Tumour-transforming multispecific proteins

Abstract

Soluble multispecific protein that binds a target peptide MHC complex on a target cell and a surface receptor on an immune cell, incorporating a half life extending region (eg., Fc region) attached via a cleavable linker. Inclusion in the linker of a substrate sequence for a protease specific to the tumour microenvironment selectively releases an active portion of the protein from the half life extending region at the tumour site. Release of the active complex increases its potency for mediating T cell signalling at an immune synapse with tumour cells presenting the target pMHC.

Description

[0001] TUMOUR-TRANSFORMING MULTISPECIFIC PROTEINS

[0002] Field of the Invention

[0003] 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.

[0004] 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. MHC 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). MHC 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.

[0005] 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.

[0006] 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 scFv to 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):980-9872012, Oates & Jakobsen, Oncolmmunology 2(2):e22891 2013). Tebentafusp, an ImmTAC containing a TCR 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.

[0007] 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.

[0008] 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. 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.

[0009] 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 influence 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.

[0010] 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 W02019 / 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).

[0011] Dickopf, Georges & Brinkmann (Computational and Structural Biotechnology Journal 18:1221-12272020) 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.

[0012] 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 for T 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.

[0013] W02020 / 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 W02020 / 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 W02020 / 157211.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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. of the Invention

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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 or a 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.

[0022] The protease cleavage site(s) located within the linker(s) connecting the half life extending region to the active complex may be found only within said linker(s) and not elsewhere in the multispecific protein. There are preferably no cleavage sites for the protease (or, optionally, for any protease) between the pMHC binding arm and the immune cell engager arm, and / or within any part of the active complex. The one or more protease cleavage sites of the cleavable linker(s) are thus preferably unique to the cleavable linker(s), being found only in the linker(s) between the half life extending region and the active complex. The multispecific protein may comprise other linkers, such as within or between domains of antibody molecules and TCR molecules as described herein, which are not substrates for the protease and are not for in-vivo cleavage. For example, the immune cell engager arm may be linked to the pMHC binding arm by a flexible peptide linker, e.g., GGGGS (SEQ ID NO: 7).

[0023] 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.

[0024] 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.

[0025] 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 may be increased by 10 % or more, 20 % or more, 30 % or more, 40 % or more, or 50 % or more. 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.

[0026] 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.

[0027] 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 to be tested, 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.

[0028] 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 CD35. It may bind the TCR, 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 or anti-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.

[0029] 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 or CD28 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.

[0030] 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.

[0031] 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 PRAM E and MAGE-A4.

[0032] 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.

[0033] 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., tumour microenvironment, 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.

[0034] 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.

[0035] 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.

[0036] 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 Co domain and a second polypeptide comprising an N terminal Vp domain and a C terminal Cp domain.

[0037] 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 scFv and domain antibodies like VHH, have a compact form well-suited to inclusion in the free active complex.

[0038] 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 US10130721 B2. The immune cell engager may be linked to the variable domain of a TCR alpha chain or TCR beta chain. The immune cell engager scFv may be linked at its C terminus to the N terminus of a TCR molecule (e.g., to the TCR beta variable domain of a TCR molecule), optionally via a flexible linker, e.g., a G4S linker comprising GGGGS (SEQ ID NO: 7) or multiples thereof. The active complex may comprise a first polypeptide comprising: anti-CD3 scFv, TOR beta variable domain, TOR beta constant domain, and a second polypeptide comprising: TOR alpha variable domain, TOR alpha constant domain.

[0039] 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.

[0040] 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 may be human Fc. 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. Optionally, the upper hinge is truncated. The sequence of the Fc (e.g., first and / or second Fc CH2-CH3, preferably both) may commence with an N terminal human IgG hinge sequence DKTHTCPPCP (SEQ ID NO: 8).

[0041] An Fc region may be engineered to include mutations from wild type. For example, it may be engineered to reduce effector functions such as binding to FcyRIII and / or ADCC. A preferred example is effector null human lgG1. A human lgG1 may be engineered to include “LALA” mutations (L234A, L235A).

[0042] The two chains of the Fc may be homodimeric or heterodimeric. A heterodimeric Fc may be engineered to include mutations that promote heterodimerisation, such as knobs-into-holes mutations or charge-pair mutations, which favour pairing of the two different Fc chains and reduce the propensity for Fc homodimerisation. In the context of asymmetric multispecific molecules that dimerise via an Fc region, it is advantageous to include such mutations in order to promote correct molecular assembly and reduces the formation unwanted homodimers. Other Fc engineering features may be included to enhance developability and / or facilitate purification in industrial manufacture, such as mutations that alter binding to Protein A. An example Fc region is a human lgG1 Fc with one or more, optionally all, of the following mutations: truncated upper hinge, LALA (L234A, L235A) mutations, N297A mutation (removes glycosylation), knob-in-hole mutation, H>R and Y>F Protein A binding mutations in the Fc knob arm. The Fc may have an N terminal hinge sequence SEQ ID NO: 8.

[0043] 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 some embodiments, the cleavable linker comprising the protease cleavage site is located above the upper hinge (or truncated upper hinge) of the Fc region. In other embodiments the protease digestion site may be inserted or engineered within the Fc region below the disulphide linked Cys residues (Figure 3).

[0044] The approach of the present invention may be exploited to improve potency of molecules such as those described in W02020 / 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.

[0045] 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.

[0046] A multispecific protein may comprise: a first polypeptide comprising, from N to C terminus, scFv (immune cell engager - optionally VH-VL or VL-VH) - TCR Vp - TCR Cp, a second polypeptide comprising, from N to C terminus, TCR Va - TCR Ca, cleavable linker, first Fc chain, and a third polypeptide comprising the second Fc chain (Figure 4).

[0047] Figure 2 illustrates an example multispecific protein, 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.

