). A molecular format that enables high affinity binding at low target copy number, and potently induces immune cell signalling, has a potentially heightened risk of off-target reactivity and toxicity, requiring careful pre-clinical screening. Additionally, the format of the molecule must also render it suitable for industrial production - for example it may need to be efficiently expressed in a recombinant host cell, purified, formulated and stored. Research in the biopharmaceutical industry has been directed to designing a molecular format for a multispecific synaptic briding molecule that delivers across all these areas. Summary of the Invention The present invention relates to soluble multispecific proteins that bind a target pMHC on a target cell and a surface receptor on an immune cell, which incorporate a half life extending region attached via a cleavable linker. The proteins may be employed for recruiting an immune cell to a target cell presenting a target pMHC complex and / or to stimulate intracellular signalling from the surface receptor in the immune cell in the presence of the target cell, for example to induce or inhibit immune cell-mediated killing of target cells. The half life extending region confers a beneficial serum half life in vivo by reducing clearance of the multispecific protein from the circulation, but is cleavable to release an active complex comprising a pMHC binding arm and an immune cell engager arm. The active complex, when released from the half life extending region, has a higher potency for mediating formation of a tight immune synapse owing to its smaller, more compact structure. Cleavage of the linker may occur in the target tissue (e.g., tumour microenvironment), for example by engineering the linker to comprise a substrate for an enzyme that is specific to or enriched in the target tissue. The cleavable linker may comprise a substrate for one or more proteases that are selectively found in target tissue such as the tumour micro-environment. This further enhances the safety and selectivity of the molecule in vivo, as it transforms to its more potent form when in the proximity of the target cells, while remaining in the uncleaved, half life extended form when in the circulation and wider tissue environment. The combined advantages of extended half life, high potency, and improved safety may thus be provided by a compact and independently functional multispecific molecule fused to a half life extending region (e.g., Fc) by a digestible linker. Accordingly, in a first aspect the present invention provides a soluble multispecific protein, wherein the protein comprises an active complex comprising a pMHC binding arm and an immune cell engager arm, and a half life extending region, characterised in that the active complex is connected to the half life extending region by a cleavable linker comprising one or more protease cleavage sites, whereby free active complex is releasable from the half life extending region by protease cleavage of the linker. The cleavable linker connects the half life extending region to the pMHC binding arm and / or to the immune cell engaging arm. The linker may be linear or branched. For example, a single linker may connect the half life extending region to the active complex, e.g., to the pMHC binding arm. Both arms of the active complex are on one side of a cleavage site in the linker, so that cleavage of a single protease site in the linker releases free active complex (Figure 2). Alternatively, separate parallel linkers ora branched linker may connect the pMHC binding arm and immune cell engager arm respectively to the half life extending region. Digestion of parallel linkers, or branches of a linker, is required to release free active complex (Figure 3). This requires two cuts, involving more complex kinetics but with the potential to enhance safety and tissue specificity. Where multiple protease cleavage sites are included, these are optionally cleavage sites for multiple different proteases. Proteases may be of the same or different protease class. The protease is preferably enriched in the target cell environment. It may be a tumour specific protease. Preferably, the protease is secreted in the tumour micro-environment, optionally by the target cell. A protease is optionally selected from any of the following classes: metalloprotease serine protease cysteine protease threonine protease aspartic protease. Examples of serine proteases are granzyme (e.g., granzyme B, granzyme A, granzyme K), KLK (e.g., KLK2, KLK3, KLK4), FAP, DPP, PEP, uPA, CatG, ELANE and matriptase. Cleavage of the linker removes the half life extending region and releases the free active complex, which has an enhanced potency for stimulating immune cell receptor signalling. The multispecific protein may be active both before and after protease cleavage to release free active complex, i.e., the uncleaved form may already be capable of mediating immune synapse formation and stimulating signalling from the immune cell receptor. Activity may be measured in an in vitro assay with target cells presenting target pMHC. The multispecific protein may exhibit dose dependent activity for stimulating immune cells in the absence of protease. Activity is measured relative to negative control assay in which the test multispecific protein is absent. Negative control may include a multispecific protein with a pMHC binding arm that does not bind pMHC presented by the target cell. Free active complex may be determined to have greater potency than uncleaved multispecific protein in the assay, e.g., at least 2-fold greater, at least 5-fold greater, or at least 10-fold greater. Potency in an assay may be quantified as EC50, which is the concentration of the test molecule producing half-maximal response. The EC50 of the uncleaved protein is thus greater, e.g., at least 2-fold greater, at least 5-fold greater or at least 10-fold greater than the EC50 of the free active complex. The uncleaved soluble multispecific protein has a longer in vivo half life than the free active complex. The soluble multispecific protein may have an in vivo half life at least 2-fold, at least 5-fold, at least 10-fold greater than that of the free active complex. Suitable in vitro assays for measuring potency of immune cell activation are detailed elsewhere herein. An assay may comprise providing immune cells and target cells in vitro, incubating the immune cells and target cells with a soluble multispecific protein as defined in any of claims 1 to 37, in the presence or absence of protease, and detecting and comparing sigalling in the immune cells in the presence vs absence of protease, wherein increased signalling in the immune cells is detected in the presence of the protease. The immune cell engager arm binds a surface receptor of an immune cell. Binding of the immune cell surface receptor at an immune synapse activates intracellular signalling in the immune cell. In some embodiments, the immune cell is a T cell. The immune cell engager arm may bind the TCR-CD3 complex. It may bind CD3 polypeptide, e.g., CD3e, CD3y and / or CD36. It may bind the TOR, e.g., a TCR constant domain. However, care should be taken that the immune cell engager arm does not recognise the pMHC binding arm, otherwise the multispecific protein will be prone to aggregation. Alternatively the immune