[0048] The cleavable linker may connect an N terminus of the half life extending region (e.g., Fc) to a C terminus of the active complex (e.g., active complex comprising anti-CD3 scFv and a TCR molecule, wherein the C terminus of the scFv is fused to the N terminus of the TCR beta variable domain through a linker peptide). Preferably, Fc is linked via the protease cleavable linker to the N or C terminus of an alpha chain of a TCR molecule of the active complex. In one embodiment, the N terminus of the upper hinge of an Fc region is linked to the C terminus of the alpha constant domain of a TCR molecule via the cleavable linker (Figure 8A). In preferred embodiments, the TCR alpha constant domain includes its native C terminal cysteine residue which forms a disulphide bond with the native C terminal cysteine residue of the TCR beta constant domain, and the cleavable linker is linked to the C terminal cysteine of the TCR alpha constant domain. The cleavable linker may be fused between the C terminal cysteine residue of the TCR constant domain (e.g., TCR alpha constant domain) and the hinge of the Fc. The linker may be fused to an Fc N terminal sequence SEQ ID NO: 8.

[0049] Alternatively, the cleavable linker may connect a C terminus of the half life extending region (e.g., Fc) to an N terminus of the active complex (e.g., active complex comprising anti- CD3 scFv and a TCR molecule, wherein the C terminus of the scFv is fused to the N terminus of the TCR beta variable domain through a linker peptide). In one embodiment, the N terminus of the immune cell engager moiety (e.g., anti-CD3 scFv) is linked to the C terminus of an Fc via the cleavable linker (Figure 8B). A multispecific protein may comprise a first polypeptide comprising, from N to C terminus, a first Fc CH2-CH3 (including hinge region with upper hinge), cleavable linker, scFv, TCR beta variable domain, TCR beta constant domain, and a second polypeptide comprising, from N to C terminus, a TCR alpha variable domain and a TCR alpha constant domain, and a third polypeptide comprising a second Fc CH2-CH3 (including hinge region with upper hinge). As described elsewhere herein, the TCR alpha and beta domains pair, and the first and second Fc pair, forming the assembled protein (Figure 8B). In another embodiment, the N terminus of the alpha variable domain of a TCR molecule is linked to the C terminus of an Fc via the cleavable linker (Figure 8C). A multispecific protein may comprise a first polypeptide comprising, from N to C terminus, a first Fc CH2-CH3 (including hinge region with upper hinge), a cleavable linker, a TCR alpha variable domain and a TCR alpha constant domain, a second polypeptide comprising scFv, TCR beta variable domain and TCR beta constant domain, and a third polypeptide comprising a second Fc-CH2-CH3 (including hinge region with upper hinge). As described elsewhere herein, the TCR alpha and beta domains pair, and the first and second Fc pair, forming the assembled protein (Figure 8C).

[0050] In some embodiments, additional moieties are attached to the half life extending region. For example, cytokines such as I FNy, may be fused to the half life extending region, optionally in multiple copies. Release of I FNy 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.

[0051] 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).

[0052] 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.

[0053] Brief Description of the Drawings

[0054] 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.

[0055] 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.

[0056] 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.

[0057] Figure 4 illustrates a soluble multispecific protein of the invention undergoing protease digestion to release an active complex and separate Fc region. The protein is a functional TCR- TCE with half life extension feature provided through the inclusion of an Fc region. Conditional cleavage separates the key TCR-TCE elements from the half life extending region. The figure was created with BioRender.

[0058] Figure 5 shows denaturing SDS-PAGE analysis of undigested and protease digested soluble multispecific proteins.

[0059] Figure 6 shows results of dual binding ELISA comparing binding strength between undigested and protease digested soluble multispecific proteins.

[0060] Figure 7 shows data from cytotoxicity assay comparing the cell killing potential of undigested and digested soluble multispecific proteins against SK-MEL-5 cells.

[0061] Figure 8 shows embodiments of the invention in which the half life extending region and active complex are interconnected via a cleavable linker at different positions.

[0062] Figure 9 shows RNA expression of ADAM 15 in transcripts per million in human cancer and healthy cells.

[0063] Figure 10 shows RNA expression of MMP9 in transcripts per million in human cancer and healthy cells.

[0064] Figure 11 shows RNA expression of MMP14 in transcripts per million in human cancer and healthy cells. Figure 12 shows RNA expression of MMP15 in transcripts per million in human cancer and healthy cells.

[0065] Figure 13 shows RNA expression of ST14 in transcripts per million in human cancer and healthy cells.

[0066] Figure 14 shows RNA expression of CTSS in transcripts per million in human cancer and healthy cells.

[0067] Detailed

[0068] TCR molecules

[0069] In humans, the native T cell receptor comprises two transmembrane polypeptides, which may be an op pair or a y<5 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:CD3b£:CD3v£:CD3^, which assemble in a 1: 1:1:1 stoichiometry. The CD3E, CD3y and CD35 subunits are transmembrane proteins each containing a single immune receptor tyrosine-based activation motif (ITAM), and CD3 is cytosolic and contains three ITAMs.

[0070] 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.

[0071] 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 (Co 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.

[0072] 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.

[0073] 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. Paired TCR constant domains are preferably linked by one or more disulphide bonds. Optionally, the Cp constant domain is covalently linked to the Ca constant domain by the native TCR diS bond in the C terminal region of the Ca and Cp, linking C95 in the alpha chain and C131 in the beta chain (IMGT numbering). Inclusion of the C95a- C131P disulphide bond, which is naturally present in the human TCR constant region, assists in stabilising the native conformation of the TCR molecule. In some embodiments, this native disulphide bond is removed and may be replaced by a non-native disulphide bond at an alternative location. In other embodiments, a non-native disulphide bond is added. 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 or TRBC2. 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 Thr 48 of TRAC and position Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulphide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 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 op 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): 260-2662003.

[0074] 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 W02017 / 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 W02004 / 033685; W098 / 39482; W001 / 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).

[0075] 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.

[0076] The TCR molecule may be a human TCR molecule. Where constant domains are present these are preferably human constant domains. Thus, a Co constant domain (TRAC) may be human Co. A Cp constant domain (TRBC) may be human TRBC1 or human TRBC2.

[0077] Antibody molecules

[0078] 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 betasheet, 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.

[0079] 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.

[0080] 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.

[0081] 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 for their construction and use were described by Holliger & Hudson, Nature Biotechnology 23(9): 1126-1 136 (2005).

[0082] 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.

[0083] 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™".

[0084] The antibody molecule may be a human antibody molecule. Thus, where constant domains are present these are preferably human constant domains.

[0085] Engagement of immune cells

[0086] The immune cell engager arm represents means for binding and activating an immune cell surface receptor. It may be a binding protein such as an antibody molecule. It may be a binding domain of a natural ligand of the immune cell surface receptor, or all or part of an extracellular region of a natural ligand that is a transmembrane protein. A large number of immune cell engaging antibodies are known in the art and may be adapted for use in multispecific molecules, or further such antibodies may be generated as desired. The immune cell engager arm may bind a surface receptor on a T cell, e.g., cytotoxic CD8+ or CD4+ T cell, inducing intracellular signalling in the T cell leading to T cell killing of the target cell. The immune cell engager may bind and activate the TCR-CD3 complex on the T cell. It may be an antibody molecule comprising the VH and VL domains of BMA031 or a variant thereof such as BMA031v36 (WO2021 / 023657, WO2021 / 023658). The immune cell engager arm may bind CD3, e.g., CD3E, CD3y and / or CD35. It may bind CD35E heterodimer and / or CD3y£ heterodimer. The immune cell engager arm may be an anti-CD3 antibody molecule. Trinklein et al (MABS 11(4):639-652 2019) evaluated anti-CD3 antibodies for use in T cell engaging bispecific antibodies and described anti-CD3 antibodies of “F2” clonotype, which induced tumour cell killing by T cells but with a relatively low accompanying level of cytokine release. Robertson et al (Clinical and Experimental Immunology 215:105-119 2024) also reviewed properties of anti-CD3 antibodies suitable for use in immune cell engaging multispecific proteins, and found that optimal affinity binding to CD3 is a function of the affinity of pMHC binding in the bispecific. In bispecifics comprising TCRs with picomolar affinity for pMHC, an anti-CD3 arm of moderate affinity was optimal, whereas in bispecifics with weaker TCRs (lower affinity for pMHC), a stronger anti-CD3 affinity was required for comparable potency.

[0087] An anti-CD3 antibody molecule may comprise the anti-CD3 VH and VL domain of any of the following: OKT3, UCHT1, UCHT1v17 (WO2021 / 023657), L2K, TR66, 12F6, SP34 (WO2016 / 110576), brenetafusp, tebentafusp, blinatumomab, catumaxomab, duvortuxizumab, epcoritamab, ertumaxomab, glofitamab, mosunetuzumab, odronextamab, pasotuxizumab, solitomab, or an engineered variant thereof. An anti-CD3 antibody molecule may comprise an anti-CD3 VH and VL domain disclosed or referenced within WO2024 / 038193 (for example scFv U0 or scFv U28), WO2022 / 233956, WO2021 / 023658, US2021 / 0032370, WO2021 / 023657, W02020 / 157210, WO2018 / 052503, WO2016 / 110576, WO2015 / 095392 or W01992 / 022653.

[0088] Although bispecific antibodies with anti-CD3 specificity were originally developed to recruit CD8+ CTL to kill tumour cells, other immune cells such as y<5 T cells, natural killer T (NKT) cells and CD4+ T cells also express CD3 and could be activated for target cell killing.

[0089] The immune cell engager arm may bind the TCR, e.g., a TCR constant domain. WO2024 / 081381 (Marengo) described a multispecific protein for redirecting T cells, in which the T cell engaging arm bound to the TCR beta variable domain.

[0090] In other embodiments, the immune cell engager arm binds CD28. CD28 is a stimulatory receptor expressed on T cells. Binding of CD28 induces intracellular signalling and activates T cell killing of the target cell. The immune cell engager arm may be an anti-CD28 antibody molecule. Examples of anti-CD28 antibodies have been described (Correnti et al., Leukemia 32:1239-12432018; Wu et al., Nature Cancer 1(1):86-98 2020). In other embodiments, the immune cell engager arm binds a surface receptor on a natural killer (NK) cell, inducing intracellular signalling and activating NK cell killing of the target cell. Surface receptors on NK cells include Fc receptors such as FCYRIH (CD16), e.g., FcyRIHA (CD16A). The immune cell engager arm may be an anti-CD16 antibody molecule, e.g., anti- CD16A. It may be an antibody molecule comprising the anti-CD16 VH and VL domain of AFM13 (Rothe et al., Blood 125(26) :4024-4031 2014) or 2B1 (Weiner et al., Cancer Res 55(20):4586- 4593 1995), or an engineered variant thereof.

[0091] Induction of intracellular signalling in the immune cell occurs in the context of engagement at the immune synapse, wherein the multispecific molecule binds the target cell and the immune cell within the immune synapse. Binding to the immune cell surface receptor activates intracellular signalling in the immune cell. A cytotoxic cell is activated to kill the proximal target cell. An activated cytotoxic cell (e.g., CTL or NK cell) releases cytokines and cytolytic molecules leading to target cell death. Released cytokines also recruit and activate other immune cells, amplifying the immune response.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] Preferably the maximum inhibition obtained in reporter assays is greater than 50%, for example 80% or more.

[0099] 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.

[0100] 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.

[0101] Binding to antigen may be unaffected by the presence or absence of the half life extending region, and thus binding to antigen by the immune cell engager moiety and pMHC binding arm of the active complex may be the same before and after protease digestion of the cleavable linker.

[0102] Methods of determining and quantifying binding to antigen are known in the art. A dual binding ELISA may be used to determine simultaneous engagement of both the immune cell receptor and the target pMHC. A soluble multispecific protein may be capable of simultaneous binding to both the immune cell receptor and to the target pMHC, e.g., as measured using dual binding ELISA. Dual binding ELISA may be used to confirm that antigen binding sites are available for antigen binding. Dual binding ELISA may be used to confirm that antigen binding is not inhibited by the half life extending region. An example assay protocol for dual binding ELISA is provided in Example 5. Half life extending region

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Tissue-; ;ific activation

[0107] 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).

[0108] The linker may comprise a sustrate seguence for a protease that is secreted by, or enriched in the proximity of, the target cells, such as a tumour specific protease.

[0109] 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 reguire 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.

[0110] There are many different proteases present in the tumollr microenvironment, such as matrix metal loproteases (MMPs), a disintegrin and metalloproteases (ADAMs), kallikreins (KLKs), cathepsins (e.g., cathepsin S), transmembrane serine proteases (TMPRSSs) and granzymes (GZMs), serving a wide range of functions. They differ in the type of secreting cells, abundance, tissue specificity and substrate preference. Proteases that are up-regulated in TME are preferred, in order to achieve TME-specific or TME-enriched cleavage of the drug, thereby enhancing efficacy as well as specificity of immune cell engagement with target cells. The protease may be expressed by tumour cells or by one or multiple stromal cell types.