cell engager arm may bind another activating surface receptor such as CD28. The immune cell engager arm may be an anti-CD3 oranti-CD28 antibody molecule. The immune cell may be a cytotoxic immune cell, optionally a cytotoxic T cell. The immune cell may be a cytotoxic CD8+ T effector cell. The cell may be a natural killer (NK) cell. Surface receptors on NK cells include Fc receptors such as FcyRIII (CD16). The immune cell engager arm may be an anti-CD16 antibody molecule. Functionally, the immune cell engaging arm is an agonist of the surface receptor, thus it activates intracellular signalling in the immune cell. Binding to CD3 orCD28 activates intracellular signalling in a cytotoxic T cell, resulting in release of cytokines and cytolytic molecules, which kill the proximal target cell. Binding to an Fc receptor on an NK cell, e.g., FcyRIII, activates intracellular signalling in the NK cell, resulting in an ADCC response. Target cells are cells that present on their surface the target pMHC complex recognised by the pMHC binding arm of the multispecific molecule, and which are to be engaged by the immune cells in the present invention. In some embodiments, the target cell is a tumour cell, e.g., a malignant cell of a solid tumour such as sarcoma, carcinoma or lymphoma. The target pMHC may be preferentially expressed on disease-associated cells (e.g., tumour cells) relative to other tissues of the body. Its presentation may be restricted to such cells, i.e., the target pMHC may not be presented on other cells in the patient. Examples of tumour associated peptides that may be presented on pMHC include peptide fragments of PRAME and MAGE-A4. Multispecific proteins of the invention are suitable for targeting pMHC at a wide range of copy number. Signalling at the immune synapse mediated by the free active complex may be effective even at low density of target pMHC. Free active complex may generate a potent immune cell response in the presence of target cells, including at low levels of pMHC presentation typical of cancer cells. Copy number of target pMHC per cell may be in the range of 1 to 10 or 1 to 100, e.g., 5 to 10, 5 to 50, or 50 to 100. In addition to the combined benefits of high potency and extended half life, the invention may provide advantages in terms of its safety profile and in the range of addressable molecular targets. As the specificity of the free active complex is enhanced by the specific or enriched presence of the protease in the target tissue (e.g., TME), the multispecific protein format of the present invention has a reduced risk of undesired binding to its target pMHC when the target pMHC is outside the target tissue (e.g., “on-target, off-tumour” binding). This provides an improved safety profile for the molecule, with potentially fewer side effects in patients. Owing to their enhanced specificity, proteins of the invention may offer the possibility of targeting antigens that are not safely targetable using less selective binders. Thus, the invention may be used to target antigens that are expressed both in normal healthy tissue and in tumour tissue, even where the difference in expression level between normal and tumour tissue is less than ideal. This expands the potential target space, offering the possibility of bringing immune therapeutics to an increased number of pathologies. A target pMHC that is relatively densely presented on the target cell can be effectively engaged by a binder of relatively low affinity, compared with the affinity required for effective engagement with a low density target pMHC. The use of low affinity (e.g., affinity less than double digit picomolar) binders further improves the safety profile of the therapeutic molecule, by reducing the risk of off-target binding. By contrast, when a binding protein is strongly affinity matured to ensure very high affinity for its target, this can carry a risk of the selectivity for binding being reduced. The pMHC binding arm comprises a binding site that recognises a target pMHC on a target cell. Examples of pMHC binding molecules are known, including TCRs and TCR-mimetic antibodies. Thus, the pMHC binding arm may comprise a TCR molecule or an antibody molecule. The pMHC binding arm may comprise all or part of a TCR extracellular domain comprising a pMHC binding site. It may comprise TCR variable regions (optionally a TCR alpha variable domain and a TCR beta variable domain) which pair to provide the pMHC binding site. Optionally the pMHC binding arm further comprises one or more TCR constant regions, e.g., it may comprise paired TCR variable domains and paired TCR constant regions. An example pMHC binding arm comprises a dimer of first and second TCR polypeptide chains, each comprising an N terminal variable domain and a C terminal constant domain, wherein the two variable domains pair to provide the pMHC binding site, and wherein the two constant domains pair, optionally wherein the two constant domains are inter-linked via one or more disulphide bonds. The TCR molecule may be a heterodimer of TCRa and TCRp polypeptide chains, i.e., a first polypeptide comprising an N terminal Va domain and a C terminal Ca domain and a second polypeptide comprising an N terminal vp domain and a C terminal Cp domain. The immune cell engager arm comprises a binding site that recognises a surface receptor on an immune cell. The immune cell engager arm may comprise an antibody molecule, e.g., an antibody scFv, such as an anti-CD3 scFv. Single chain antibodies, such as scFvand domain antibodies like VHH, have a compact form well-suited to inclusion in the free active complex. The active complex may comprise an scFv immune cell engager arm linked to a pMHC binding arm comprising a first TCR polypeptide comprising a TCR variable domain and a TCR constant domain and a second TCR polypeptide comprising a TCR variable domain and a TCR constant domain. The active complex may comprise a fusion protein In which the C terminus of the immune cell engager is linked to the N terminus of a TCR molecule, for example as described in WO2010 / 133828, incorporated herein by reference. The active complex may comprise a first polypeptide comprising, from N to C terminus, an immune cell engager antibody molecule (optionally scFv), a first TCR variable domain and a first TCR constant domain, and a second polypeptide comprising, from N to C terminus, a second TCR variable domain and a second TCR constant domain. The active complex may be an ImmTAC, e.g., as claimed in US10130721B2. The half life extending region may comprise an antibody Fc region, serum albumin, a serum albumin binding peptide or other half life extension moiety such as a PEGylated domain. An antibody Fc (fragment crystallisable) region represents the tail region of an antibody molecule comprising antibody heavy chain constant regions CH2 and CH3, distal from the antigen-binding variable regions of the antibody. Fc has two polypeptide chains, each comprising CH2-CH3, which dimerise and are covalently linked by disulphide bonds. Naturally occurring or engineered forms of Fc may be utilised. Fc domains from immunoglobulin subclasses lgG1, lgG2 and lgG4 bind to and undergo FcRn mediated recycling, affording a long circulatory half-life (3 - 4 weeks), thus extending the half life of the multispecific protein of the invention. The interaction of IgG with FcRn