[0111] The protease may be a metalloprotease (e.g., MMP), a serine protease, a cysteine protease, a threonine protease, or an aspartic protease. Examples of serine proteases are granzyme (e.g., granzyme B, granzyme A, granzyme K), KLK (e.g., KLK2, KLK3, KLK4), fibroblast activation protein (FAP), DPP, PEP, uPA, CatG, ELANE and matriptase. Granzymes are of particular interest as they are released during T / NK-dependent cellular cytotoxicity against cancer cells expressing the target pMHC. Immune cell activation against target pMHC may thus activate granzyme cleavage of the linker and release of the active complex, leading to triggering and / or amplification of drug activity locally within the target tissue. This represents an advantageous self-triggering and / or self-amplifying mode of action, which would be less affected by variability of the TME.

[0112] Expression of RNA encoding a protease of interest may be compared across human healthy and cancer tissues, for example using information from public data sets, to identify proteases for which an elevated RNA level is observed in cancer samples. Examples of proteases that have elevanted RNA in human cancers, relative to healthy tissues, are ADAM 15 (Figure 9), MMP9 (Figure 10), MMP14 (Figure 11), MMP15 (Figure 12), matriptase (ST14) (Figure 13) and cathepsin S (CTSS) (Figure 14).

[0113] To maximize cleavage efficiency and / or applicability, the linker sequence may contain one or multiple cleavage sites specifically or degeneratively sensitive to the selected TME proteases. Owing to degeneracy of protease recognition motifs, a sequence may be engineered to be recognised by multiple different proteases (e.g., multiple TME proteases).

[0114] Examples of protease cleavage sites include:

[0115] Recognition sequence VGPDAG (SEQ ID NO: 1) for granzyme B Recognition sequence LSGRSD (SEQ ID NO: 2) for ST14 (matriptase) Recognition sequence PLGLAG (SEQ ID NO: 3) for MMP. The cleavable linker may contain one or multiple tandemly arranged protease cleavable sites, each with flexible flanking sequence. Examples of cleavable linkers SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 are shown in the Examples.

[0116] Protease cleavable linkers have been described before in other contexts of conditional activation of therapeutic proteins, and may be adopted for use in the present invention. For example, the cleavable linker in a multispecific protein of the present invention may be a linker comprising a protease cleavage site, as disclosed in any of the following documents, incorporated herein by reference:

[0117] WO2013 / 192546 described an activatable antibody, comprising a non-binding steric moiety (such as albumin) connected to the antibody via a cleavable linker.

[0118] WO2015 / 066279 described an activatable anti-EGFR antibody having a masked light chain, where the light chain of the antibody was coupled to a spacer sequence and a masking moiety via a protease-cleavable linker.

[0119] WQ2024 / 040247 described protease-activated IFN proproteins comprising a tumourtargeting moiety and an IFN moiety, in which the IFN moiety was sterically hindered from binding to its receptor through attachment to an Fc moiety via a protease-cleavable linker. IFN was fused within an antibody molecule, between the CH1 and CH2 domains of the heavy chain, with a protease-cleavable linker on one or both sides of the IFN. Exemplary proteases for substrate cleavage are shown in Table A of WO2024 / 040247, incorporated by reference herein. Exemplary substrate sequences that are cleavable by a tumour protease and can be incorporated into the protease-cleavable linkers are shown as SU1 to SLI151 in Table B of WQ2024 / 040247, incorporated by reference herein. A cleavable linker may further comprise a spacer sequence of any of SP1 to SP38 exemplified in Table C of WO2024 / 040247, incorporated by reference herein. A cleavable linker may comprise a linker sequence of any of PCL1 to PCL95 exemplified in Table D of WQ2024 / 040247, incorporated by reference herein.

[0120] WO2022 / 125566 described a multispecific protein in which two antigen-binding proteins (A1 and A2) were each linked via tumour protease-cleavable linkers (L1 and L2) to masking peptides (P1 and P2), and to half life extending antibody H1 , in a structure P2-L2-A2-A1-L1-P1- H1.

[0121] Therapeutic use

[0122] A method 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.

[0123] 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.

[0124] Clauses

[0125] The following numbered clauses represent aspects of the invention.

[0126] 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.

[0127] 2. A protein according to clause 1, wherein the cleavable linker connects the half life extending region to the pMHC binding arm.

[0128] 3. A protein according to clause 1, wherein the cleavable linker connects the half life extending region to the immune cell engaging arm.

[0129] 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.

[0130] 5. A protein according to any preceding clause, wherein the cleavable linker comprises a single protease cleavage site.

[0131] 6. A protein according to any of clauses 1 to 4, wherein the cleavable linker comprises multiple protease cleavage sites.

[0132] 7. A protein according to clause 6, wherein the cleavable linker comprises cleavage sites for multiple proteases.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 12. A protein according to clause 11, wherein the cleavable linker comprises a serine protease cleavage site.

[0137] 13. A protein according to clause 12, wherein the protease is granzyme B, granzyme A or granzyme K.

[0138] 14. A protein according to clause 12, wherein the protease is KLK2, KLK3 or KLK4.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 18. A protein according to any preceding clause, wherein the immune cell is a cytotoxic immune cell, optionally a T cell.

[0143] 19. A protein according to any preceding clause, wherein the immune cell is a cytotoxic CD8+ T effector cell.

[0144] 20. A protein according to any preceding clause, wherein the immune cell engager arm binds CD3 or CD28.

[0145] 21. A protein according to any preceding clause, wherein the target cell is a cancer cell of a solid tumour.

[0146] 22. A protein according to clause 21, wherein the solid tumour is sarcoma, carcinoma or lymphoma.

[0147] 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.

[0148] 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.

[0149] 25. A protein according to clause 24, wherein the pMHC binding arm comprises paired TCR variable domains and paired TCR constant regions.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 30. A protein according to any preceding clause, wherein the immune cell engager arm comprises a single chain antibody molecule.

[0155] 31. A protein according to clause 30, wherein the antibody molecule is an scFv.

[0156] 32. A protein according to any of clauses 1 to 28, wherein the antibody molecule is a single domain antibody.

[0157] 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.

[0158] 34. A protein according to any preceding clause, wherein the half life extending region comprises an antibody Fc.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 39. Nucleic acid encoding a soluble multispecific protein according to any of clauses 1 to 37.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 44. A method of producing a soluble multispecific protein according to any of clauses 1 to

[0168] 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.

[0169] 1A. 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, and wherein 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.

[0170] 2A. A protein according to clause 1A, wherein the pMHC binding arm comprises a binding site for the pMHC complex and the immune cell engager arm comprises a binding site for a surface receptor on the immune cell, wherein the said binding sites are not masked. 3A. A protein according to clause 1A, wherein the cleavable linker connects the half life extending region to the pMHC binding arm.

[0171] 4A. A protein according to clause 1A, wherein the cleavable linker connects the half life extending region to the immune cell engaging arm.

[0172] 5A. A protein according to clause 1A, wherein the cleavable linker connects the half life extending region to the pMHC binding arm and the immune cell engaging arm.

[0173] 6A. A protein according to any preceding clause, wherein the cleavable linker comprises a single protease cleavage site.

[0174] 7A. A protein according to any of clauses 1A to 5A, wherein the cleavable linker comprises multiple protease cleavage sites, optionally cleavage sites for multiple proteases.

[0175] 8A. A protein according to clause 7A, 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.

[0176] 9A. A protein according to clause 7A, wherein the cleavable linker comprises serial protease cleavage sites, whereby cleavage at any one of said protease cleavage sites releases free active complex.

[0177] 10A. A protein according to any preceding clause, wherein the protease is secreted in the tumour micro-environment.

[0178] 11 A. A protein according to any preceding clause, wherein the protease is a metal I oprotease, serine protease (e.g., granzyme, KLK, FAP, DPP, PEP, uPA, CatG, ELANE or matriptases), cysteine protease, threonine protease or aspartic protease.

[0179] 12A. A protein according to any preceding clause, 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.

[0180] 13A. 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 for stimulating immune cells in an assay with target cells presenting target pMHC.

[0181] 14A. 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.

[0182] 15A. A protein according to any preceding clause, wherein the immune cell is a cytotoxic immune cell, optionally a T cell.

[0183] 16A. A protein according to any preceding clause, wherein the immune cell engager arm binds CD3.

[0184] 17A. A protein according to any preceding clause, wherein the target cell is a cancer cell of a solid tumour. 18A. 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.

[0185] 19A. A protein according to clause 17A, 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.

[0186] 20A. 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. 21A. A protein according to clause 20A, wherein the immune cell engager arm comprises a single chain antibody molecule, optionally scFv or a single domain antibody.

[0187] 22A. A protein according to any preceding clause, 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.

[0188] 23A. A protein according to any preceding clause, wherein the half life extending region comprises an antibody Fc.

[0189] 24A. A protein according to clause 23A, wherein the Fc includes a hinge region.

[0190] 25A. A protein according to clause 24A, wherein the protease cleavage site is N terminal to the hinge region of the Fc.

[0191] 26A. A protein according to any of clauses 23A to 25A, 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 the half life extending region comprises 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.

[0192] 27A. A protein according to clause 26A, wherein the protein comprises a first polypeptide comprising, from N to C terminus, anti-CD3 scFv, TCR beta variable domain, TCR beta constant domain, a second polypeptide comprising, from N to C terminus, TCR alpha variable domain, TCR alpha constant domain, cleavable linker, first Fc CH2-CH3, and a third polypeptide comprising second Fc CH2-CH3.

[0193] 28A. A protein according to any preceding clause, wherein the cleavable linker is fused to a C terminal cysteine residue of a TCR constant domain (e.g., alpha constant domain) and to an N terminal hinge sequence (e.g., SEQ ID NO: 8) of an Fc.

[0194] 29A. A protein according to any preceding clause, wherein the cleavable linker comprises a protease cleavage site for granzyme B, a protease cleavage site for ST14 and / or a protease cleavage site for MMP.

[0195] 30A. 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.

[0196] 31A. Nucleic acid encoding a soluble multispecific protein according to any preceding clause. 32A. A protein according to any of clauses 1A to 30A, or nucleic acid according to clause 30A, provided in isolated form in a composition comprising a pharmaceutically acceptable excipient, formulated for delivery to a patient.

[0197] 33A. A method of treating a condition in a patient, comprising administering a soluble multispecific protein as defined in any of clauses 1 A to 30A, or nucleic acid according to clause 31A, to the patient.

[0198] 34A. A soluble multispecific protein according to any of clauses 1A to 30A, or nucleic acid according to clause 31 A, for use in method of treatment of the human or animal body by therapy.

[0199] 35A. A method according to clause 33A, or a soluble multispecific protein or nucleic acid for use according to clause 34A, wherein the treatment comprises treating a solid tumour.

[0200] 36A. A population of recombinant host cells in vitro comprising nucleic acid encoding a soluble multispecific protein according to any of clauses 1A to 30A.

[0201] 37A. A method of producing a soluble multispecific protein according to any of clauses 1A to 30A, comprising culturing a population of cells according to clause 36A 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.

[0202] 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.

[0203] Comparative experiment: T cell killing assay with scFv-TCR in ImmTAC format

[0204] 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.

[0205] 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)

[0206] TDCC assay set up

[0207] 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.

[0208] Running assay program

[0209] 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 or 6) and LDH release. Green fluorescence intensity is extracted and exported and data analysis involved plotting timecourse and dose curves for each molecule tested.

[0210] Bispecific

[0211] 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 (TOR a19ka; b15), fused to the anti-CD3 antibody scFv arm of tebentafusp.

[0212] The starting concentration of bispecific molecule was set at 100 nM and serially diluted 1 in 5 to 2.5 x 10'4nM (9 dilution points) and a control well with effector and target cells only without bispecific.

[0213] Results

[0214] 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 of target cells in the assay.

[0215] Linkage of an Fc region or albumin-binding domain to an scFv-TCR bispecific “ImmTAC”, as described for example in W02020 / 157211 or WO2024 / 038193, increases its size and reduces its potency for mediating tumour cell killing by T cells.

[0216] 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.

[0217] Removal of the Fc region will improve potency in a cell killing assay.