has been localized in the Fc region covering parts of the CH2 and CH3 domains. The hinge (upper and / or lower hinge) region of the antibody may be included with the Fc region. The protease digestion site may be added N terminal to, or engineered within, a hinge region of an Fc chain, e.g., above the disulphide linked Cys residues (Figure 2). In other embodiments the protease digestion site may be inserted or engineered within the Fc region below the disulphide linked Cys residues (Figure 3). The approach of the present invention may be exploited to improve potency of molecules such as those described in WO2020 / 157211 or WO2024 / 038193, which disclose half life extended ImmTAC molecules, by introducing a protease cleavage site between the ImmTAC (active complex) and the half life extending region. For example, to improve the molecule illustrated in Figure 1A, Figure 1 B(i) or Figure 10 of WO2024 / 038193, a protease cleavage site is introduced at each point where a TCR domain is fused to an Fc chain. In one embodiment, the active complex comprises a first polypeptide comprising, from N to C terminus, an immune cell engager antibody molecule (optionally VHH or scFv), a first TCR variable domain and a first TCR constant domain, and a second polypeptide comprising, from N to C terminus, a second TCR variable domain and a second TCR constant domain, and a half life extending region comprising an Fc region, the Fc region being a dimer of first and second CH2-CH3 chains, wherein the C terminus of the second polypeptide of the active complex is connected via a cleavable linker to the N terminus of a first Fc CH2-CH3 chain, and the second Fc CH2-CH3 chain has a free N terminus. An example is illustrated in Figure 2, comprising a first polypeptide chain comprising, from N to C terminus, VH-VL (immune cell engager) - TCR Va - TCR Ca - cleavable linker - FC chain 1, a second polypeptide comprising, from N to C terminus, TCR Vp - TCR Cp, and a third polypeptide comprising Fc chain 2. In some embodiments, additional moieties are attached to the half life extending region. For example, cytokines such as IFNy, may be fused to the half life extending region, optionally in multiple copies. Release of IFNy may promote T cell activation, including cytotoxic activity. Alternatively, immune checkpoint molecules such as PD-1 may be fused to the half life extending region. For example, as described in WO2024 / 102954, Fc may be linked to PD-1, so that release of the Fc binds PD-L1 on target cells and inhibits PD-L1 mediated immune suppression signals. Such strategies may be used to further boost activity of the immune cell, especially in cytotoxic contexts where killing of target cells is desired. A further aspect of the invention relates to nucleic acid, e.g., DNA or RNA, encoding the soluble multispecific protein. Molecules of the invention, including nucleic acids and proteins, may be provided in isolated form, optionally with one or more pharmaceutically acceptable excipients. The molecules may be provided for use in methods of treating the human or animal body by therapy. Where humans are to be treated, proteins herein are preferably human in order to reduce immunogenicity concerns, the immune cell receptor targeted is a human immune cell receptor, and the MHC is human MHC. Further aspects of the invention thus relate to methods of treating a condition in a patient (e.g., human patient), comprising administering the soluble multispecific protein or its encoding nucleic acid to the patient. The condition to be treated may be a solid tumour such as sarcoma, carcinoma or lymphoma, or an infection (e.g., viral infection, optionally a tumour-associated viral infection). A population of recombinant host cells may be provided in vitro comprising nucleic acid encoding the soluble multispecific protein. The protein may be produced by a method comprising culturing the cells under conditions for expression of the protein, recovering the protein from the cell culture, and optionally purifying the protein by one or more additional steps. Purified protein may be formulated into pharmaceutical compositions comprising one or more excipients, and provided for delivery as described above. Brief Description of the Drawings Figure 1 illustrates a representation of engagement between pMHC of a target cell (above) and immune cell receptors comprising TCR, CD3 and CD8 on a T cell (below) at an immune synapse. The TCR engages the pMHC complex across the synapse between adjacent cell membranes. The class I MHC is composed of an alpha chain comprising domains a1, a2 and a3. N terminal domains a1 and a2 provide a binding site for peptide p. Beta 2 microglobulin (p2m) associates with the alpha chain. The TCR is composed of an a chain and a p chain. Each chain comprises an N terminal variable region, a constant region, a connecting region, a transmembrane region and a short cytosolic C-terminal tail. The TCR is complexed with CD3 polypeptides. Dimeric co-receptor CD8 of the T cell binds a3 domain of the MHC class I molecule. CD8 is a heterodimer of an a chain and a p chain, and is associated with intracellular signalling molecule Lek. The figure was created with BioRender. Figure 2 shows an embodiment of the invention with bispecificity in cis. The dashed line indicates an engineered digestion site that can be cut by one or more protease(s) abundant in the tumour micro-environment (e.g. KLK2 / 3 / 4 in prostate). A single cut releases free active complex. Figure 3 shows an embodiment of the invention with bispecificity in trans. The dashed line indicates an engineered digestion site that can be cut by one or more protease(s) abundant in the tumour micro-environment (e.g. KLK2 / 3 / 4 in prostate). Two cuts are required to release free active complex. Detailed Description TCR molecules In humans, the native T cell receptor comprises two transmembrane polypeptides, which may be an ap pair or a y6 pair, associated with the CD3 complex. Each of the two paired TCR polypeptide chains has an N terminal variable domain comprising three hypervariable complementarity determining regions (CDRs), a constant domain, and a connecting region linked to a transmembrane domain and C terminal cytosolic tail. TCR polypeptides lack a signal transduction domain, and instead signalling occurs through the CD3 components of an octomeric complex comprising TCRap:CD36E:CD3yE:CD3^, which assemble in a 1:1:1:1 stoichiometry. The CD3e, CD3y and CD30 subunits are transmembrane proteins each containing a single immune receptor tyrosine-based activation motif (ITAM), and CD3£ is cytosolic and contains three ITAMs. In a structure analagous to an antibody’s antigen-binding fragment (Fab) binding domain, the extracellular region of the TCR comprises two paired variable domains and two paired constant domains, and the pMHC binding site of the TCR is formed by a set of 6 CDRs, comprising three CDRs from one variable domain and three CDRs from the second variable domain, with the CDR3 loops being centrally located and primarily responsible for recognition of the peptide. A TCR molecule used in the present invention may comprise a pMHC-binding region of a TCR. To provide the protein in soluble form, the TCR molecule does not comprise the transmembrane domain or cytoplasmic tail of a full length T cell receptor. The TCR constant region may be