[0218] Example 3

[0219] Soluble multispecific proteins were composed of 3 polypeptide chains consisting of 1) anti-CD3 scFv linked to TCR beta chain; 2) TCR alpha chain linked to Fc containing knob mutation and 3) a second Fc chain containing hole mutations. The assembly of the polypeptide resulting from these 3 chains forms three functional elements as follows: immune cell engager arm - a T-cell recruiting arm in the form of an scFv targeting CD3; pMHC binding arm - TCR specific for pMHC target of interest; and half life extending region - human Fc.

[0220] The target of interest is a human pMHC complex comprising a tumour associated peptide. The immune cell engager arm and pMHC binding arm together form an active complex comprising functional elements of the TCR-TCE. To transform this protein into a conditionally cleavable format, recognition sequences for specific proteases were inserted above the Fc hinge region to allow conditional cleavage and separation of the active complex from the Fc arm (Figure 4). Three versions were generated, differing in their cleavable linker sequences, as indicated in the table below. Versions 1-3 (v1-v3) contained 1 , 2 or 3 protease recognition sequences respectively.

[0221] The protease recognition sequences used for the different proteases are as follows: Granzyme B > VGPDAG (SEQ ID NO: 1); ST14 > LSGRSD (SEQ ID NO: 2) and MMP > PLGLAG (SEQ ID NO: 3).

[0222] All three versions of the multispecific protein have: a first polypeptide comprising, from N to C terminus, anti-CD3 scFv (VH-VL), flexible linker GGGGS (SEQ ID NO: 7), TOR beta variable domain, TOR beta constant domain; a second polypeptide comprising, from N to C terminus, a TOR alpha variable domain, TOR alpha constant domain, cleavable linker (SEQ ID NO as in table), Fc containing knob mutations; and a third polypeptide comprising an Fc containing hole mutations.

[0223] The Fc of the second and third polypeptides heterodimerise, with paired knob-in-hole mutations, to form an Fc region conferring extended half life on the soluble protein.

[0224] Example 4

[0225] The protease cleavage feature of the invention was demonstrated using soluble multispecific proteins with cleavable linkers containing recognition sites for different proteases, as described in Example 3.

[0226] Proteins were expressed in Expi-HEK293 cells (Invitrogen) at 30 mL scale. In brief, the cells were cultured at 37 degrees C and allowed to reach 2.5-3.0 x 10A6 cells / mL density for the day of transfection. To 25.5 mL of cells, a premix of expifectamine and 30 ug of plasmid DNA encoding the polypeptide chains of the designed protein formats were added. The cells were incubated in a shaking incubator at 37 degrees C for 16-18 hours before adding enhancers 1 and 2 and then allowed to culture for another 6 days after which the supernatant was collected by centrifugation (10,000 rpm for 15 minutes). The collected supernatant was filtered through a 0.22 urn filter before affinity purifying using gravity columns pre-packed with 1mL Protein A beads (MabSelect SuRe LX protein A resin - Cytiva). The protein A column was prepared by 10 column volume washes with 0.5 M sodium hydroxide, followed by water and then PBS. The supernatant was then loaded onto the column and then washed with 10 column volumes of PBS. The bound proteins were eluted from the column using 12 mL of IgG elution buffer (Thermofisher) and then immediately neutralized by adding 4 mL of 1M Tris pH 8.0. The eluted protein was concentrated down to 1 mL using Amicon columns (Merck) with 30 kDa cut-off before performing a second round SEC purification and buffer exchange into PBS buffer. The resulting proteins were used for the downstream analysis.

[0227] To analyse the cleavability of formats v1, v2 and v3, recombinant proteases were used to digest the formats according to the protocol detailed below.

[0228] Materials:

[0229] 1. Recombinant Human Granzyme B (R&D, Catalog Number: 2906-SE).

[0230] 2. Recombinant Human Matriptase / ST14 Catalytic Domain (R&D, Catalog Number: 3946-SEB).

[0231] 3. Recombinant Human MMP 9 (R&D, Catalog Number: 911-MP).

[0232] 4. Recombinant Human MMP 14 / MT1 MMP (R&D, Catalog Number: 918-MP).

[0233] 5. Recombinant Human MMP 15 / MT2 MMP (R&D, Catalog Number: 916-MP).

[0234] 6. Granzyme B Assay Buffer: 50 mM Tris, pH 7.5.

[0235] 7. ST14 Assay Buffer: 50 mM Tris, 50 mM NaCI, 0.01% (v / v) Tween® 20, pH 9.0.

[0236] 8. MMP-9 Assay Buffer: 50 mM Tris, 10 mM CaCI2, 150 mM NaCI, pH 7.5.

[0237] 9. MMP-14 Assay Buffer: 50 mM Tris, 3 mM CaCI2, 1 pM ZnCI2, pH 8.5.

[0238] 10. MMP-15 Assay Buffer: 50 mM Tris, 500 mM NaCI, 5 mM CaCI2, 1 pM ZnCI2, pH 8.0.

[0239] Digestion:

[0240] 1. Format v1 digested with Granzyme B:

[0241] In a 1.5 mL Eppendorf tube add 150 pL 0.65 mg / mL Cleavable Format (Single). Add 150 pL Granzyme B Assay Buffer. Add 2 pg Recombinant Human Granzyme B (R&D, Catalog Number: 2906-SE). Incubate at 37 °C overnight.

[0242] 2. Format v2 digested with ST14:

[0243] In a 1.5mL Eppendorf tube add 120 pL 0.84 mg / mL Cleavable Format (Dual). Add 120 pL ST14 Assay Buffer. Add 2 pg Recombinant Human Matriptase / ST14 Catalytic Domain (R&D, Catalog Number: 3946-SEB). Incubate at 37 °C overnight.

[0244] 3. Format v2 digested with MMP-9:

[0245] In a 1.5mL Eppendorf tube add 120 pL 0.84 mg / mL Cleavable Format (Dual). Add 120 pL MMP-9 Assay Buffer. Add 2 pg Recombinant Human MMP 9 (R&D, Catalog Number: 911- MP). Incubate at 37 °C overnight. 4. Format v2 digested with MMP-14:

[0246] In a 1.5mL Eppendorf tube add 120 pL 0.84 mg / mL Cleavable Format (Dual). Add 120 pL MMP-14 Assay Buffer. Add 2 pg Recombinant Human MMP 14 / MT1 MMP (R&D, Catalog Number: 918-MP). Incubate at 37 °C overnight.

[0247] 5. Format v2 digested with MMP-15:

[0248] In a 1.5mL Eppendorf tube add 120 pL 0.84 mg / mL Cleavable Format (Dual). Add 120 pL MMP-15 Assay Buffer. Add 2 pg Recombinant Human MMP 15 / MT2 MMP (R&D, Catalog Number: 916-MP). Incubate at 37 °C overnight.

[0249] 6. Format v3 digested with Granzyme B:

[0250] In a 1.5mL Eppendorf tube add 125 pL 0.80 mg / mL Cleavable Format (Triple). Add 125 pL Granzyme B Assay Buffer. Add 2 pg Recombinant Human Granzyme B (R&D, Catalog Number: 2906-SE). Incubate at 37 °C overnight.

[0251] 7. Format v3 digested with ST 14:

[0252] In a 1.5mL Eppendorf tube add 125 pL 0.80 mg / mL Cleavable Format (Triple). Add 125 pL ST14 Assay Buffer. Add 2 pg Recombinant Human Matriptase / ST14 Catalytic Domain (R&D, Catalog Number: 3946-SEB). Incubate at 37 °C overnight.

[0253] 8. Format v3 digested with MM P-9:

[0254] In a 1.5mL Eppendorf tube add 125 pL 0.80 mg / mL Cleavable Format (Triple). Add 125 pL MMP-9 Assay Buffer. Add 2 pg Recombinant Human MMP 9 (R&D, Catalog Number: 911- MP). Incubate at 37 °C overnight.

[0255] 9. Format v3 digested with MMP-14:

[0256] In a 1.5mL Eppendorf tube add 125 pL 0.80 mg / mL Cleavable Format (Triple). Add 125 pL MMP-14 Assay Buffer. Add 2 pg Recombinant Human MMP 14 / MT1 MMP (R&D, Catalog Number: 918-MP). Incubate at 37 °C overnight.

[0257] 10. Format v3 digested with MMP-15:

[0258] In a 1.5mL Eppendorf tube add 125 pL 0.80 mg / mL Cleavable Format (Triple). Add 125 pL MMP-15 Assay Buffer. Add 2 pg Recombinant Human MMP 15 / MT2 MMP (R&D, Catalog Number: 916-MP). Incubate at 37 °C overnight.

[0259] The protease digested formats version 1-3 were analysed by running 2 ug of protein on denaturing SDS-PAGE gel under non-reduced conditions alongside an undigested sample (Figure 5). Blue wide range protein marker - 10-245 kDa (Cleaver Scientific) was used to size the protein fragments.

[0260] The expected molecular weight of the soluble multispecific protein format is -129.5 kDa. However, once cleaved using protease, the functional effector unit (active complex) is physically separated from the Fc element (half life extending region) and the expected molecular weight of cleaved proteins are 78.5 kDa and 51 kDa, respectively. From Figure 5, it can be seen there are varying degrees of cleavage. Format v1 containing Granzyme B recognition site has little to no cleavage under the enzyme conditions used. This was also true for v3, which also has Granzyme B recognition site. Digestion with ST14 on the other hand, whether in version 2 or version 3, yielded complete digestion as did MMP15, whereas MMP9 and MMP14 only showed partial digestion.

[0261] The protease recognition sequences used in the cleavable linker can be modified and optimised to suit individual proteases and cleavage conditions. Under the present assay conditions, MMP15 achieved full cleavage, while MMP14 cleaved only partially. The cleavable linker sequence employed in these proteins matched the MMP15 cleavage sequence motif slightly better than the MMP14 motif, suggesting that the efficiency might be very sensitive to small sequence differences. The linker could be optimised to achieve complete cleavage with MMP14. The granzyme B cleavage sequence used in the exemplified linker is also not optimal and could be improved.

[0262] Example 5

[0263] Soluble multispecific proteins described in Example 3 were confirmed to simultaneously bind CD3 and target pMHC complex. Digested proteins and undigested proteins showed dual binding to CD3 and pMHC using an ELISA binding assay according to the protocol described below. As a control, an scFv-TCR molecule representing free active complex was used in the dual-binding ELISA assay.

[0264] The dual binding ELISA showed that the undigested proteins were capable of dual binding at a binding strength comparable to the proteins digested with protease. Figure 6(i)-(iii). This effect was seen more clearly in v2 (Figure 6(H)) and v3 (Figure 6(iii)) where ST14 and MMP15 yielded complete cleavage yet the dual binding was comparable to the undigested proteins.

[0265] This confirms that the binding sites for pMHC and for CD3 were available for binding in the undigested form, and that inclusion of the Fc region did not block or sterically hinder binding.

[0266] Dual Binding ELISA Protocol:

[0267] Materials:

[0268] 1. Corning costar assay plate, 96 well, clear flat bottom, high binding (Corning, Catalog Number: REF3361).

[0269] 2. Wash buffer: 0.1% Tween (Sigma-Aldrich, Catalog Number: P9416) in PBS.

[0270] 3. Blocking buffer: 1% BSA (Sigma-Aldrich, Catalog Number: A7906) in PBS.

[0271] 4. Diluent buffer 0.1% BSA (Sigma-Aldrich, Catalog Number: A7906) in PBS.

[0272] 5. CD3-HRP (Aero Biosystems, Catalog Number: CDD-HR2W3) 6. 1-Step Ultra TMB-ELISA (Thermo Scientific, Catalog Number: REF34029)

[0273] 7. Sulphuric Acid Solution, 1M (Thermo Scientific, Catalog Number: 12933634)

[0274] Assay:

[0275] 1. Coat Corning costar assay plate with 1 pg / mL MHC-25 (diluted in PBS), 50 pL in each well. Incubate at 4 °C overnight.

[0276] 2. Wash with 200 pL ELISA wash buffer twice. Add 200 pL blocking buffer in each well. Incubate at room temperature for 1 hour.

[0277] 3. Wash with 200 pL ELISA wash buffer twice. Add 100 pL sample protein format in each well. All samples are 1 in 2 serially diluted from 10 ug / mL in diluent buffer. Each sample has 1 duplication. Incubate at room temperature for 1 hour.