truncated to remove membrane-associated and cytoplasmic portions from the C-terminal end. The pMHC binding arm may comprise all or part of a TCR extracellular domain comprising a pMHC binding site. It may comprise TCR variable regions (optionally a TCR alpha variable domain and a TCR beta variable domain) which pair to provide the pMHC binding site. The TCR molecule may further comprise one or more constant domains. A constant domain may be a constant domain of a TCRa chain (Ca domain) or of a TCRp chain (Cp domain). Alternatively a pMHC binding arm may comprise one or more antibody constant domains, which may be from an antibody light or heavy chain (e.g., CL, CH1, CH2, CH3 or CH4). A pMHC binding arm may comprise paired TCR variable domains and paired TCR constant regions. An example pMHC binding arm comprises a dimer of first and second TCR polypeptide chains, each comprising an N terminal variable domain and a C terminal constant domain, wherein the two variable domains pair to provide the pMHC binding site, and wherein the two constant domains pair, optionally wherein the two constant domains are inter-linked via one or more disulphide bonds. The TCR molecule may be a heterodimer of TCRa and TCRp polypeptide chains, i.e., a first polypeptide comprising an N terminal Va domain and a C terminal Ca domain and a second polypeptide comprising an N terminal Vp domain and a C terminal Cp domain. The amino acid sequence of the variable and constant domain may correspond to those found in nature, or they may contain one or more mutations relative to a natural protein. Such mutations may be made to increase the affinity of the pMHC binding domain for a given antigen. Additionally or alternatively, mutations may be incorporated to improve stability and manufacturability. Additional mutations may be introduced into the amino acid sequence of a constant domain relative to a natural constant domain. The constant domains may also include residues, either naturally-occurring or introduced, that allow for dimerisation by, for example, a disulphide bond between two cysteine residues. One or both of a Ca and Cp constant domain may contain mutations, substitutions or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. Alpha and beta chain constant region sequences may be modified by truncation or substitution to delete the native disulphide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 orTRBC2. Alpha and / or beta chain constant region sequence(s) may have an introduced disulphide bond between residues of the respective constant domains, as described, for example, in WO 2003 / 020763, WO 2004 / 033685 and WO 2006 / 000830, and for example, in U.S. Patent Nos. 7,329,731,7,569,664; and 8,361,794, the contents of each of which are herein incorporated by reference. Alpha and beta constant domains may be modified by substitution of cysteine residues at position Thr48 of TRAC and position Ser 57 of TRBC1 orTRBC2, the said cysteines forming a disulphide bond between the alpha and beta constant domains of the TCR. TRBC1 orTRBC2 may additionally include a cysteine to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant regions present in an ap heterodimer may be truncated at the C terminus or C termini, for example by up to 15, or up to 10, or up to 8 or fewer amino acids. The C terminus of an alpha chain extracellular constant region may be truncated by 8 amino acids. TCR sequences are described with reference to IMGT nomenclature which is widely known and accessible to those working in the TCR field. For example, see LeFranc and LeFranc "T cell Receptor Factsbook", Academic Press 2001; Lefranc, Cold Spring Harb Protoc (6):595-603 2011; Lefranc, Curr Protoc Immunol 2001 Appendix 1 : Appendix 100; and Lefranc, Leukemia 17(1): 260-266 2003. The TCR molecule may be a single chain TCR molecule. In principle, extracellular regions of the TCR alpha and beta chains may be connected by a peptide linker and recombinantly expressed to provide a soluble, single chain TCR molecule. Various TCR engineering efforts have been directed toward the production of stable single chain TCR molecules, capable of expression at high levels and recovery with good yield. WO99 / 18129 described a soluble single chain TCR in which a covalently linked TCR alpha chain portion and beta chain portion were fused to an immunoglobulin light chain constant region. It also reported that a variety of scTCR molecules could be made without fusing the Ig-CL to the molecule. One example of a soluble scTCR comprised Va, Ca fragment, peptide linker, Vp. Another example of a soluble scTCR comprised: Va domain, Ca domain or a fragment thereof, peptide linker, vp domain, and a Cp domain or fragment thereof. Innovative Targeting Solutions WO2017 / 091905 described single unpaired variable domains of TCRs as soluble binding molecules, optionally lacking constant domains or portions thereof. Oh et al, Sci Rep 9:17291 2019, described a “single variable domain TCR” or “svd TCR” comprising a p variable domain of a TCR, unpaired with an a variable domain. Single chain TCRs are further described in WO2004 / 033685; W098 / 39482; WO01 / 62908; Weidanz et al. (1998) J Immunol Methods 221 (1 -2): 59-76; Hoo et al. (1992) Proc Natl Acad Sci U S A 89(10): 4759-4763; Schodin (1996) Mol Immunol 33(9): 819-829). Other suitable TCR chain amino acid sequences are provided in WO2011001152, WO2017109496, WO2017175006 and WO2018234319, and, for example, in U.S. Patent Nos.8,519,100, 11,639,374, 11,505,590, and 11,427,624, the contents of each which are herein incorporated by reference. The TCR molecule may be a human TCR molecule. Where constant domains are present these are preferably human constant domains. Antibody molecules In humans, the most common native format of an antibody is an IgG which is a heterotetramer consisting of two identical heavy chains and two identical light chains. The heavy and light chains are made up of modular domains with a conserved secondary structure consisting of a four-stranded antiparallel beta-sheet and a three-stranded anti-parallel beta sheet, stabilised by a single disulphide bond. Antibody heavy chains each have an N terminal variable domain (VH) and 3 relatively conserved "constant" immunoglobulin domains (CH1 , CH2, CH3) while the light chains have one N terminal variable domain (VL) and one constant domains (CL). Disulphide bonds stabilise individual domains and form covalent linkages to join the four chains in a stable complex. The VL and CL of the light chain associates with VH and CH1 of the heavy chain and these elements can be expressed alone to form a Fab fragment. The CH2 and CH3 domains (also called the "Fc domain") associate with another CH2:CH3 pair to give a tetrameric Y shaped molecule with the variable domains from the heavy and light chains at the tips of the "Y". The CH2 and CH3 domains are responsible for the interactions with effector cells and complement components within the immune system. Antibody variable domains have sequence variability in their complementarity determining regions (CDRs), which are loops primarily involved in antigen recognition. A VH domain comprises VH CDR1, VH CDR2 and VH CDR3 in a VH domain framework, and a VL domain comprises VL CDR1, VL CDR2 and VL CDR3 in a VL domain framework. The bound antigen may make contact with a subset of residues within the