[0278] 4. Wash with 200 pL ELISA wash buffer twice. Add 100 pL 200 ng / mL CD3-HRP in each well. CD3-HRP are diluted in diluent buffer. Incubate at room temperature for 1 hour.

[0279] 5. Wash with 200 pL ELISA wash buffer twice. Add 100 pL 1-Step Ultra TMB-ELISA in each well. Incubate at room temperature for 3 minutes.

[0280] 6. Add 50 pL 1M Sulphuric Acid Solution in each well.

[0281] 7. Read Abs 450nm by EnVision (Serial number: 1051260), export with EnVision Workstation version 1.14.3049.1642. Data was analyzed by GraphPad Prism 9.5.0 (730).

[0282] Example 6

[0283] Soluble multispecific proteins described in Example 3 were assayed for potency for killing target cells.

[0284] To confirm that protease digestion of the soluble multispecific proteins enhanced the immune synapse formation and thus improved the targeted cell killing potency, the digested and undigested proteins v1 to v3 described in Example 3, without any further purification, were tested in T cell cytotoxicity assays for killing of target cells that present the pMHC complex recognised by the TCR of the multispecific proteins, according to the method described below.

[0285] The cell killing data (Figure 7(i) - (iii)) correlate with the protease digestion data from Example 4 (Figure 5). With protein samples where there was efficient cleavage, such as v2 and v3 with ST14 and MMP15, a significant enhancement in killing potency was observed compared with the respective undigested protein. Conversely, where there was little or no cleavage, for example with Granzyme B, MMP9 and MMP14, there was little or only marginal improvement in killing potency verses the undigested protein. In general, the half life extended proteins showed lower potency compared with the free active complex represented by an scFv-TCR (“ImmTAC” format), and protease digestion of the cleavable linker enhanced their potency. Extent of protease digestion correlated with potency, indicating that release of the active complex from the half life extending region restored a potency comparable to that of the control scFv-TCR active complex.

[0286] The data in these Examples highlight the comparable dual CD3 and pMHC binding properties of the full-length and the cleaved soluble multispecific proteins. In cell killing assays where the protein is cleaved, a significant enhancement in killing potency can be observed. This enhancement can be attributed to the smaller size of the functional protein unit available after cleavage, which is capable of forming more efficient immune synapse than the full-length protein with the half life extending region, resulting in increased potency.

[0287] Cell killing assay protocol:

[0288] Target cells: SK-MEL 5 (HTB-70, ATCC) are an adherent melanoma-derived cell line that is grown in complete Eagle MEM and is passaged twice a week at 1:4. (Complete E-MEM - E-MEM, 10%FBS, 2mM Glutamine or Glutamax, 5ml Penicillin-Streptomycin).

[0289] The day prior to running the assay, PBMCs (5 million per 96 well plate) are thawed as required in complete RPMI (RPMI, 10% FBS, 2mM Glutamax, 2mM Pyruvate, 1% MEM, 1% HEPES, 1% P / S) and allow to recover overnight. Target cell lines are trypsinised and seeded in flatbottom transparent 96 well plate at 1.104 cells per well in 100 uL complete media. On the day of the assay, bispecific is serial diluted in complete RPMI at 4X concentration in 100 ul final volume. 100uL of PBMC at 1.106 c / mL is transferred on the bispecific plates. 100 ul of bispecific : effector mix is transferred onto target plates. This creates a ratio of 1:5 of Target to Effector.

[0290] Cytotoxicity is evaluated using a luminescence-based ATP assay which measures remaining viable target cells 48 h later. Briefly, culture plates are washed 3 times with PBS 1X and an equal volume of PBS and CellTiter Gio 2.0 (G9241 , Promega) reagent is added to each well. Plates are incubated at room temperature in the dark for 10 minutes before luminescence signal is read according to the manufacturer’s instruction. Luminescence signal is plotted against bispecific concentration to obtain a dose-response curve from which an EC50 value can be calculated.

Claims

Claims1. 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, wherein the pMHC binding arm comprises a binding site for the pMHC complex and the immune cell engager arm comprises a binding site for a surface receptor on the immune cell, wherein the said binding sites are not masked, and wherein 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 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 greater than 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, wherein the Fc includes a hinge region, e.g., wherein the Fc has N terminal sequence SEQ ID NO: 8.

24. A protein according to claim 23, wherein the protease cleavage site is N terminal to the hinge region of the Fc.

25. A protein according to any of claims 22 to 24, 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 the half life extending region comprises 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.

26. A protein according to claim 25, wherein the protein comprises a first polypeptide comprising, from N to C terminus, anti-CD3 scFv, TCR beta variable domain, TCR beta constant domain,a second polypeptide comprising, from N to C terminus, TCR alpha variable domain, TCR alpha constant domain, cleavable linker, first Fc CH2-CH3, and a third polypeptide comprising second Fc CH2-CH3.

27. A protein according to any preceding claim, wherein the cleavable linker is fused to a C terminal cysteine residue of a TCR constant domain (e.g., alpha constant domain) and to an N terminal hinge sequence (e.g., SEQ ID NO: 8) of an Fc.

28. A protein according to any preceding claim, wherein the cleavable linker comprises a protease cleavage site for granzyme B, a protease cleavage site for ST14 and / or a protease cleavage site for MMP.

29. 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.

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

31. A protein according to any of claims 1 to 29, or nucleic acid according to claim 30, provided in isolated form in a composition comprising a pharmaceutically acceptable excipient, formulated for delivery to a patient.

32. A method of treating a condition in a patient, comprising administering a soluble multispecific protein as defined in any of claims 1 to 29, or nucleic acid according to claim 30, to the patient.

33. A soluble multispecific protein according to any of claims 1 to 29, or nucleic acid according to claim 30, for use in method of treatment of the human or animal body by therapy.

34. A method according to claim 31 , or a soluble multispecific protein or nucleic acid for use according to claim 32, wherein the treatment comprises treating a solid tumour.

35. A population of recombinant host cells in vitro comprising nucleic acid encoding a soluble multispecific protein according to any of claims 1 to 29.

36. A method of producing a soluble multispecific protein according to any of claims 1 to 29, comprising culturing a population of cells according to claim 35 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.

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