six CDRs, for example in one or more particular CDRs such as the heavy chain CDR3 and / or light chain CDR3. An antibody molecule used in the present invention comprises an antigen-binding region of an antibody. It may be an antibody fragment and / or a synthetic antibody construct. The antibody molecule may comprise an antibody VH and / or VL domain. It may be monovalent for binding to its antigen. The antibody molecule may be a Fab, consisting of a dimer of VH:CH1 and an antibody light chain (VL:CL). It may be a single chain antibody molecule. The antibody molecule may be a single chain Fv (scFv), consisting of VH and VL fragments fused by a flexible linker. In the scFv molecule, the VH and VL domains form a VH-VL pair in which the complementarity determining regions of the VH and VL come together to form an antigen binding site. Antibody fragments that comprise an antibody antigen-binding site include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment, which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) scFv, wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) a diabody, a multivalent or multispecific fragment constructed by gene fusion (WO94 / 13804). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains. Various other antibody molecules including one or more antibody antigen-binding sites have been engineered, including for example Fab2, Fab3, diabodies, triabodies, tetrabodies and minibodies (small immune proteins). Antibody molecules and methods fortheir construction and use were described by Holliger &Hudson, Nature Biotechnology 23(9): 1126-1 136 (2005). Other examples of binding fragments are Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region, and Fab'-SH, which is a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group. A dAb (domain antibody) is a small monomeric antigen-binding fragment of an antibody, namely the variable region of an antibody heavy or light chain. VH dAbs occur naturally in camelids (e.g., camel, llama) and may be produced by immunizing a camelid with a target antigen, isolating antigen-specific B cells and directly cloning dAb genes from individual B cells. dAbs are also producible in cell culture. Their small size, good solubility and temperature stability makes them particularly physiologically useful and suitable for selection and affinity maturation. Camelid VH dAbs are being developed for therapeutic use under the name "nanobodies™". The antibody molecule may be a human antibody molecule. Thus, where constant domains are present these are preferably human constant domains. Potency of active complex Potency of a molecule may be determined in an assay of immune synapse engagement, such as an assay of target cell killing by immune cells or an immune cell reporter gene assay. Intracellular signalling from the immune cell receptor may be detected and quantified using immune cells engineered to generate a detectable product downstream of the signalling pathway, e.g., a Jurkat cell NFAT reporter assay. Depending on immune cell type, signalling may alternatively be measured as the release of activation markers (e.g., IFNy), immune cell proliferation, or killing of target cells. With cytotoxic immune cells such as CTL, activation of the immune cell receptor (e.g., by CD3 engaging arm of the multispecific protein) may be measured as killing of target cells in an in vitro cell killing assay. An in vitro assay of target cell killing by T effector cells (“T cell killing assay”) may comprise: providing target cells (e.g., a tumour cell line) presenting target pMHC providing T effector cells (e.g., PBMC or isolated CD8+ T cells) incubating the cells with a dilution series of the multispecific protein detecting killing of the target cells by the T effector cells; and calculating EC50 for the killing of the target cells. EC50 is quantified by plotting a dose response curve and finding the concentration of the test molecule producing half-maximal response. A full dose-response curve should be obtained, in which the 2 highest and 2 lowest points have reached a plateau. Parameters will be optimised for the assay according to the target cells and effector cells employed. A ratio of 5:1 for effector cells to T cells may be utilised, and the cells incubated for between 48 - 96 hours (e.g., 72 hours). Cells may be co-cultured in a 1:1 mix of RPMI and DMEM. The cell killing assay may be performed with a tumour cell line presenting the target pMHC. A375-GFP is a tumour cell line which presents a pMHC complex of a MAGEA4 peptide. Copy number of target pMHC per target cell in the in vitro assay may be in the range of 1 to 10 or 1 to 100, e.g., 5 to 10, 5 to 50, or 50 to 100. A potent response may be one with an EC50 value in the nM - pM range, for example 500 nM or lower, e.g., 1 nM or lower, or 500 pM or lower. EC50 may be 100 pM or lower, 50 pM or lower, 40 pM or lower, 30 pM or lower, 20 pM or lower or 10 pM or lower. Preferably the maximum inhibition obtained in reporter assays is greater than 50%, for example 80% or more. Active complex may have a higher potency than the uncleaved soluble multispecific protein for inducing target-dependent signalling in the immune cell. The EC50 of the uncleaved protein may be greater, e.g., at least 2-fold greater, at least 5-fold greater or at least 10-fold greater than the EC50 of the free active complex. For increased safety, the active complex may optionally be provided with a masked binding site (pMHC binding site and / or immune cell receptor binding site). Examples such as masking peptides are known in the art and have been described for example for protecting anti-CD3 binding sites (e.g., Janux WO2022 / 125566). However, due to the enhanced safety profile of the molecule conferred by a site-specific cleavable linker, such masking technology is often not required. In many embodiments, therefore, no masking is present and binding sites of the active complex are available for antigen binding. Half life extending region The uncleaved soluble multispecific protein has a longer in vivo half life than the free active complex. Half life is the length of time taken for the concentration of the protein to decrease to half of its starting dose in the body. Half life of a molecule may be determined by methods known in the art. Concentration is conveniently measured as serum / plasma concentration in peripheral blood, representing half life of the molecule in circulation. The multispecific protein may have an in vivo half life similar to comparator Fc-fusion molecules such as the “TCER” format described by Immatics and / or comparable to that of an IgG antibody. The pharmacokinetics (PK) of the protein will depend on the specificity of the protease(s) for the cleavage site and on the availability of the protease(s) outside the target tissue (e.g., TME). The half life of a multispecific protein can be measured by pharmacokinetic studies, e.g., as described by Kim et al., Eur J of Immunol 24:542 1994. According to this method, radiolabelled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example at 3 minutes to 72 hours after the injection. Alternatively, unlabelled protein of the disclosure can be injected and its plasma concentration periodically measured using an ELISA. The clearance curve thus obtained should be biphasic, that is, an alpha phase and beta phase. For the determination of the in vivo half life of the protein, the clearance rate in beta phase is calculated and compared with that of the wild type or unmodified protein. Tissue-specific activation by proteases Removal of the half life extending region preferably occurs at the local site, within the target tissue, e.g., tumour microenvironment. Cleavage occurring at the site of action, e.g., in a tumour microenvironment in which the target antigen is presented, increases the activity of the multispecific molecule at the local site. The molecule is therefore in its half life extended form (e.g., Fc-fusion form) in circulation, but is converted to a more potent compact format once at the target site (e.g., tumour microenvironment). The linker may comprise a sustrate sequence for a protease that is secreted by, or enriched in the proximity of, the target cells, such as a tumour specific protease. Proteases are known that are relatively highly expressed, and / or are relatively active, within the target tissue (e.g., tumour microenvironment) compared with the blood. As many tumours require proteases to remodel the extracellular matrix, invade tissues or evade / suppress immune response, the presence of the protease component is directly linked with the tumour pathology. Active tumours are therefore likely to retain a continuing protease activity, rendering them susceptible to enhanced targeting by means of the present invention. Therapeutic use A methods of treating a condition in a patient (e.g., human patient) comprises administering the soluble multispecific protein to the patient. The condition to be treated may be a solid tumour such as sarcoma, carcinoma or lymphoma, or an infection (e.g., viral infection, optionally a tumour-associated viral infection). Copy number of target pMHC per target cell in the disease tissue, e.g., solid tumour, may be in the range of 1 to 10 or 1 to 100, e.g., 5 to 10, 5 to 50, or 50 to 100. Therapy may be provided within a hospital environment, for which the molecules may be formulated for delivery in sterile aqueous solution, optionally provided within a bag connected to a drip line for intravenous infusion to a patient, either by flow under gravity (optionally with regulated flow) or in connection with an infusion pump to control flow rate and / or dose. The following numbered clauses represent aspects of the invention. 1. A soluble multispecific protein comprising an active complex comprising a pMHC binding arm and an immune cell engager arm, and a half life extending region, characterised in that the active complex is connected to the half life extending region by a cleavable linker comprising a protease cleavage site, whereby free active complex is releasable from the half life extending region by protease cleavage of the linker. 2. A protein according to clause 1, wherein the cleavable linker connects the half life extending region to the pMHC binding arm. 3. A protein according to clause 1, wherein the cleavable linker connects the half life extending region to the immune cell engaging arm. 4. A protein according to clause 1, wherein the cleavable linker connects the half life extending region to the pMHC binding arm and the immune cell engaging arm. 5. A protein according to any preceding clause, wherein the cleavable linker comprises a single protease cleavage site. 6. A protein according to any of clauses 1 to 4, wherein the cleavable linker comprises multiple protease cleavage sites. 7. A protein according to clause 6, wherein the cleavable linker comprises cleavage sites for multiple proteases. 8. A protein according to clause 6 or clause 7, wherein the cleavable linker comprises parallel protease cleavage sites, whereby release of free active complex requires cleavage of at least two of said protease cleavage sites. 9. A protein according to clause 6 or clause 7, wherein the cleavable linker comprises serial protease cleavage sites, whereby cleavage at any one of said protease cleavage sites releases free active complex. 10. A protein according to any preceding clause, wherein the protease is secreted in the tumour micro-environment. 11. A protein according to any preceding clause, wherein the protease is a metalloprotease, serine protease (e.g., granzyme, KLK, FAP, DPP, PEP, uPA, CatG, ELANE or matriptases), cysteine protease, threonine protease or aspartic protease. 12. A protein according to clause 11, wherein the cleavable linker comprises a serine protease cleavage site. 13. A protein according to clause 12, wherein the protease is granzyme B, granzyme A or granzyme K. 14. A protein according to clause 12, wherein the protease is KLK2, KLK3 or KLK4. 15. A protein according to any preceding clause, wherein the protein exhibits dose dependent activity in an immune cell killing assay with target cells presenting target pMHC, in the absence of protease. 16. A protein according to any preceding clause, wherein the free active complex has a potency at least 2-fold greater than uncleaved soluble multispecific protein in an immune cell killing assay with target cells presenting target pMHC. 17. A protein according to any preceding clause, wherein the soluble multispecific protein has an in vivo half life at least 10-fold greater than that of the free active complex. 18. A protein according to any preceding clause, wherein the immune cell is a cytotoxic immune cell, optionally a T cell. 19. A protein according to any preceding clause, wherein the Immune cell is a cytotoxic CD8+ T effector cell. 20. A protein according to any preceding clause, wherein the immune cell engager arm binds CD3 or CD28. 21. A protein according to any preceding clause, wherein the target cell is a cancer cell of a solid tumour. 22. A protein according to clause 21, wherein the solid tumour is sarcoma, carcinoma or lymphoma. 23. A protein according to any preceding clause, wherein the pMHC binding arm comprises all or part of a TCR extracellular domain comprising a pMHC binding site. 24. A protein according to clause 23, wherein the pMHC binding arm comprises TCR variable regions (optionally a TCR alpha variable domain and a TCR beta variable domain) which pair to provide the pMHC binding site. 25. A protein according to clause 24, wherein the pMHC binding arm comprises paired TCR variable domains and paired TCR constant regions. 26. A protein according to clause 25, wherein the pMHC binding arm comprises a dimer of first and second TCR polypeptide chains, each comprising an N terminal variable domain and a C terminal constant domain, wherein the two variable domains pair to provide the pMHC binding site, and wherein the two constant domains pair, optionally wherein the two constant domains are inter-linked via one or more disulphide bonds. 27. A protein according to any of clauses 1 to 22, wherein the pMHC binding arm comprises an antibody molecule comprising a binding site for the target pMHC. 28. A protein according to any preceding clause, wherein the immune cell engager arm comprises an antibody molecule comprising a binding site for an immune cell surface receptor. 29. A protein according to clause 27 or clause 28, wherein the antibody molecule comprises an antibody VH domain and an antibody VL domain, wherein the VH domain and VL domain pair to provide the binding site. 30. A protein according to any preceding clause, wherein the immune cell engager arm comprises a single chain antibody molecule. 31. A protein according to clause 30, wherein the antibody molecule is an scFv. 32. A protein according to any of clauses 1 to 28, wherein the antibody molecule is a single domain antibody. 33. A protein according to any of clauses 1 to 30, wherein the active complex comprises a first polypeptide comprising, from N to C terminus, an immune cell engager antibody molecule (optionally scFv), a first TCR variable domain and a first TCR constant domain, and a second polypeptide comprising, from N to C terminus, a second TCR variable domain and a second TCR constant domain. 34. A protein according to any preceding clause, wherein the half life extending region comprises an antibody Fc. 35. A protein according to clause 34, wherein the active complex comprises a first polypeptide comprising, from N to C terminus, an immune cell engager antibody molecule (optionally VHH or scFv), a first TCR variable domain and a first TCR constant domain, and a second polypeptide comprising, from N to C terminus, a second TCR variable domain and a second TCR constant domain, and a half life extending region comprising an Fc region, the Fc region being a dimer of first and second CH2-CH3 chains, wherein the C terminus of the second polypeptide of the active complex is connected via a cleavable linker to the N terminus of a first Fc CH2-CH3 chain, and the second Fc CH2-CH3 chain has a free N terminus. 36. A protein according to any preceding clause, wherein the immune cell engager arm binds CD3 and the target cell is a solid tumour cell presenting a pMHC complex comprising a PRAME peptide. 37. A protein according to any preceding clause, comprising one or more cytokines (e.g., IFNy) or immune checkpoint molecules (e.g., PD-1) linked to the half life extending region. 38. An in vitro method of recruiting an immune cell to a target cell presenting a target pMHC complex, comprising providing immune cells and target cells in vitro, incubating the immune cells and target cells with a soluble multispecific protein as defined in any of clauses 1 to 37, in the presence or absence of protease, and detecting and comparing sigalling in the immune cells in the presence vs absence of protease, wherein increased signalling in the immune cells is detected in the presence of the protease. 39. Nucleic acid encoding a soluble multispecific protein according to any of clauses 1 to 37. 40. A method of treating a condition in a patient, comprising administering a soluble multispecific protein as defined in any of clauses 1 to 37, or its encoding nucleic acid, to the patient. 41. A soluble multispecific protein according to any of clauses 1 to 37, or its encoding nucleic acid, for use in method of treatment of the human or animal body by therapy. 42. A method according to clause 40 or a soluble multispecific protein or nucleic acid for use according to clause 41, wherein the treatment comprises treating a solid tumour. 43. A population of recombinant host cells in vitro comprising nucleic acid encoding a soluble multispecific protein according to any of clauses 1 to 37. 44. A method of producing a soluble multispecific protein according to any of clauses 1 to 37, comprising culturing a population of cells according to clause 43 under conditions for expression of the protein, recovering the protein from the cell culture, and optionally purifying the protein by one or more additional steps. Examples Example 1 The immune synapse is a tight and busy space. A stable and closely-packed TCR-pMHC microcluster is key to forming an active immune synapse. A T cell engaging TCR bispecific molecule with a small, compact structure is best suited for effectively stimulating TCR signalling. The molecule should be able to be accommodated within the inter-membrane distance of approximately 15 mM. This will permit and promote TCR-dependent cellular cytotoxicity, via tight packing of multiple TCR-pMHC forming the immune synapse. Comparative experiment: T cell killing assay with scFv-TCR in ImmTAC format A bispecific molecule is tested in an in vitro cell killing assay, using A375-GFP as target cells for killing by PBMC or purified human CD8+T cells as effectors. A375-GFP (P20122, Innoprot) is an adherent melanoma-derived cell line that stably expresses tGFP and presents a pMHC with a MAGE-A4 peptide. It is grown in complete DMEM and is passaged twice a week at 1:8 or 1:10. (Complete DMEM - DMEM, 10% FBS, 2 mM Glutamine or Glutamax, 5 ml Penicillin-Streptomycin) TDCC assay set up The day prior to running the assay, PBMC or CD8 T cells vials (5 million per 96 well plate) are thawed as required in complete RPMI (RPMI, 10% FBS, 2 mM Glutamax, 2 mM Pyruvate, 1% MEM, 1% HEPES, 1% P / S) and allow to recover overnight. A375-GFP is trypsinised and seeded in flatbottom transparent 96 well plate at 1.104 cells per well in 100 uL complete DMEM. Outer wells of the plate are excluded and filled with 200uL of PBS or RPMI. On the day of the assay, the test bispecific molecule is serial diluted in complete RPMI at 4X concentration in 100 ul final volume. 100 uL of effector cells (PBMC or CD8+ T cells) at 1.106 c / mL are transferred on the bispecific plates. This dilutes the bispecific at 2X in a total volume of 200 uL. 100 ul of bispecific:effector mix is transferred onto A375-GFP plates. This creates a ratio of 1:5 of Target to Effector and dilutes the bispecific to 1X. This also provides the right medium for both cell types to grow. Running assay program Plates are loaded in a live cell analysis incubator (Incucyte S3) and the instrument is programmed to scan 4 images / well every 12h until manually stopped. At the end of the experiments, supernatant may be collect to run further complementary assay such as cytokine ELISA (Interferon gamma, Tumour necrosis factor, Interleukin 2 or6) and LDH release. Green fluorescence intensity is extracted and exported and data analysis involved plotting timecourse and dose curves for each molecule tested. Bispecific The bispecific scFv-TCR is provided in the format of an ImmTAC as described by Immunocore (WO2010 / 133828). It comprises a soluble TCR extracellular domain that binds a target pMHC from MAGE-A4 as described in WO2017 / 175006 (TCR a19ka; b15), fused to the anti-CD3 antibody scFv arm of tebentafusp. The starting concentration of bispecific molecule was set at 100 nM and serially diluted 1 in 5 to 2.5 x 10 4 nM (9 dilution points) and a control well with effector and target cells only 5 without bispecific. Results Dose curves were obtained for the bispecific with A375-GFP and PBMC / CD8 T cells as effector. The bispecific had a mean EC50 of 0.0025 nM with both types of effector cell. The bispecific is a potent mediator of immune synapse formation and produces effective T cell killing 10 of target cells in the assay. Example 2 Linkage of an Fc region oralbumin-binding domain to an scFv-TCR bispecific “ImmTAC”, as described for example in WO2020 / 157211 or WO2024 / 038193, increases its size and reduces its potency for mediating tumour cell killing by T cells. 15 An Fc region can be linked to the bispecific molecule via a cleavable linker comprising a protease cleavage site. Protease digestion releases the Fc and generates free scFv-TCR (active complex in “ImmTAC” format). Figure 2. Removal of the Fc region will improve potency in a cell killing assay.

Claims

1. A soluble multispecific protein for recruiting an immune cell to a target cell presenting a target peptide major histocompatibility complex (pMHC complex), wherein the protein comprises an active complex comprising a pMHC binding arm and an immune cell engager arm, and a half life extending region, whereinthe active complex is connected to the half life extending region by a cleavable linker comprising a protease cleavage site, whereby free active complex is releasable from the half life extending region by protease cleavage of the linker.

2. A protein according to claim 1, wherein the cleavable linker connects the half life extending region to the pMHC binding arm.

3. A protein according to claim 1, wherein the cleavable linker connects the half life extending region to the immune cell engaging arm.

4. A protein according to claim 1, wherein the cleavable linker connects the half life extending region to the pMHC binding arm and the immune cell engaging arm.

5. A protein according to any preceding claim, wherein the cleavable linker comprises a single protease cleavage site.

6. A protein according to any of claims 1 to 4, wherein the cleavable linker comprises multiple protease cleavage sites, optionally cleavage sites for multiple proteases.

7. A protein according to claim 6, wherein the cleavable linker comprises parallel protease cleavage sites, whereby release of free active complex requires cleavage of at least two of said protease cleavage sites.

8. A protein according to claim 6, wherein the cleavable linker comprises serial protease cleavage sites, whereby cleavage at any one of said protease cleavage sites releases free active complex.

9. A protein according to any preceding claim, wherein the protease is secreted in the tumour micro-environment.

10. A protein according to any preceding claim, wherein the protease is a metalloprotease, serine protease (e.g., granzyme, KLK, FAP, DPP, PEP, uPA, CatG, ELANE or matriptases), cysteine protease, threonine protease or aspartic protease.

11. A protein according to any preceding claim, wherein the protein exhibits dose dependent activity for stimulating immune cells in an assay with target cells presenting target pMHC, in the absence of protease.

12. A protein according to any preceding claim, wherein the free active complex has a potency at least 2-fold greater than uncleaved soluble multispecific protein for stimulating immune cells in an assay with target cells presenting target pMHC.

13. A protein according to any preceding claim, wherein the soluble multispecific protein has an in vivo half life at least 10-fold greaterthan that of the free active complex.

14. A protein according to any preceding claim, wherein the immune cell is a cytotoxic immune cell, optionally a T cell.

15. A protein according to any preceding claim, wherein the immune cell engager arm binds CD3.

16. A protein according to any preceding claim, wherein the target cell is a cancer cell of a solid tumour.

17. A protein according to any preceding claim, wherein the pMHC binding arm comprises all or part of a TCR extracellular domain comprising a pMHC binding site.

18. A protein according to claim 17, wherein the pMHC binding arm comprises a dimer of first and second TCR polypeptide chains, each comprising an N terminal variable domain and a C terminal constant domain, wherein the two variable domains pair to provide the pMHC binding site, and wherein the two constant domains pair, optionally wherein the two constant domains are inter-linked via one or more disulphide bonds.

19. A protein according to any preceding claim, wherein the immune cell engager arm comprises an antibody molecule comprising a binding site for an immune cell surface receptor.

20. A protein according to claim 19, wherein the immune cell engager arm comprises a single chain antibody molecule, optionally scFv or a single domain antibody.

21. A protein according to any preceding claim, wherein the active complex comprises a first polypeptide comprising, from N to C terminus, an immune cell engager antibody molecule (optionally scFv), a first TCR variable domain and a first TCR constant domain, and a second polypeptide comprising, from N to C terminus, a second TCR variable domain and a second TCR constant domain.

22. A protein according to any preceding claim, wherein the half life extending region comprises an antibody Fc.

23. A protein according to claim 22, whereinthe active complex comprises a first polypeptide comprising, from N to C terminus, an immune cell engager antibody molecule (optionally VHH or scFv), a first TCR variable domain and a first TCR constant domain, and a second polypeptide comprising, from N to C terminus, a second TCR variable domain and a second TCR constant domain, anda half life extending region comprising an Fc region, the Fc region being a dimer of first and second CH2-CH3 chains, whereinthe C terminus of the second polypeptide of the active complex is connected via a cleavable linker to the N terminus of a first Fc CH2-CH3 chain, and the second Fc CH2-CH3 chain has a free N terminus.

24. A protein according to any preceding claim, wherein the immune cell engager arm binds CD3 and the target cell is a solid tumour cell presenting a pMHC complex comprising a PRAME peptide.

25. Nucleic acid encoding a soluble multispecific protein according to any preceding claim.

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